纳米载体介导的Livin RNAi治疗大肠癌的实验研究
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摘要
大肠癌是临床上常见的消化道肿瘤之一,且大肠癌的发病率呈逐年上升。而肿瘤的发生是细胞增殖与凋亡失衡所致,凋亡抑制是肿瘤发生的重要机制。凋亡的调节机制非常复杂,近年发现的凋亡抑制蛋白家族(IAP)是一类重要的分子水平的抗凋亡调节因子,Livin是新近发现的一个IAP家族成员,研究表明,Livin在多数肿瘤中表达,而在除胎盘外的大多数终末分化组织中低表达或不表达。在大多数研究livin在肿瘤中的表达中,认为高表达的Livin mRNA和蛋白是肿瘤进展的预兆标记,与肿瘤的发生、发展及预后相关。因此许多种以IAP为靶点的抗肿瘤研究多聚焦于Livin,通过调控Livin基因也许可以为肿瘤的治疗提供新靶点。Livin与大肠癌发生、发展、转移及预后等关系及针对Livin治疗大肠癌研究目前国内外还未见详细报道。
RNA干扰技术(RNAi)是近年发展起来的新技术,RNAi引发的基因沉默作用是特异的和有效的,具有抑制作用强、稳定性高、细胞摄取相对容易等优点,为肿瘤的基因治疗提供新的、有前途的手段。是当前基因治疗研究最广为应用的基因沉默工具。小干扰RNA(siRNA)选择性抑制基因表达使基因治疗达到最大的特异性,但是弱小的细胞内导入能力、有限的血液稳定性及非特异的免疫原性阻碍了siRNA基因治疗的发展。目前,国内外的研究都没能有效解决将siRNA导入体内转染细胞的效率低下的问题。
基因转运需要合适的载体,理想的基因转运系统,应具有较高的基因转染效率、良好的靶向性和生物相容性、较高的稳定性及生物可降解性。目前的病毒载体和非病毒脂质体载体都存在不同程度的缺陷,因而寻找一种理想的基因载体就成为当务之急。纳米基因转运体的研制是目前国际纳米生物和肿瘤基因治疗研究领域重要的前沿性课题。纳米颗粒体积小,可在血管中随血液循环、透过血管壁进入各个脏器的细胞中,作为新型非病毒型基因载体能有效介导DNA的转导,并使其在细胞内高水平的表达,从而为基因表达、功能研究及基因治疗提供了新的技术和手段。壳聚糖(CS)是一种天然多聚糖,已有研究表明,壳聚糖纳米粒有良好的生物相容性和生物可降解性,且可通过修饰改性增加其靶向性和缓释性,是一种良好的基因载体。我们拟通过接枝共聚的方法将亲水无毒的聚乙二醇(PEG)链段引入壳聚糖链段中以增加其水溶性和缓释性,从而增加基因转染效率。
基于以上背景和已有的研究成果,我们先用RT-PCR和Westernblot方法分别从mRNA和蛋白质水平检测Livin基因的表达情况及与大肠肿瘤特性的相关性。结果显示,在所有的癌旁和正常组织中,Livin均不表达,而在肿瘤组织中,阳性表达率却达45.5%,两种方法的检测结果一致,说明Livin是大肠癌的一种标志物,这不仅有助于大肠癌的诊断,而且可以为大肠癌的生物治疗提供依据;分析Livin表达与结直肠癌患者主要临床参数之间的关系后发现,Livin在结直肠癌中表达情况与年龄、性别、肿瘤大小、肿瘤部位、组织学类型、淋巴是否转移、Dukes分期均无显著相关性,而与患者远处转移及放化疗密切相关(p＜0.05),提示Livin的表达可以影响结直肠癌的生物学行为,使其更易发生远处转移等恶性行为,是预后不良的指征之一;在放化疗后肿瘤组织中Livin表达也明显升高,提示其与肿瘤的耐药性有关。可能为大肠癌的诊断和预后判断提供新的分子生物学指标,同时也为下一步的肿瘤基因治疗提供了实验基础。
在第二部分,我们设计、合成了Livin shRNA,与pGenesil-1质粒载体链接构建重组质粒,通过酶切电泳、基因测序证实是否正确构建,通过转染高表达Livin的大肠癌HT-29细胞检测Livin mRNA下降水平,筛选出最佳的shRNA。结果重组质粒pGenesil-shRNA经酶切电泳、基因测序证明寡核苷酸片段成功插入预计位点,且序列与我们设计合成的完全一致,说明我们成功地构建shRNA真核表达载体pGenesil-shRNA重组质粒;重组质粒载体转染HT-29细胞后,肿瘤细胞livin mRNA含量较转染前及对照组均有明显的下降(p＜0.01),说明我们制备的shRNA能有效抑制Livin基因的表达,其中尤以Livin1抑制作用明显,抑制率达到66%。故在后续研究中,我们均以Livin1作为实验模板,探讨针对Livin基因的RNAi对肿瘤的治疗作用。
第三部分先通过接枝共聚的方法制备壳聚糖-聚乙二醇(CS-PEG)纳米粒,用透射电镜、激光粒度分析仪考察其形态、粒径、ζ电位,MTT法考察其细胞毒性,不同的基因/纳米粒材料比制备基因纳米复合物,通过其形态、粒径、ζ电位、载药量、包封率、基因保护实验考察药物的理化特性,转染后EGFP蛋白表达和LivinmRNA抑制率比较考察基因转染效果。结果制备的空白纳米粒粒径约60nm,细胞毒性较脂质体要低;在基因:纳米粒为1:5时(质量比)制备的基因纳米复合物粒径约为120nm,ζ电位为6.6mV,包封率和载药量分别为79.4%和32.3%,对基因有较好的保护作用,选此条件下制备的基因纳米复合物进行基因转染,持续作用时间较脂质体和裸基因均要长,转染72h后的Livin mRNA抑制率较两对照组明显增强(分别为p＜0.05和p＜0.01)。故后续研究中以此基因纳米复合物进行基因治疗研究。
第四部分为纳米载体介导的RNAi治疗结肠癌研究。培养HT-29结肠癌细胞后,以基因纳米复合物转染,72h后用RT-PCR和westernblot检测Livin mRNA和蛋白质抑制率,用流式细胞仪及荧光显微镜检测细胞凋亡情况,从体外考察基因治疗效果;建立裸鼠结肠癌动物模型,以基因纳米复合物作瘤内多次注射,绘制肿瘤生长曲线,24d后切取肿瘤检测体积抑制率和瘤重抑制率,组织切片TUNEL法检测细胞凋亡指数(AI),从动物在体模型考察基因治疗效果。结果发现试验组Livin mRNA和蛋白质的抑制率为78%和76%,较假性治疗组显著增强(p＜0.01);试验组的肿瘤生长明显减慢,瘤重抑制率和体积抑制率分别为74%和80%,较假性治疗组明显增强(p＜0.01);切片组织的凋亡细胞较对照组明显增多,AI增高(p＜0.01)。说明我们制备的CS-PEG纳米载体介导的Livin shRNA对Livin高表达的结肠癌有很好的治疗效果。
本研究仅为“863”课题的一部分,为大肠癌基因诊断和基因治疗新策略提供了一个初步的实验基础,其开发潜能及应用于临床本课题组还会进行更深一步的实验研究。
Colorectal carcinoma is one of the most frequent digestive canal cancers in clinic and the incidence rate goes up year by year.The occurrence of tumorous is as to the disequilibrium between cell proliferation and apoptosis.Apoptosis inhibition is an important mechanism of tumorous occurrence.The regulate mechanism of apoptosis is very complex.Inhibitor of apoptosis proteins(IAPs)are important anti-apoptosis regulatory factors of molecular level and Livin is a novel member of IAPs.Research found that Livin express highly in most tumors but lowly or negatively in most terminal differentiation tissue except for placenta.In most research about Livin expression in cancer,high expression of Livin mRNA and protein is considered the prognostic sign of cancer development,which is related with the incidence,devolepment and prognosis.So many researchs considered Livin as a target for therapy tumor focused on IAP.The cancer therapy may be found a new target by regulate Livin.Though research found that Livin has important significance in some tumors,so far the relationship of Livin with incidence,development,metastasis and prognosis and gene therapy for colorectal carcinoma has not detail report domestic and abroad.
RNA interference(RNAi)is a new technology developed in recent years.RNAi can be induced by small interference RNA(siRNA),plasmid or virus carrier that express short hairpin RNA(shRNA).The role of gene silencing of RNAi is specific and effective,which has some merit such as powerful inhibitory action,good stability and easily absorbed by cell,and provide a new and potent method for gene therapy of tumor.RNAi has becaming a widely used tool for gene silencing in current gene therapy research.SiRNA selectively inhibit gene express make gene therapy achieve maximal specificity.But puny introduction capability into cell, definite blood stability and non-specific immunogenicity hinder siRNA development for gene therapy.Researchs both domestic and abroad have not efficiently solved the problem of transfection efficiency low of siRNA.
Gene transfection need suitable carrier.An ideal gene transport system should has good gene transfection rate,favourable target and biocompatibility,fine stability and biological degradation.The viral vector and non-viral vector at present have different defect to some extent.So to find a ideal gene carrier is very critical.The research of nanoparticles for gene carrier is an impotant advancing front topic in fields of nano-biology and tumor gene therapy in international.Because of minute volume,nanoparticles can circulate in blood vessel,enter the cells of every organ through vessel wall,mediate DNA transfection efficiently and make it express highly in cell as a novel non-viral carrier.Which provides new technology and method for gene express,research of fuction and therapy.Chitosan(CS)is a natural polycose,which has good biocompatibility and biological degradation.Researchs indicate that CS is a very good gene carrier,which has better target and slow-release if it be modified and reshaped.Through inoculation and copolymerization,the atoxic polyethylene glycol(PEG)is inserted into CS strand to increase target and slow-release,at last increase gene transfection efficience.
Based on the background and achievement above,we detected the express of Livin from mRNA and protein level by RT-PCR and western blot and investigated the correspond with characteristic of colorectal carcinoma at first.The result indicated that Livin negative expressed in all para-cancerous tissue and normal tissue,but the express rate attained 45.5%in tumor.It had the same result by two methods.So it hinted that Livin is a marker for cancer of colon,which not only help for the diagnosis but also supply evidence for the biotherapy of it.Analyzed the relationship of Livin express with main clinic parameter we found that there was no significant correlation between livin expression with age, sex,tumor size,tumor location,histological type,lymphnode metastasis and Dukes classification(P＞0.05),however,livin expression was strongly correlated with distant metastasis and radio-/chemotherapy(P＜0.05).It hinted that the express of Livin can affect the biological behaviour of colorectal cancer and make incident of malignant behavior such as metastasis,which is one of the indications of unfavourable prognosis.The express of Livin is also increased remarkably after radiochemotherapy, which indicated that it related with drug resistance for tumor.So that it could supply a new parameter of molecular biology for diagnosis and prognosis.At the same time supplied experiment base for the next research of gene therapy.
In the second part,we designed and synthetized Livin shRNA,then linked with pGenesil-1 plasmid vector to construct recombinant plasmid, at last verified that the correct plasmid vector had been construct by enzyme-cutting electro-phoresis and gene sequencing.The plasmid was transfected into HT-29 cell that express highly of Livin and the Livin mRNA was detected to select the optimal shRNA.At last it verified that oligonucleotides were correctly inserted into the recombinant plasmid pGenesil-shRNA by enzyme-cutting electro-phoresis and gene sequencing and the sequence was completely the same as designed,which also illustrated that the eukaryotic expression vector was constructed correctly.After the recombinant plasmid vector was transfected into HT-29 cell,livin mRNA level of cancer cell was remarkably decreased than before transfection and control group(p＜0.01).It verified that the shRNA prepared can inhibit the express of Livin efficiently and the role of livinl is more obvious,the inhibition rate of which was 66 percent.So in the next research,Livinl was selected as experiment template to explore the effect of Livin RNAi for cancer therapy.
In the third part is preparing gene-nanoparticle compound.Chitosanpolyethylene glycol particle(CS-PEG)was synthesized firstly through stem grafting and copoly-merizing,then characteristics of nanoparticle was detected by evaluating shape,size and zeta electric potential and cytotoxicity was detected by MTT.Different ratio of gene/nanoparticle was used to prepared gene-nanoparticle compound.Then shape,size,zeta electric potential,envelopment rate and carry drug rate were inspected to evaluate characteristics of drug and expresses of EGFP protein and Livin mRNA were detected to evaluate effect of gene transfection.At last the blank nanoparticle size and zeta electric potential were 60 nm and 37mV respectively,the cytotoxicity was inferior to liposome.In conditions of 1:5 for gene/nanoparticle,the size,zeta electric potential,envelopment rate and carry drug rate of DNA-NP prepared were about 120nm,6.6my, 79.4%and 32.3%respectively.Which also had very good protection to gene.Selected the DNA-NP prepared in accordance with the ratio to continue gene transfection experiment,the persistence time was longer than both of liposome and bare gene,the inbition rate after 72h of transfection was reinforced significantly(p＜0.05 and p＜0.01 respectively).So the next step we selected it to do research of gene therapy.
The forth part is research of nanoparticle vector mediated RNAi to therapy colon cancer.At first HT-29 cell was cultivated,then the DNA-NP was transfected into it,after 72h detected inbition rate of Livin mRNA and protein by RT-PCR and western blot,detected apoptosis by flow cytometry and fluorescence microscope to inspect gene therapy efficacy in vitro.Established naked mouse colon carcinoma animal model, injected DNA-NP into tumor multiple,drew the growth curve of tumor,took out the tumor to detect volume inhibition ratio and weight inhibition ratio after 24 d,detected apoptotic index(AI)by histological section and TUNEL dyeing to inspect gene therapy role in animal cancer model.Results indicated that the inhibition ratio of Livin mRNA and protein of test group were about 78 and 76 percent respectively,which increased remarkably than spurious therapy groups(p＜0.01).In test group tumor grew slow remarkably,the mean volume inhibition ratio and weight inhibition ratio were 74 and 80 percent,which also increased significantly than contral groups(p＜0.01),apoptosis cell was significantly more than contral groups in tissue section and AI was increased(p＜0.01).Which indicated that the CS-PEG nanoparticle vector mediated Livin shRNA has very good therapeutic efficacy to colon cancer which Livin express is high.
The present research is just a part of National "863" High-technology Project,and supply a primary experiment basis for colorectal carcinoma gene diagnosis and gene therapy new strategy.The exploitation potentiality and clinical application will have more profound research.
RNA干扰技术(RNAi)是近年发展起来的新技术,RNAi引发的基因沉默作用是特异的和有效的,具有抑制作用强、稳定性高、细胞摄取相对容易等优点,为肿瘤的基因治疗提供新的、有前途的手段。是当前基因治疗研究最广为应用的基因沉默工具。小干扰RNA(siRNA)选择性抑制基因表达使基因治疗达到最大的特异性,但是弱小的细胞内导入能力、有限的血液稳定性及非特异的免疫原性阻碍了siRNA基因治疗的发展。目前,国内外的研究都没能有效解决将siRNA导入体内转染细胞的效率低下的问题。
基因转运需要合适的载体,理想的基因转运系统,应具有较高的基因转染效率、良好的靶向性和生物相容性、较高的稳定性及生物可降解性。目前的病毒载体和非病毒脂质体载体都存在不同程度的缺陷,因而寻找一种理想的基因载体就成为当务之急。纳米基因转运体的研制是目前国际纳米生物和肿瘤基因治疗研究领域重要的前沿性课题。纳米颗粒体积小,可在血管中随血液循环、透过血管壁进入各个脏器的细胞中,作为新型非病毒型基因载体能有效介导DNA的转导,并使其在细胞内高水平的表达,从而为基因表达、功能研究及基因治疗提供了新的技术和手段。壳聚糖(CS)是一种天然多聚糖,已有研究表明,壳聚糖纳米粒有良好的生物相容性和生物可降解性,且可通过修饰改性增加其靶向性和缓释性,是一种良好的基因载体。我们拟通过接枝共聚的方法将亲水无毒的聚乙二醇(PEG)链段引入壳聚糖链段中以增加其水溶性和缓释性,从而增加基因转染效率。
基于以上背景和已有的研究成果,我们先用RT-PCR和Westernblot方法分别从mRNA和蛋白质水平检测Livin基因的表达情况及与大肠肿瘤特性的相关性。结果显示,在所有的癌旁和正常组织中,Livin均不表达,而在肿瘤组织中,阳性表达率却达45.5%,两种方法的检测结果一致,说明Livin是大肠癌的一种标志物,这不仅有助于大肠癌的诊断,而且可以为大肠癌的生物治疗提供依据;分析Livin表达与结直肠癌患者主要临床参数之间的关系后发现,Livin在结直肠癌中表达情况与年龄、性别、肿瘤大小、肿瘤部位、组织学类型、淋巴是否转移、Dukes分期均无显著相关性,而与患者远处转移及放化疗密切相关(p＜0.05),提示Livin的表达可以影响结直肠癌的生物学行为,使其更易发生远处转移等恶性行为,是预后不良的指征之一;在放化疗后肿瘤组织中Livin表达也明显升高,提示其与肿瘤的耐药性有关。可能为大肠癌的诊断和预后判断提供新的分子生物学指标,同时也为下一步的肿瘤基因治疗提供了实验基础。
在第二部分,我们设计、合成了Livin shRNA,与pGenesil-1质粒载体链接构建重组质粒,通过酶切电泳、基因测序证实是否正确构建,通过转染高表达Livin的大肠癌HT-29细胞检测Livin mRNA下降水平,筛选出最佳的shRNA。结果重组质粒pGenesil-shRNA经酶切电泳、基因测序证明寡核苷酸片段成功插入预计位点,且序列与我们设计合成的完全一致,说明我们成功地构建shRNA真核表达载体pGenesil-shRNA重组质粒;重组质粒载体转染HT-29细胞后,肿瘤细胞livin mRNA含量较转染前及对照组均有明显的下降(p＜0.01),说明我们制备的shRNA能有效抑制Livin基因的表达,其中尤以Livin1抑制作用明显,抑制率达到66%。故在后续研究中,我们均以Livin1作为实验模板,探讨针对Livin基因的RNAi对肿瘤的治疗作用。
第三部分先通过接枝共聚的方法制备壳聚糖-聚乙二醇(CS-PEG)纳米粒,用透射电镜、激光粒度分析仪考察其形态、粒径、ζ电位,MTT法考察其细胞毒性,不同的基因/纳米粒材料比制备基因纳米复合物,通过其形态、粒径、ζ电位、载药量、包封率、基因保护实验考察药物的理化特性,转染后EGFP蛋白表达和LivinmRNA抑制率比较考察基因转染效果。结果制备的空白纳米粒粒径约60nm,细胞毒性较脂质体要低;在基因:纳米粒为1:5时(质量比)制备的基因纳米复合物粒径约为120nm,ζ电位为6.6mV,包封率和载药量分别为79.4%和32.3%,对基因有较好的保护作用,选此条件下制备的基因纳米复合物进行基因转染,持续作用时间较脂质体和裸基因均要长,转染72h后的Livin mRNA抑制率较两对照组明显增强(分别为p＜0.05和p＜0.01)。故后续研究中以此基因纳米复合物进行基因治疗研究。
第四部分为纳米载体介导的RNAi治疗结肠癌研究。培养HT-29结肠癌细胞后,以基因纳米复合物转染,72h后用RT-PCR和westernblot检测Livin mRNA和蛋白质抑制率,用流式细胞仪及荧光显微镜检测细胞凋亡情况,从体外考察基因治疗效果;建立裸鼠结肠癌动物模型,以基因纳米复合物作瘤内多次注射,绘制肿瘤生长曲线,24d后切取肿瘤检测体积抑制率和瘤重抑制率,组织切片TUNEL法检测细胞凋亡指数(AI),从动物在体模型考察基因治疗效果。结果发现试验组Livin mRNA和蛋白质的抑制率为78%和76%,较假性治疗组显著增强(p＜0.01);试验组的肿瘤生长明显减慢,瘤重抑制率和体积抑制率分别为74%和80%,较假性治疗组明显增强(p＜0.01);切片组织的凋亡细胞较对照组明显增多,AI增高(p＜0.01)。说明我们制备的CS-PEG纳米载体介导的Livin shRNA对Livin高表达的结肠癌有很好的治疗效果。
本研究仅为“863”课题的一部分,为大肠癌基因诊断和基因治疗新策略提供了一个初步的实验基础,其开发潜能及应用于临床本课题组还会进行更深一步的实验研究。
Colorectal carcinoma is one of the most frequent digestive canal cancers in clinic and the incidence rate goes up year by year.The occurrence of tumorous is as to the disequilibrium between cell proliferation and apoptosis.Apoptosis inhibition is an important mechanism of tumorous occurrence.The regulate mechanism of apoptosis is very complex.Inhibitor of apoptosis proteins(IAPs)are important anti-apoptosis regulatory factors of molecular level and Livin is a novel member of IAPs.Research found that Livin express highly in most tumors but lowly or negatively in most terminal differentiation tissue except for placenta.In most research about Livin expression in cancer,high expression of Livin mRNA and protein is considered the prognostic sign of cancer development,which is related with the incidence,devolepment and prognosis.So many researchs considered Livin as a target for therapy tumor focused on IAP.The cancer therapy may be found a new target by regulate Livin.Though research found that Livin has important significance in some tumors,so far the relationship of Livin with incidence,development,metastasis and prognosis and gene therapy for colorectal carcinoma has not detail report domestic and abroad.
RNA interference(RNAi)is a new technology developed in recent years.RNAi can be induced by small interference RNA(siRNA),plasmid or virus carrier that express short hairpin RNA(shRNA).The role of gene silencing of RNAi is specific and effective,which has some merit such as powerful inhibitory action,good stability and easily absorbed by cell,and provide a new and potent method for gene therapy of tumor.RNAi has becaming a widely used tool for gene silencing in current gene therapy research.SiRNA selectively inhibit gene express make gene therapy achieve maximal specificity.But puny introduction capability into cell, definite blood stability and non-specific immunogenicity hinder siRNA development for gene therapy.Researchs both domestic and abroad have not efficiently solved the problem of transfection efficiency low of siRNA.
Gene transfection need suitable carrier.An ideal gene transport system should has good gene transfection rate,favourable target and biocompatibility,fine stability and biological degradation.The viral vector and non-viral vector at present have different defect to some extent.So to find a ideal gene carrier is very critical.The research of nanoparticles for gene carrier is an impotant advancing front topic in fields of nano-biology and tumor gene therapy in international.Because of minute volume,nanoparticles can circulate in blood vessel,enter the cells of every organ through vessel wall,mediate DNA transfection efficiently and make it express highly in cell as a novel non-viral carrier.Which provides new technology and method for gene express,research of fuction and therapy.Chitosan(CS)is a natural polycose,which has good biocompatibility and biological degradation.Researchs indicate that CS is a very good gene carrier,which has better target and slow-release if it be modified and reshaped.Through inoculation and copolymerization,the atoxic polyethylene glycol(PEG)is inserted into CS strand to increase target and slow-release,at last increase gene transfection efficience.
Based on the background and achievement above,we detected the express of Livin from mRNA and protein level by RT-PCR and western blot and investigated the correspond with characteristic of colorectal carcinoma at first.The result indicated that Livin negative expressed in all para-cancerous tissue and normal tissue,but the express rate attained 45.5%in tumor.It had the same result by two methods.So it hinted that Livin is a marker for cancer of colon,which not only help for the diagnosis but also supply evidence for the biotherapy of it.Analyzed the relationship of Livin express with main clinic parameter we found that there was no significant correlation between livin expression with age, sex,tumor size,tumor location,histological type,lymphnode metastasis and Dukes classification(P＞0.05),however,livin expression was strongly correlated with distant metastasis and radio-/chemotherapy(P＜0.05).It hinted that the express of Livin can affect the biological behaviour of colorectal cancer and make incident of malignant behavior such as metastasis,which is one of the indications of unfavourable prognosis.The express of Livin is also increased remarkably after radiochemotherapy, which indicated that it related with drug resistance for tumor.So that it could supply a new parameter of molecular biology for diagnosis and prognosis.At the same time supplied experiment base for the next research of gene therapy.
In the second part,we designed and synthetized Livin shRNA,then linked with pGenesil-1 plasmid vector to construct recombinant plasmid, at last verified that the correct plasmid vector had been construct by enzyme-cutting electro-phoresis and gene sequencing.The plasmid was transfected into HT-29 cell that express highly of Livin and the Livin mRNA was detected to select the optimal shRNA.At last it verified that oligonucleotides were correctly inserted into the recombinant plasmid pGenesil-shRNA by enzyme-cutting electro-phoresis and gene sequencing and the sequence was completely the same as designed,which also illustrated that the eukaryotic expression vector was constructed correctly.After the recombinant plasmid vector was transfected into HT-29 cell,livin mRNA level of cancer cell was remarkably decreased than before transfection and control group(p＜0.01).It verified that the shRNA prepared can inhibit the express of Livin efficiently and the role of livinl is more obvious,the inhibition rate of which was 66 percent.So in the next research,Livinl was selected as experiment template to explore the effect of Livin RNAi for cancer therapy.
In the third part is preparing gene-nanoparticle compound.Chitosanpolyethylene glycol particle(CS-PEG)was synthesized firstly through stem grafting and copoly-merizing,then characteristics of nanoparticle was detected by evaluating shape,size and zeta electric potential and cytotoxicity was detected by MTT.Different ratio of gene/nanoparticle was used to prepared gene-nanoparticle compound.Then shape,size,zeta electric potential,envelopment rate and carry drug rate were inspected to evaluate characteristics of drug and expresses of EGFP protein and Livin mRNA were detected to evaluate effect of gene transfection.At last the blank nanoparticle size and zeta electric potential were 60 nm and 37mV respectively,the cytotoxicity was inferior to liposome.In conditions of 1:5 for gene/nanoparticle,the size,zeta electric potential,envelopment rate and carry drug rate of DNA-NP prepared were about 120nm,6.6my, 79.4%and 32.3%respectively.Which also had very good protection to gene.Selected the DNA-NP prepared in accordance with the ratio to continue gene transfection experiment,the persistence time was longer than both of liposome and bare gene,the inbition rate after 72h of transfection was reinforced significantly(p＜0.05 and p＜0.01 respectively).So the next step we selected it to do research of gene therapy.
The forth part is research of nanoparticle vector mediated RNAi to therapy colon cancer.At first HT-29 cell was cultivated,then the DNA-NP was transfected into it,after 72h detected inbition rate of Livin mRNA and protein by RT-PCR and western blot,detected apoptosis by flow cytometry and fluorescence microscope to inspect gene therapy efficacy in vitro.Established naked mouse colon carcinoma animal model, injected DNA-NP into tumor multiple,drew the growth curve of tumor,took out the tumor to detect volume inhibition ratio and weight inhibition ratio after 24 d,detected apoptotic index(AI)by histological section and TUNEL dyeing to inspect gene therapy role in animal cancer model.Results indicated that the inhibition ratio of Livin mRNA and protein of test group were about 78 and 76 percent respectively,which increased remarkably than spurious therapy groups(p＜0.01).In test group tumor grew slow remarkably,the mean volume inhibition ratio and weight inhibition ratio were 74 and 80 percent,which also increased significantly than contral groups(p＜0.01),apoptosis cell was significantly more than contral groups in tissue section and AI was increased(p＜0.01).Which indicated that the CS-PEG nanoparticle vector mediated Livin shRNA has very good therapeutic efficacy to colon cancer which Livin express is high.
The present research is just a part of National "863" High-technology Project,and supply a primary experiment basis for colorectal carcinoma gene diagnosis and gene therapy new strategy.The exploitation potentiality and clinical application will have more profound research.
引文
[1] Fischer U, Schulze OK. New approaches and therapeutics targeting apoptosis in disease[J]. Pharmacol Rev, 2005,57 (2),187-215.
[2] Yang Y ,Yu X. Regulation of apoptosis: the ubiquitous way[J]. FASEB J, 2003,17 (8),790-799.
[3] Carter BZ, Gronda M, Wang Z, et al. Small molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells[J]. Blood, 2005,105 (10),4043-4050.
[4] Zhang M, Latham DE, Delaney MA,et al. Survivin mediates resistance to antiandrogen therapy in prostate cancer[J]. Oncogene,2005,24 (15), 2474-2482.
[5] Kim EH, Kim SU, Choi KS. Rottlerin sensitizes glioma cells to TRAIL-induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP[J]. Oncogene, 2005, 24 (46),838-849.
[6] Demarchi F ,Brancolini C. Altering protein turnover in tumor cells: new opportunities for anti-cancer therapies[J]. Drug Resist Updat, 2005,8 (6),359-368.
[7] Watson R , Fitzpatrick JM. Targeting apoptosis in prostate cancer: focus on caspases and inhibitors of apoptosis proteins[J]. BJU Int 2005,96 (2),30-34.
[8] Roy N, Mahadevan MS, Mclean M,et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy[J]. Cell, 1995,80(1): 167-178.
[9] Rothe M, Pan MG, Henzel WJ, Ayres T M ,et al. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins[J ]. Cell, 1995,83(7): 1243-1252.
[10] Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes[J ]. Nature ,1996,379 (6563):349-353.
[11] Ambrosini G, Adida C , Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J ]. Nat Med, 1997,3 (8) : 917-921.
[12] Chen Z, Naito M, Hori S,et al. A human IAP-family gene, apollon, expressed in human brain cancer cells[J]. Biochem. Biophys Res Commun, 1999,264 (3):847-854.
[13] Richter BW, Mir SS, Eiben LJ, et al. Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family[J ]. Mol Cell Biol, 2001,21 (13): 4292-4301.
[14] Kasof GM, Gomes BC. Livin ,a novel inhibitor of apoptosis protein family member[J ] .Biol Chem,2001 ,276 (5) :3238 - 3246.
[15] Lacass EC, Baird S, Komeluk RG, et al. The inhibitors of apoptosis (IAPs) and their emerging role in cancer [J]. Oncogene, 1998,17 (25) :3247 - 3259.
[16] Vucic D, Stennicke HR, Pisabarro MT ,et al . ML- IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas [J ] . Curr Biol, 2000 ,10(21) :1359-1366.
[17] Boaz N ,Yaqoub A ,Vered B ,et al. caspase-mediated cleavage convert s livin from an antiapoptotic to a proapoptotic factor: implications for drug-resistant melanoma[J ]. Cancer Res ,2003 , 63 (10) :6340
[18] Ashhab Y, Alian A , Polliack A ,et al. Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue dist ribution pattern [J] .FEBS Lett ,2001 ,495 (4) :56
[19] Lin JH, Deng G, Huang Q ,et al. KIAP, a novel member of the inhibitor of apoptosis protein family[J]. Biochem Biophys Res Commun, 2000,279 (6), 820-831.
[20] Yagihashi A, Asanuma K, Tsuji N, et al. Detection of anti-Livin antibody in gastrointestinal cancer patients[J]. Clin Chem 2003; 49:1206-1208.
[21] Deveraux QL, Reed JC.IAP family proteins-suppressors of apoptosis[J ]. Genes Dev, 1999,13(3):239-252.
[22] Miller L.An exegesis of IAPs: salvation and surprises from BIR motifs[J ]. Trends Cell Biol, 1999,9 (8):323-328.
[23] Song Z, Yao X, Wu M. Direct interaction between Survivin and Smac is essential for the anti-apoptotic activity of Survivin during Taxol-induced apoptosis[J]. J Biol Chem, 2003 ; 278 (25): 23130-23140
[24] Maier JK, Lahoua Z, Gendron NH et al. The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7[J]. J Neuro Sci ,2002 ;22 (6) :2035-2043
[25] Liu Z,Sun C, Olejniczak ET, et al. Structural basis for binding of SMAC/DIABLO to the XIAP BIR3 domain[J]. Nature, 2000,408 (6815): 1004-1008.
[26] Wu G, Chai J, Suber TL, et al. Structural basis of IAP recognition by SMAC/DIABLO[J],Nature,2000,408(6815):1008-1012.
[27]Chai J,Du C,Wu JW,et al.Structural and biochemical basis of apoptotic activation by SMAC/DIABLO[J].Nature,2000,406(6798):855-862.
[28]Vucic D,Franklin MC,Wallweber HJ,et al.Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor:imp lications for SMAC-dependent anti-apop totic activity ofML2IAP[J].Biochem J,2005,385(Pt 1):11-20.
[29]Vucic D,Deshayes K,Ackerly H,et al.SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP)[J].J Biol Chem,2002;277(14):12275-12279
[30]Kasof GM,Gomes BC.Livin,a novel inhibitor of apoptosis protein family member[J].Biol Chem,2001,276(5):3238-3246.
[31]Vucic D,Stennicke HR,Pisabarro MT,et al.ML-IAP,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas[J].Curr Biol,2000,10(21):1359-1366.
[32]Hariu H,Hirohashi Y,Torigoe T,et al.Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family,Livin/ML-IAP in lung cancer[J].Clin Cancer Res.2005;11(3):1000-1009
[33]Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of livin,survivin and other apoptosis-related genes in the progression of superficial bladder cancer[J].Ann Oncol,2003,14(1):85-90.
[34]李显文,杨罗艳,王红珊,等.凋亡抑制蛋白Livin在膀胱移行细胞癌中的表达及临床意义[J].临床泌尿外科杂志,2006,21(3):216-220.
[35]孙建国,廖荣霞,陈正堂,等.Livin异构体基因转染对肺腺癌A549细胞生长及放化疗敏感性的影响[J].中华结核和呼吸杂志,2005,28(12):836-840.
[36]Crnkovic-Mertons I,Hoppe-Seyler F,Butz K.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the Livin / ML-IAP / KIAP gene[J].Oncogene,2003.22(51):8330-8336.
[37]Zhang Q,Xiong J,Jin Y,et al.CC chemokine ligand 25 enhances restance to apoptosis in CD4+ T cells from patients with T-cell-lineage acute and chronic lymhocytie leukemia by means of Livin activation[J].Cancer Res,2004,64(20):7579-7587.
[38]Zhang Q,Xiong J,Jin Y.et al.Selectively frequent expression of CXCR5enhances resistance to apoptoeis in CD8(+)CD34(+)T cells from patients with T-cell-lineage acute lymphocytic leukemia[J].Oneogene,2005,24(4):573-584.
[1] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic intederenee by double—stranded RNA in Caenorhabdltis eIegatm[J]. Nature. 1998. 391: 806-811.
[2] Song E ,Lee S K,Wang J ,Ince N ,et al. RNA i nterference targeting Fas protects mice from fulminanth epatitis[J].Nat Med,2003,9(3):347-351
[3] Brummelkamp T R,Bernards R ,Agami R .Stable suppression of tumor igenicity by virus-mediate dRNA interference [J]. Cancer Cell,2002;2(3):245-249
[4] Paul CP, Good PD, Winer L, et al. Efective expression of small interfering RNA in human cells[J]. Nat Biotechnol, 2002, 20(5): 505—508.
[5] Sui G, Soohoo C, Afarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells[J]. Proc Nail Acad sci USA,2002, 99(8): 5515-5520.
[6] Yu JY. DeRuiter SL, Turner DL. RNA interference by expression of short-intefering and hairpin RNAs in mammalian cells[J]. Proc Natl Acad Sci USA, 2002. 99(9): 6047—6052.
[7] Yu JY,DeRuiter SL,Tumer D L.RNA interference by expression of short-interfering RNAs and haifpin RNAs in mammalian cells[J]. Proc Natl Acad Sci UsA,2002, 99(9): 6047-6052.
[8] Haqnon GJ,Conklin DS. RNA interference by shorthairpin RNAs expressed in vertebrate cells[J]. Methods Mol Biol, 2004, 257: 255-266.
[9] Uitel K, Nalto Y, Takabashi F, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference[J]. Nueleic Acids Res,2004,32: 936-948.
[10] Hsieh AC, Bo R, Manola J, et al. A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens[J]. Nucleic Acids Res, 2004, 32: 893-901.
[11] Uchida H. TanakaT, Sasaki K, et al. Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo[J]. Mol Ther, 2004, 10(1): 162-171.
[12] Mazor M,KawanoY, Zhu H, et al. Inhibition of glycogen synthase kinase-3 represses androgen receptor activity and prostate cancer cell growth[J]. Oneogene, 2004,23(47): 7882-7892.
[13]Wilda M,Fuchs U,Wossmarm W,et al.Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference(RNAi)[J].Oncogene,2002,21(37):5716-5721
[14]Qiu S,Adema CM,Lane T.A computational study of off-target effects of RNA interference[J].Nucleic Acids Res,2005,33(6):1834-1847.
[15]Miller LK.An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif[J].J Virol,1993,67(4):2168-2174.
[16]Hakhyun Ka,Joan S,Hunt.Temporal and spatial paterns of Expression of Inhibitors of apoptosis in human placentas[J].Am J Pathol,2003,163(2);413-422
[17]Zhang HD,Yuan SJ,Chen HP,et al.Induction effects of antisense phosphorothioate oligodeoxynucleotides of livin mRNA on apoptosis in MCF-7cells[J].Chin J clin Pharmacol Them,2004,9(12):1353-1356.
[18]Hannon GJ.RNA interference[J].Nature,2002,418:244-251.
[19]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic intederenee by double-stranded RNA in Caenorhabdltis elegatm[J].Nature,1998,391:806-811.
[20]Caplen NJ.A new approach to the inhibition of gene express[J].Trends Biotechnol,2002.20:49-51.
[21]Uprichard S L.The therapeutic potential of RNA interference.FEBS Lett,2005,579(26):5996-6007.
[22]Elbashir SM,Harbort h J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cell lines[J].Nature,2001,411:494
[23]McManus MT,Sharp PA.Gene silencing in mammalians by small interfering RNAs[J].Nat Rev Gene,2002,3:737
[24][美]J.萨姆布鲁克,D.W.拉萨尔,著,黄培堂译.分子克隆实验指南(第三版)(下册)[M].北京:科学技术出版社,2002:1595-1598.
[25][美]J.萨姆布鲁克,D.W.拉萨尔,著,黄培堂译.分子克隆实验指南(第三版)(上册)[M].北京:科学技术出版社,2002:96-99.
[26]Lewis KE,Stephens C,Shahidi MM,et al.Delay in starting treatment for tuberculosis in east London[J].Commun Dis Public Health,2003,6(2):133
[27]Lienhardt C,Rowley J,Manneh K,et al.Factors affecting time delay to treatment in a tuberculosis control programme in a sub-Saharan African country:the experience of t he Gambia[J].Int J Tuberc Lung Dis,2001,5(3):233
[28] Demissie M , Lindtjorn B , Berhane Y. Patient and health service delay in the diagnosis of pulmonary tuberculosis in Ethiopia. [J] BMC Public Health ,2002,2 (1) :23
[29] Hariu H , Hirohashi Y, Torigoe T , et al. Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family , Livin/ ML2IAP in lung cancer[J ]. Clin Cancer Res ,2005 ,11 (3): 1000-1009
[30] Crnkovic M, Wagener N, Semzow J,et,al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J].Cell Mol Life Sci, 2007,64 (9): 1137-1144
[31] Nykallen A, Haley B, Zamore PD. A1P requirements and small interfering RNA structure in the RNA interference pathway. Cell, 2001, 107(3): 309-321.
[1]Wu Q,Moyana T,Xiang J.Cancer gene therapy by adenovirus-mediated gene transfer.Curr Gene Ther 2001;1(1):101-122
[2]Lai CM,Lai YK,Rakoczy PE.Adenovirus and adeno-associated virus vectors.DNA Cell Biol.2002;21(12):895-913.
[3]郭荣,邹萍,陆华中.非病毒型纳米载体在基因治疗中的研究现状及展望.国外医学生物医学工程分册,2002,25(2):77-81.
[4]Pierre L,Sylvie F,Noufrssa O,eta l.Gene delivery systems:Bridging the gap between Recombinant viruses and artificial vectors.Adv Drug Delve.1998(30):5-11.
[5]Truong L,Walsh W,Scot M,et al.Gene transfer by DNA-gelatin nanospheres.Arch Biochem Biophys.1999(361):47-56.
[6]Fox JL.Gene therapy safety issues come to fore.Nat Biotechnol.1999;17(12):1153.
[7]Wu X,Li Y,Crise B,Burgess SM.Transcription start regions in the human genome are favored targets for MLV integration.Science.2003;300(5626):1749-1751.
[8]Sun X,Zhang HW,Zhang ZR.Transfection efficiency of pORF lacZ plasmid lipopolyplex to hepatocytes and hepatoma cells.World J Gastroenterol.2004;10(4):531-534.
[9]胡云霞,原续波,张晓金,等.阳离子多聚物纳米基因载体系统的研究进展.北京生物医学工程,2003,22(4):299-302.
[10]Colin W,Pouton S,Leonard W,et al.Key issues in non-viral gene delivery.Adv Drug Delve.1998(34):3-19.
[11]Cristiano R J.Targeted,non-viral gene delivery for cancer gene therapy.Front Biosci.19 98(3):D1161-D1170.
[12]Anderson W EHuman gene therapy.Nature.1998,392(6679 Suppl),25-30.
[13]Richardson SCW,Kolbe HVJ,Duncan R,et al.Potential of low molecular mass chitosan as a DNA delivery system:biocompatibility,body distribution and ability to complex and protect DNA.Int J Pharm,1999,178(2):231-243.
[14]Mao HQ,Roy K,Troung-Le VL,et al.Chitosan-DNA nanopaticles as gene carriers:synthesis,characterization and transfection efficiency.J Controlled Release,2001,70(3):399-421.
[15]Lambert G,Fattal E,Couvreur P.Nanoparticμlate systems for the delivery of antisense oligonucleotides.Adv Drug Deliv Rev,2001,47(1):99-112.
[16]Zhu SG,Lu WB,Xiang JJ,et al.A novel nonviviral-nanoparticle gene vector:poly-lysine silica nanoparticales[J].Chin Sci Bμll,2002,47(8):654-658.
[17]汪杨,吴伟.隐形纳米粒的体内靶向性[J].中国药学杂志,2004,39(1):7-11.
[18]Peraeehia M T,Fattal E,Desmaele D.Stealth PEGylalted polyeyanoaerylate nanopartides for intravenous adminstration and splenic targeting[i].J Controlled Release,1999,60:121-130.
[19]Gref R,Ialek M,Qudlee P,et al.'Stealth' corono-core nanopartides surface modified by polyethylene glycol(PEG):influenees the corona(PEG chain length and surface density)and the core composition on phacocytic uptake and plasma protein adsorption[J].Colloids Surf B Biointerface,2000,18(3～4):301-308.
[20]Richardson SCW,Kolbe HVJ,Duncan R,et al.Potential of low molecμlar mass chitosan as a DNA delivery system:biocompatibility,body distribution and ability to complex and protect DNA[J].Int J Pharm,1999,178(2):231-243.
[21]Mao HQ,Roy K,Troung LeVL,et al.Chitosan-DNA nanopaticles as gene carriers:synthesis,characterization and transfection efficiency[J].J Controlled Release,2001,70(3):399-421.
[22]Maclaughlin FC,Mumper RJ,Wang J,et al.Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery.J Controlled Release,1998,56(1-3):259-272
[23]Kneuer C,Ehrhardt C,Bakowsky H,et al.The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes:a comparative study.J Nanosci Nanotechnol,2006,6(9-10):2776-2782.
[24]Alexakis T,Boadi DK,Quong D,et al.Mieroeneapsμlation of DNA within alginate microspheres and crosslinked chitosan membi'anes for in vivo application[J].Appl Biochem Biotechnol,1995,50(1):93-106.
[25]Mumper R J,Wang J,Claspell J M,et al.In:Proc Intl Symp Controlled,Rel Bioact Mater,1995,23:178.
[26]Mohapatra SS.Mucosal gene expression vaccine:a novel vaccine strategy for respiratory syncytial virus[J].Pediatr Inject Dis,2003,22(2):S100-S104.
[27]Iqbal M,Lin W,Jabbal-Gill I,et al.Nasal delivery of chitosan-DNA plasmid expressing cpitopesot respiratory syncytial virus(RSV)induces protective CTL responses in BALB/c nfice[J].Vaccine,2003,21(13-14):1478-1485.
[28]Li YH,Fan MW,Bian Z,et al.Chitosan-DNA microparticles as mucosal delivery system:synthesis,characterization and release in vitro[J].Clin Med J,2005,118(11):936-941.
[29]Vanessa Carla Furtado Mosqueira,Philippe Legrand,Annette Gulik et al.Biomaterials[J],2001,22:2967-2979
[30]Mosqueira VC,Legrand P,Gulik A,et al.Relationship between complement activation,cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.Biomaterials,2001,22(22):2967-2979.
[31]K Park,YH Park,BA Shin,ESC hoi,YR Park,T Akaike,CS Cho,Galactosylated chitosan-graft-dextran as hepatocyte-targetting DNA carrier.J Control Release,2000,69:97-108.
[32]FC MacLaughlin,RJ Mumper,et al.Rolland Chitosan and depolymerized chitosan oligomers as condensing carriers in vivo plamid delivery.J Control Release.1998,56:259-272.
[33]林友文,许晨.甲壳素及其衍生物的医学应用.福建医科大学学报,1999,2:226-228.
[34]张阳德,孙颖.一种新型的非病毒基因转运载体.纳米粒基因转运体.中国现代医学杂志,2003,13(21):148-151.
[35]朱诗国,李桂源.纳米基因转运体-原理,研制与应用.生物化学与生物物理进展,2002,29(6):868-871.
[36]基因表达技术[M].李育阳主编.科学出版社,2001:197-199
[37]Maclaughlin FC,Mumper RJ,Wang J,et al.Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery.J Controlled Release,1998,56(1-3):259-272
[38]Kneuer C,Ehrhardt C,Bakowsky H,et al.The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes:a comparative study.J Nanosci Nanotechnol,2006,6(9-10):2776-2782.
[39]Mosqueira VC,Legrand P,Gulik A,et al.Relationship between complement activation,cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.Biomaterials,2001,22(22):2967-2979.
[40]Leong KW,Mao HQ,Truong-LeVL,Roy K,Walsh SM,AugustJ T.DNA-poly-cation nanospherea as non-viral gene delivery vehicles.J Control Release 1998,30;53(1-3);183-93.
[41]Leong KW,Jiang X,Dai H,et al.Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions.J Gene Med,2006,8(4):477-487
[42]TaanoRM,Florea BI,Geldof M,et al.Quatemized chitesan oligomem as novel gene delivery vectors in epithelial cell lines.Biomatefials,2001,23(1):153.
[43]Illum L,Jabbal Gill I,Hinchcliffe M,et al.Chitosan as a novel nasal delivery system for vaccines.Adv Drug Deliver Rev,2001,51(1-3):81-96.
[44]Gill DR,Southern KW,Moford KA,et al.A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis.Gene Ther.1997(4):199-209.
[1] Steller H.Mechanisms and genes of ellularsuiocide [J].Science, 1995;267(10),14 45 -1 44 9 .
[2] Mattson M P.Apoptosis in neurodegenerative disorders.Nat Rev Mol Cell Biol,2000 ,1:1 20-129.
[3] Rathmell JC ,Thompson C B.P thways of apoptosis in lymphocyted evelopment,homeostasis,and disease.Cell,2002,109 Suppl:97 -107
[4] Reed CJ.Apoptosis and cancer: strategies for integratingp rogrammed cell death.Semin Hematol,2000,37 :9-16.
[5] LaCasse EC,Baird S,Korneluk RG,et al .The inhibitors of apoptosis(I A-Ps)and their emerging role in cancer.Oncogene,1998,17 :3247-3259.
[6] Thompson CB.Apoptosis in the pathogenesis and treatment of disease[J]. Science,1995,267(10):1456-1462.
[7] Deveraux Q L and Reed J C.IAP family proteins-suppressors of apoptosis[J]. Genes Dev, 1999,13 (3):239-252.
[8] Miller L. An exegesis of IAPs: salvation and surprises from BIR motifs[J]. Trends Cell Biol, 1999,9 (5):323-328.
[9] Richter B W, Mir S S, Eiben L J, et al. Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family[J ]. Mol Cell Biol, 2001,21 (13): 4292-4301.
[10] Liu Z, Sun C, Olejniczak E T, et al. Structural basis for binding of Smac/ DIABLO to the XIAP BIR3 domain[J ]. Nature, 2000,408 (6815): 1004-1008.
[11] Wu G, Chai J, Suber T L, et al. Structural basis of IAP recognition by SMAC/DIABLO[J], Nature, 2000,408 (6815):1008-1012.
[12] Kasof GM,Gomes BC. Livin ,a novel inhibitor of apoptosis protein family member[J ] .Biol Chem,2001,276 (5) :3238 - 3246.
[13] Vucic D ,Stennicke HR ,Pisabarro MT ,et al . ML- IAP ,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas [J ] . Curr Biol, 2000 ,10(21) :1359-1366.
[14] Ashhab Y, Alian A , Polliack A ,et al. Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue dist ribution pattern [ J ] .FEBS Lett ,2001,495 (4) :56
[15]Lin JH,Deng G,Huang Q,et al.KIAP,a novel member of the inhibitor of apoptosis protein family[J].Biochem Biophys Res Commun,2000,279(6),820-831.
[16]Zhang M,Latham D E,Delaney M A,et al.Survivin mediates resistance to antiandrogen therapy in prostate cancer[J].Oncogene,2005,24(15),2474-2482.
[17]Vucic D,Deshayes K,Ackerly H et al SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP)[J].J Biol Chem,2002;277(14):12275-9
[18]Chai J,Du C,Wu J W,et al.Structural and biochemical basis of apoptotic activation by SMAC/DIABLO[J].Nature,2000,406(6798):855-862.
[19]Mads H A,Sine R,Jurgen C.et al.The melanoma inhibitor of apoptosis protein:a target forspontaneous cytotoxic T cell responses[J].J Invest Dermatol,2004,122(2):392
[20]Schmollinger JC,Dranoff G.Targeting melanoma inhibitor of apoptosis protein with cancer immunotherapy[J].Apoptosis,2004,9:309
[21]Vucic D,Deshayes K,Ackerly H et al.SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP)[J].J Biol Chem,2002;277(14):12275-9
[22]Hariu H,Hirohashi Y,Torigoe T,et al.Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family,Livin/ML2IAP in lung cancer[J].Clin Cancer Res,2005,11(3):1000-1009
[23]Schimmer A D,Dalili S,Batey R A,et al.Targeting XIAP for the treatment of malignancy[J].Cell Death Differ,2006,13(2)179-188.
[24]Schimmer A D,Dalili S.Targeting the IAP family of caspase inhibitors as an emerging therapeutic strategy[J].Hematology,2005,16(5),215-219.
[25]张华东,袁守军,陈惠鹏,等.Livin mRNA的反义核酸诱导MCF27乳癌细胞凋亡作用[J].中国临床药理学与治疗学,2004,9(12):1353-1156.
[26]Crnkovic M,Felix H S,Karin B.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene[J].Oncogene,2003,53(11):8330
[27]Crnkovic M,Wagener N,Semzow J,et,al.Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J].Cell Mol Life Sci,2007,64(9):1137-1144
[28]Brand S.Antisense-RNA regulation and RNA interference.Biochem Bophys Acta,2002,1575:15-25.
[29] Agrawal N,Dasaradhi PV,Mohmmed A ,et al.RNA interference: biology, mechanism, and applications. Microbiol Mol Rev,2003,67:657-685.
[30] Downward J .RNA interference[M].BMJ,2004,328:1245-1248
[31]Carete JE,Overmeer RM,Schagen FH,et al. Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells.Cancer Res,2004,64:2663-2667.
[32] Scherr M,Morgen MA,Eder M. Gene silencing mediated by small interfering RNAs inmammalian cells. Curr Med Chem,2003,10:245-256.
[33] Caplen NJ.RNAi as a gene therapy approach.Expert Opin Biol Ther, 2003,3:575-586.
[1]Fischer U,Schulze O K.New approaches and therapeutics targeting apoptosis in disease[J].Pharmacol Rev,2005,57(2),187-215.
[2]Yang Y,Yu X.Regulation of apoptosis:the ubiquitous way[J].FASEB J,2003,17(8),790-799.
[3]Carter BZ,Gronda M,Wang Z,et al.Small molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells[J].Blood,2005,105(10),4043-4050.
[4] Zhang M, Latham D E, Delaney M A,et al. Survivin mediates resistance to antiandrogen therapy in prostate cancer[J]. Oncogene,2005,24 (15), 2474-2482.
[5] Kim E H, Kim S U, Choi K S. Rottlerin sensitizes glioma cells to TRAIL-induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP[J]. Oncogene, 2005, 24 (46),838-849.
[6] Demarchi F ,Brancolini C. Altering protein turnover in tumor cells: new opportunities for anti-cancer therapies[J]. Drug Resist Updat, 2005,8 (6),359-368.
[7] Watson R , Fitzpatrick J M. Targeting apoptosis in prostate cancer: focus on caspases and inhibitors of apoptosis proteins[J]. BJU Int 2005,96 (2),30-34.
[8] Roy N, Mahadevan M S, McLean M,et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy[J]. Cell, 1995,80(1):167-178.
[9] Rothe M, Pan M G, Henzel W J, Ayres T M ,et al. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins[J ]. Cell, 1995,83(7):1243-1252.
[10] Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes[J ]. Nature ,1996,379 (6563):349-353.
[11] Ambrosini G, Adida C , Altieri D C. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J ]. Nat Med, 1997,3 (8) : 917-921.
[12] Chen Z, Naito M, Hori S,et al. A human IAP-family gene, apollon, expressed in human brain cancer cells[J]. Biochem. Biophys Res Commun, 1999,264 (3):847-854.
[13] Richter B W, Mir S S, Eiben L J, et al. Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family [J ]. Mol Cell Biol, 2001,21 (13): 4292-4301.
[14] Kasof GM,Gomes BC. Livin ,a novel inhibitor of apoptosis protein family member[J ] .Biol Chem,2001 ,276 (5) :3238 - 3246.
[15] Lacass EC ,Baird S , Komeluk RG, et al . The inhibitors of apoptosis (IAPs) and their emerging role in cancer [J ]. Oncogene ,1998 ,17 (25) :3247 - 3259.
[16] Vucic D ,Stennicke HR ,Pisabarro MT ,et al . ML- IAP ,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas [J ] . Curr Biol, 2000 ,10(21) :1359 -1366.
[17]Boaz N,Yaqoub A,Vered B,et al.caspase-mediated cleavage convert s livin f rom an antiapoptotic to a proapoptotic factor:implications for drug-resistant melanoma[J].Cancer Res,2003,63(10):6340
[18]Ashhab Y,Alian A,Polliack A,et al.Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue dist ribution pattern[J].FEBS Lett,2001,495(4):56
[19]Lin JH,Deng G,Huang Q,et al.KIAP,a novel member of the inhibitor of apoptosis protein family[J].Biochem Biophys Res Commun,2000,279(6),820-831.
[20]Schmollinger JC,Vonderheide RH,Hoar KM,et al.Melanoma inhibitor of apoptosis protein(ML2IAP)is a target for immune-mediated tumor destruction [J].Proc Natl Acad SciUSA,2003,100(6):3398-3403.
[21]Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of livin,survivin and other apoptosis related genes in the progression of superficial bladder cancer[J].Ann Oncol,2003,14(1):85-90.
[22]李显文;杨罗艳;王红珊,等.凋亡抑制蛋白Livin在膀胱移行细胞癌中的表达及临床意义[J].临床泌尿外科杂志,2006,21(3):216-220..
[23]Kitamura H,Honma I,Torigoe T,et al.Expression of livin in renal cell carci noma and detection of anti-livin autoantibody in patients[J].Urology,2007,70(1):38-42
[24]宋希双;张志伟;车翔宇,等.凋亡抑制基因Livin在膀胱癌中的表达及意义[J].中华泌尿外科杂志,2006,27:37-39.
[25]何远桥;曾甫清;鞠文.凋亡抑制因子Livin在膀胱移行细胞癌组织中的表达及意义[J].临床泌尿外科杂志,2006,21(6):458-460.
[26]Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of livin,survivin and other apoptosis-related genes in the progression of superficial bladder cancer[J].Ann Oncol,2003,14(1):85-90.
[27]Yagihashi A,Asanuma K,Kobayashi D,et al.Detection of autoantibodies to Livin and survivin in Sera from lung cancer patients[J].Lung Cancer,2005,48(2):217-221.
[28]Tanabe H,Yagihashi A,Ysuji N,et al.Expression of Survivin mRNA and Livin mRNA in non-small-cell lung cancer[J].Lung Cancer,2004,46(3):299-304.
[29]孙建国,廖荣霞,陈正堂,等.一种新的凋亡抑制蛋白Livin在非小细胞肺癌组 织中的表达[J].重庆医学,2004,33(7):982
[30]Kim DK,Alvarado CS,Abramowsky CR,et al.Expression of inhibitor of apoptosis protein(IAP)Livin by neuroblastoma cells:correlation with prognostic factors and outcome[J].PediatrDev Pathol,2005,8(6):621-629.
[31]向欣,黄正松,石忠松,等.脑星形细胞瘤L ivin基因表达与细胞增殖的关系[J].中华实验外科杂志,2005,22(9):1141-1146.
[32]Qiuping Z,Jei X,Youxin J,et al.CC chemokine ligand 25 enhances resistance to apoptosis in CD4+T cells from patients with T cell lineage acute and chronic lymphocytic leukemia by means of Livin activation[J].Cancer Res,2004,64(20):7579-7587.
[33]Choi J,Hwang Y K,Sung,Ki W,et al.Expression of Livin,an antiapoptotic protein,is an independent favorable prognostic factor in childhood acute lymphoblastic leukemia[J].Blood,2007,109(2):471-477
[34]Yagihashi A,Ohmura T,Asanuma K,et al.Detection of autoantibodies to survivin and Livin in sera from patients with breast cancer[J].Clin Chim Acta,2005,362(1-2):125-130.
[35]Yagihashi A,Asanuma K,Tsuji N,et al.Detection of anti-Livin antibody in gastrointestinal cancer patients[J].Clin Chem,2003,49(7):1206-1208.
[36]Yagihashi A,Asanuma K,Nakamura M,et al.Detection of anti-Survivin antibody in gastrointestinal cancer patients[J].Clin Chem,2001,47(7):1729-1736
[37]Deveraux Q L and Reed J C.IAP family proteins-suppressors of apoptosis[J].Genes Dev,1999,13(3):239-252.
[38]Miller L.An exegesis of IAPs:salvation and surprises from BIR motifs[J].Trends Cell Biol,1999,9(5):323-328.
[39]Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biol Chem,1998,273(14):7787-7790.
[40]Sun C,Cai M,Gunasekera A H,et al.NMR structure and mutagenesis of the inhibitor of apoptosis protein XIAP[J].Nature,1999,401(6755):818-822.
[41]甄海宁,章翔.新的凋亡抑制因子Livin[J].第四军医大学学报,2004,25(19):1822-1823.
[42]Sanna MG,da Silva Correia J,DucreyO,et al.IAP suppression of apoptosis involves distinct mechanisms:the TAK1/JNK1 signaling cascade and Caspase inhibition[J].Mol Cell Biol,2002,22(6):1754-1766.
[43] Vucic D, Franklin MC,Wallweber HJ, et al. Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: imp lications for Smac-dependent anti-apop totic activity of ML-IAP [ J ]. Biochem J, 2005, 385 (Pt 1): 11 -20.
[44] Vucic D , Deshayes K, Ackerly H et al . SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP) [J] . J Biol Chem, 2002 ;277 (14): 12275-9
[45] Crnkovic M I, Semzow J , Hoppe S F, et al. Isoform specific silencing of the Livin gene by RNA interference defines Livin beta as key mediator of apoptosis inhibition in HeLa cells [ J ]. J Mol Med. 2006, 84 (3): 232 -240.
[46] Eckelman B P , Salvesen G S.The human anti apoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases[J ]. J Biol Chem, 2006,281(12): 3254-3260.
[47] Samuel T, Welsh K, Lober T,et al.Distinct BIR domains of cIAP1 mediate binding to and ubiquitination of tumor necrosis factor receptor-associated factor 2 and second mitochondrial activator of caspases[J]. J Biol Chem., 2006,281 (2): 1080-1090.
[48] Riedl S J, Renatus M, Schwarzenbacher R,et al.Structural basis for the inhibition of caspase-3 by XIAP[J].Cell, 2001,104 (5):791-800.
[49] Liu Z, Sun C, Olejniczak E T, et al. Structural basis for binding of Smac/ DIABLO to the XIAP BIR3 domain[J ]. Nature, 2000,408 (6815): 1004-1008.
[50] Riedl S J, Renatus M, Schwarzenbacher R,et al.Structural basis for the inhibition of caspase-3 by XIAP[J].Cell, 2001,104 (5):791-800.
[51] Liu Z, Sun C, Olejniczak E T, et al. Structural basis for binding of Smac/ DIABLO to the XIAP BIR3 domain[J ]. Nature, 2000,408 (6815): 1004-1008.
[52] Wu G, Chai J, Suber T L, et al. Structural basis of IAP recognition by SMAC/DIABLO[J], Nature, 2000,408 (6815): 1008-1012.
[53] Chai J, Du C, Wu J W, et al. Structural and biochemical basis of apoptotic activation by SMAC/DIABLO[J].Nature, 2000,406 (6798):855-862.
[54] Yang Q H ,Du C. Smac/ DIABLO selectively reduces the levels of c-IAP1 and c-IAP2 but not that of XIAP and livin in HeLa cells [ J ] . J Biol Chem , 2004 , 279 (17): 16963
[55] Morita T,Yoshida K.RNAi provides a new tool for functional analyses of the mammalian genes.Tanpakushitsu Kakusan Koso,2002;47:1839-1845
[56] Franklin M C, Kadkhodayan S,Ackerly H, et al. Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis(ML-IAP)[J].Biochem-istry,2003,42(27):8223-8231.
[57]Stucki J W,Simon H U,Mathematical modeling of the regulation of caspase-3activation and degradation[J].J Theor Biol,2005,234(1):123-131.
[58]Crnkovic M,Felix H S,Karin B.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene[J].Oncogene,2003,53(11):8330
[59]Mads H A,Sine R,Jurgen C.et al.The melanoma inhibitor of apoptosis protein:a target forspontaneous cytotoxic T cell responses[J].J Invest Dermatol,2004,122(2):392
[60]Schmollinger JC,Dranoff G.Targeting melanoma inhibitor of apoptosis protein with cancer immunotherapy[J].Apoptosis,2004,9:309
[61]Takeuchi H;Morton,DL;Elashoff D;et al.Survivin expression by metastatic melanoma predicts poor disease outcome in patients receiving adjuvant polyvalent vaccine.Int J Cancer,2005,117(6):1032-1038
[62]Hariu H,Hirohashi Y,Torigoe Y,et al.Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family,Livin/ML2IAP in lung cancer[J].Clin Cancer Res,2005,11(3):1000-1009
[63]Schmolinger JC,Vonderheide RH,Hoar KM,et al.Melanoma inhibitor of apoptosis protein(ML-IAP)is a target for immune-mediated tumor destruction[J].Proc Natl Acad Sci USA,2003,100(6):3398-3403.
[64]Schimmer A D,Dalili S,Batey R A,et al.Targeting XIAP for the treatment of malignancy[J].Cell Death Differ,2006,13(2)179-188.
[65]Schimmer A D,Dalili S.Targeting the IAP family of caspase inhibitors as an emerging therapeutic strategy[J].Hematology,2005,16(5),215-219.
[66]张华东,袁守军,陈惠鹏,等.Livin mRNA的反义核酸诱导MCF27乳癌细胞凋亡作用[J].中国临床药理学与治疗学,2004,9(12):1353-1156.
[67]Chi K N,Gleave M E.Antisense approaches in prostate cancer[J].Expert Opin Biol Ther,2004,4(6),927-936.
[68]Puri N,Eller M S,Byers H R,et al.Telomere-based DNA damage responses:a new approach to melanoma.FASEB J,2004,18(12),1373-1381.
[69]Izquierdo M.Short interfering RNAs as a tool for cancer gene therapy[J].Cancer Gene Ther,2005,12(8),217-227.
[70]Hatano M,Mizuno M,Yoshida J.Enhancement of C2-ceramide antitumor activity by small interfering RNA on X chromosome-linked inhibitor of apoptosis protein in resistant human glioma cells[J]. Neurosurg J, 2004,101 (1),119-127.
[71] Zhang Q , Zhang Z F, Rao JY, et al.Treatment with siRNA and antisense oligonucleotides targeted to HIF-1 alpha induced apoptosis in human tongue squamous cell carcinomas[J]. Int J Cancer, 2004,111 (6), 849-857.
[72] Yamaguchi Y, Shiraki K, Fuke H, et al.Targeting of X-linked inhibitor of apoptosis protein or survivin by short interfering RNAs sensitize hepatoma cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic agent-induced cell death[J]. Oncol. Rep,2005,14 (5), 1311-1316.
[73] Yonesaka K, Tamura K, Kurata T,et al. Small interfering RNA targeting surviv in sensitizes lung cancer cell with mutant p53 to adriamycin[J].Int J Cancer,2006 ,118 (4), 812-820.
[74] Crnkovic M, Wagener N, Semzow J,et,al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J].Cell Mol Life Sci, 2007,64 (9): 1137-1144
[2] Yang Y ,Yu X. Regulation of apoptosis: the ubiquitous way[J]. FASEB J, 2003,17 (8),790-799.
[3] Carter BZ, Gronda M, Wang Z, et al. Small molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells[J]. Blood, 2005,105 (10),4043-4050.
[4] Zhang M, Latham DE, Delaney MA,et al. Survivin mediates resistance to antiandrogen therapy in prostate cancer[J]. Oncogene,2005,24 (15), 2474-2482.
[5] Kim EH, Kim SU, Choi KS. Rottlerin sensitizes glioma cells to TRAIL-induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP[J]. Oncogene, 2005, 24 (46),838-849.
[6] Demarchi F ,Brancolini C. Altering protein turnover in tumor cells: new opportunities for anti-cancer therapies[J]. Drug Resist Updat, 2005,8 (6),359-368.
[7] Watson R , Fitzpatrick JM. Targeting apoptosis in prostate cancer: focus on caspases and inhibitors of apoptosis proteins[J]. BJU Int 2005,96 (2),30-34.
[8] Roy N, Mahadevan MS, Mclean M,et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy[J]. Cell, 1995,80(1): 167-178.
[9] Rothe M, Pan MG, Henzel WJ, Ayres T M ,et al. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins[J ]. Cell, 1995,83(7): 1243-1252.
[10] Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes[J ]. Nature ,1996,379 (6563):349-353.
[11] Ambrosini G, Adida C , Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J ]. Nat Med, 1997,3 (8) : 917-921.
[12] Chen Z, Naito M, Hori S,et al. A human IAP-family gene, apollon, expressed in human brain cancer cells[J]. Biochem. Biophys Res Commun, 1999,264 (3):847-854.
[13] Richter BW, Mir SS, Eiben LJ, et al. Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family[J ]. Mol Cell Biol, 2001,21 (13): 4292-4301.
[14] Kasof GM, Gomes BC. Livin ,a novel inhibitor of apoptosis protein family member[J ] .Biol Chem,2001 ,276 (5) :3238 - 3246.
[15] Lacass EC, Baird S, Komeluk RG, et al. The inhibitors of apoptosis (IAPs) and their emerging role in cancer [J]. Oncogene, 1998,17 (25) :3247 - 3259.
[16] Vucic D, Stennicke HR, Pisabarro MT ,et al . ML- IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas [J ] . Curr Biol, 2000 ,10(21) :1359-1366.
[17] Boaz N ,Yaqoub A ,Vered B ,et al. caspase-mediated cleavage convert s livin from an antiapoptotic to a proapoptotic factor: implications for drug-resistant melanoma[J ]. Cancer Res ,2003 , 63 (10) :6340
[18] Ashhab Y, Alian A , Polliack A ,et al. Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue dist ribution pattern [J] .FEBS Lett ,2001 ,495 (4) :56
[19] Lin JH, Deng G, Huang Q ,et al. KIAP, a novel member of the inhibitor of apoptosis protein family[J]. Biochem Biophys Res Commun, 2000,279 (6), 820-831.
[20] Yagihashi A, Asanuma K, Tsuji N, et al. Detection of anti-Livin antibody in gastrointestinal cancer patients[J]. Clin Chem 2003; 49:1206-1208.
[21] Deveraux QL, Reed JC.IAP family proteins-suppressors of apoptosis[J ]. Genes Dev, 1999,13(3):239-252.
[22] Miller L.An exegesis of IAPs: salvation and surprises from BIR motifs[J ]. Trends Cell Biol, 1999,9 (8):323-328.
[23] Song Z, Yao X, Wu M. Direct interaction between Survivin and Smac is essential for the anti-apoptotic activity of Survivin during Taxol-induced apoptosis[J]. J Biol Chem, 2003 ; 278 (25): 23130-23140
[24] Maier JK, Lahoua Z, Gendron NH et al. The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7[J]. J Neuro Sci ,2002 ;22 (6) :2035-2043
[25] Liu Z,Sun C, Olejniczak ET, et al. Structural basis for binding of SMAC/DIABLO to the XIAP BIR3 domain[J]. Nature, 2000,408 (6815): 1004-1008.
[26] Wu G, Chai J, Suber TL, et al. Structural basis of IAP recognition by SMAC/DIABLO[J],Nature,2000,408(6815):1008-1012.
[27]Chai J,Du C,Wu JW,et al.Structural and biochemical basis of apoptotic activation by SMAC/DIABLO[J].Nature,2000,406(6798):855-862.
[28]Vucic D,Franklin MC,Wallweber HJ,et al.Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor:imp lications for SMAC-dependent anti-apop totic activity ofML2IAP[J].Biochem J,2005,385(Pt 1):11-20.
[29]Vucic D,Deshayes K,Ackerly H,et al.SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP)[J].J Biol Chem,2002;277(14):12275-12279
[30]Kasof GM,Gomes BC.Livin,a novel inhibitor of apoptosis protein family member[J].Biol Chem,2001,276(5):3238-3246.
[31]Vucic D,Stennicke HR,Pisabarro MT,et al.ML-IAP,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas[J].Curr Biol,2000,10(21):1359-1366.
[32]Hariu H,Hirohashi Y,Torigoe T,et al.Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family,Livin/ML-IAP in lung cancer[J].Clin Cancer Res.2005;11(3):1000-1009
[33]Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of livin,survivin and other apoptosis-related genes in the progression of superficial bladder cancer[J].Ann Oncol,2003,14(1):85-90.
[34]李显文,杨罗艳,王红珊,等.凋亡抑制蛋白Livin在膀胱移行细胞癌中的表达及临床意义[J].临床泌尿外科杂志,2006,21(3):216-220.
[35]孙建国,廖荣霞,陈正堂,等.Livin异构体基因转染对肺腺癌A549细胞生长及放化疗敏感性的影响[J].中华结核和呼吸杂志,2005,28(12):836-840.
[36]Crnkovic-Mertons I,Hoppe-Seyler F,Butz K.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the Livin / ML-IAP / KIAP gene[J].Oncogene,2003.22(51):8330-8336.
[37]Zhang Q,Xiong J,Jin Y,et al.CC chemokine ligand 25 enhances restance to apoptosis in CD4+ T cells from patients with T-cell-lineage acute and chronic lymhocytie leukemia by means of Livin activation[J].Cancer Res,2004,64(20):7579-7587.
[38]Zhang Q,Xiong J,Jin Y.et al.Selectively frequent expression of CXCR5enhances resistance to apoptoeis in CD8(+)CD34(+)T cells from patients with T-cell-lineage acute lymphocytic leukemia[J].Oneogene,2005,24(4):573-584.
[1] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic intederenee by double—stranded RNA in Caenorhabdltis eIegatm[J]. Nature. 1998. 391: 806-811.
[2] Song E ,Lee S K,Wang J ,Ince N ,et al. RNA i nterference targeting Fas protects mice from fulminanth epatitis[J].Nat Med,2003,9(3):347-351
[3] Brummelkamp T R,Bernards R ,Agami R .Stable suppression of tumor igenicity by virus-mediate dRNA interference [J]. Cancer Cell,2002;2(3):245-249
[4] Paul CP, Good PD, Winer L, et al. Efective expression of small interfering RNA in human cells[J]. Nat Biotechnol, 2002, 20(5): 505—508.
[5] Sui G, Soohoo C, Afarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells[J]. Proc Nail Acad sci USA,2002, 99(8): 5515-5520.
[6] Yu JY. DeRuiter SL, Turner DL. RNA interference by expression of short-intefering and hairpin RNAs in mammalian cells[J]. Proc Natl Acad Sci USA, 2002. 99(9): 6047—6052.
[7] Yu JY,DeRuiter SL,Tumer D L.RNA interference by expression of short-interfering RNAs and haifpin RNAs in mammalian cells[J]. Proc Natl Acad Sci UsA,2002, 99(9): 6047-6052.
[8] Haqnon GJ,Conklin DS. RNA interference by shorthairpin RNAs expressed in vertebrate cells[J]. Methods Mol Biol, 2004, 257: 255-266.
[9] Uitel K, Nalto Y, Takabashi F, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference[J]. Nueleic Acids Res,2004,32: 936-948.
[10] Hsieh AC, Bo R, Manola J, et al. A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens[J]. Nucleic Acids Res, 2004, 32: 893-901.
[11] Uchida H. TanakaT, Sasaki K, et al. Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo[J]. Mol Ther, 2004, 10(1): 162-171.
[12] Mazor M,KawanoY, Zhu H, et al. Inhibition of glycogen synthase kinase-3 represses androgen receptor activity and prostate cancer cell growth[J]. Oneogene, 2004,23(47): 7882-7892.
[13]Wilda M,Fuchs U,Wossmarm W,et al.Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference(RNAi)[J].Oncogene,2002,21(37):5716-5721
[14]Qiu S,Adema CM,Lane T.A computational study of off-target effects of RNA interference[J].Nucleic Acids Res,2005,33(6):1834-1847.
[15]Miller LK.An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif[J].J Virol,1993,67(4):2168-2174.
[16]Hakhyun Ka,Joan S,Hunt.Temporal and spatial paterns of Expression of Inhibitors of apoptosis in human placentas[J].Am J Pathol,2003,163(2);413-422
[17]Zhang HD,Yuan SJ,Chen HP,et al.Induction effects of antisense phosphorothioate oligodeoxynucleotides of livin mRNA on apoptosis in MCF-7cells[J].Chin J clin Pharmacol Them,2004,9(12):1353-1356.
[18]Hannon GJ.RNA interference[J].Nature,2002,418:244-251.
[19]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic intederenee by double-stranded RNA in Caenorhabdltis elegatm[J].Nature,1998,391:806-811.
[20]Caplen NJ.A new approach to the inhibition of gene express[J].Trends Biotechnol,2002.20:49-51.
[21]Uprichard S L.The therapeutic potential of RNA interference.FEBS Lett,2005,579(26):5996-6007.
[22]Elbashir SM,Harbort h J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cell lines[J].Nature,2001,411:494
[23]McManus MT,Sharp PA.Gene silencing in mammalians by small interfering RNAs[J].Nat Rev Gene,2002,3:737
[24][美]J.萨姆布鲁克,D.W.拉萨尔,著,黄培堂译.分子克隆实验指南(第三版)(下册)[M].北京:科学技术出版社,2002:1595-1598.
[25][美]J.萨姆布鲁克,D.W.拉萨尔,著,黄培堂译.分子克隆实验指南(第三版)(上册)[M].北京:科学技术出版社,2002:96-99.
[26]Lewis KE,Stephens C,Shahidi MM,et al.Delay in starting treatment for tuberculosis in east London[J].Commun Dis Public Health,2003,6(2):133
[27]Lienhardt C,Rowley J,Manneh K,et al.Factors affecting time delay to treatment in a tuberculosis control programme in a sub-Saharan African country:the experience of t he Gambia[J].Int J Tuberc Lung Dis,2001,5(3):233
[28] Demissie M , Lindtjorn B , Berhane Y. Patient and health service delay in the diagnosis of pulmonary tuberculosis in Ethiopia. [J] BMC Public Health ,2002,2 (1) :23
[29] Hariu H , Hirohashi Y, Torigoe T , et al. Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family , Livin/ ML2IAP in lung cancer[J ]. Clin Cancer Res ,2005 ,11 (3): 1000-1009
[30] Crnkovic M, Wagener N, Semzow J,et,al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J].Cell Mol Life Sci, 2007,64 (9): 1137-1144
[31] Nykallen A, Haley B, Zamore PD. A1P requirements and small interfering RNA structure in the RNA interference pathway. Cell, 2001, 107(3): 309-321.
[1]Wu Q,Moyana T,Xiang J.Cancer gene therapy by adenovirus-mediated gene transfer.Curr Gene Ther 2001;1(1):101-122
[2]Lai CM,Lai YK,Rakoczy PE.Adenovirus and adeno-associated virus vectors.DNA Cell Biol.2002;21(12):895-913.
[3]郭荣,邹萍,陆华中.非病毒型纳米载体在基因治疗中的研究现状及展望.国外医学生物医学工程分册,2002,25(2):77-81.
[4]Pierre L,Sylvie F,Noufrssa O,eta l.Gene delivery systems:Bridging the gap between Recombinant viruses and artificial vectors.Adv Drug Delve.1998(30):5-11.
[5]Truong L,Walsh W,Scot M,et al.Gene transfer by DNA-gelatin nanospheres.Arch Biochem Biophys.1999(361):47-56.
[6]Fox JL.Gene therapy safety issues come to fore.Nat Biotechnol.1999;17(12):1153.
[7]Wu X,Li Y,Crise B,Burgess SM.Transcription start regions in the human genome are favored targets for MLV integration.Science.2003;300(5626):1749-1751.
[8]Sun X,Zhang HW,Zhang ZR.Transfection efficiency of pORF lacZ plasmid lipopolyplex to hepatocytes and hepatoma cells.World J Gastroenterol.2004;10(4):531-534.
[9]胡云霞,原续波,张晓金,等.阳离子多聚物纳米基因载体系统的研究进展.北京生物医学工程,2003,22(4):299-302.
[10]Colin W,Pouton S,Leonard W,et al.Key issues in non-viral gene delivery.Adv Drug Delve.1998(34):3-19.
[11]Cristiano R J.Targeted,non-viral gene delivery for cancer gene therapy.Front Biosci.19 98(3):D1161-D1170.
[12]Anderson W EHuman gene therapy.Nature.1998,392(6679 Suppl),25-30.
[13]Richardson SCW,Kolbe HVJ,Duncan R,et al.Potential of low molecular mass chitosan as a DNA delivery system:biocompatibility,body distribution and ability to complex and protect DNA.Int J Pharm,1999,178(2):231-243.
[14]Mao HQ,Roy K,Troung-Le VL,et al.Chitosan-DNA nanopaticles as gene carriers:synthesis,characterization and transfection efficiency.J Controlled Release,2001,70(3):399-421.
[15]Lambert G,Fattal E,Couvreur P.Nanoparticμlate systems for the delivery of antisense oligonucleotides.Adv Drug Deliv Rev,2001,47(1):99-112.
[16]Zhu SG,Lu WB,Xiang JJ,et al.A novel nonviviral-nanoparticle gene vector:poly-lysine silica nanoparticales[J].Chin Sci Bμll,2002,47(8):654-658.
[17]汪杨,吴伟.隐形纳米粒的体内靶向性[J].中国药学杂志,2004,39(1):7-11.
[18]Peraeehia M T,Fattal E,Desmaele D.Stealth PEGylalted polyeyanoaerylate nanopartides for intravenous adminstration and splenic targeting[i].J Controlled Release,1999,60:121-130.
[19]Gref R,Ialek M,Qudlee P,et al.'Stealth' corono-core nanopartides surface modified by polyethylene glycol(PEG):influenees the corona(PEG chain length and surface density)and the core composition on phacocytic uptake and plasma protein adsorption[J].Colloids Surf B Biointerface,2000,18(3～4):301-308.
[20]Richardson SCW,Kolbe HVJ,Duncan R,et al.Potential of low molecμlar mass chitosan as a DNA delivery system:biocompatibility,body distribution and ability to complex and protect DNA[J].Int J Pharm,1999,178(2):231-243.
[21]Mao HQ,Roy K,Troung LeVL,et al.Chitosan-DNA nanopaticles as gene carriers:synthesis,characterization and transfection efficiency[J].J Controlled Release,2001,70(3):399-421.
[22]Maclaughlin FC,Mumper RJ,Wang J,et al.Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery.J Controlled Release,1998,56(1-3):259-272
[23]Kneuer C,Ehrhardt C,Bakowsky H,et al.The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes:a comparative study.J Nanosci Nanotechnol,2006,6(9-10):2776-2782.
[24]Alexakis T,Boadi DK,Quong D,et al.Mieroeneapsμlation of DNA within alginate microspheres and crosslinked chitosan membi'anes for in vivo application[J].Appl Biochem Biotechnol,1995,50(1):93-106.
[25]Mumper R J,Wang J,Claspell J M,et al.In:Proc Intl Symp Controlled,Rel Bioact Mater,1995,23:178.
[26]Mohapatra SS.Mucosal gene expression vaccine:a novel vaccine strategy for respiratory syncytial virus[J].Pediatr Inject Dis,2003,22(2):S100-S104.
[27]Iqbal M,Lin W,Jabbal-Gill I,et al.Nasal delivery of chitosan-DNA plasmid expressing cpitopesot respiratory syncytial virus(RSV)induces protective CTL responses in BALB/c nfice[J].Vaccine,2003,21(13-14):1478-1485.
[28]Li YH,Fan MW,Bian Z,et al.Chitosan-DNA microparticles as mucosal delivery system:synthesis,characterization and release in vitro[J].Clin Med J,2005,118(11):936-941.
[29]Vanessa Carla Furtado Mosqueira,Philippe Legrand,Annette Gulik et al.Biomaterials[J],2001,22:2967-2979
[30]Mosqueira VC,Legrand P,Gulik A,et al.Relationship between complement activation,cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.Biomaterials,2001,22(22):2967-2979.
[31]K Park,YH Park,BA Shin,ESC hoi,YR Park,T Akaike,CS Cho,Galactosylated chitosan-graft-dextran as hepatocyte-targetting DNA carrier.J Control Release,2000,69:97-108.
[32]FC MacLaughlin,RJ Mumper,et al.Rolland Chitosan and depolymerized chitosan oligomers as condensing carriers in vivo plamid delivery.J Control Release.1998,56:259-272.
[33]林友文,许晨.甲壳素及其衍生物的医学应用.福建医科大学学报,1999,2:226-228.
[34]张阳德,孙颖.一种新型的非病毒基因转运载体.纳米粒基因转运体.中国现代医学杂志,2003,13(21):148-151.
[35]朱诗国,李桂源.纳米基因转运体-原理,研制与应用.生物化学与生物物理进展,2002,29(6):868-871.
[36]基因表达技术[M].李育阳主编.科学出版社,2001:197-199
[37]Maclaughlin FC,Mumper RJ,Wang J,et al.Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery.J Controlled Release,1998,56(1-3):259-272
[38]Kneuer C,Ehrhardt C,Bakowsky H,et al.The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes:a comparative study.J Nanosci Nanotechnol,2006,6(9-10):2776-2782.
[39]Mosqueira VC,Legrand P,Gulik A,et al.Relationship between complement activation,cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.Biomaterials,2001,22(22):2967-2979.
[40]Leong KW,Mao HQ,Truong-LeVL,Roy K,Walsh SM,AugustJ T.DNA-poly-cation nanospherea as non-viral gene delivery vehicles.J Control Release 1998,30;53(1-3);183-93.
[41]Leong KW,Jiang X,Dai H,et al.Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions.J Gene Med,2006,8(4):477-487
[42]TaanoRM,Florea BI,Geldof M,et al.Quatemized chitesan oligomem as novel gene delivery vectors in epithelial cell lines.Biomatefials,2001,23(1):153.
[43]Illum L,Jabbal Gill I,Hinchcliffe M,et al.Chitosan as a novel nasal delivery system for vaccines.Adv Drug Deliver Rev,2001,51(1-3):81-96.
[44]Gill DR,Southern KW,Moford KA,et al.A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis.Gene Ther.1997(4):199-209.
[1] Steller H.Mechanisms and genes of ellularsuiocide [J].Science, 1995;267(10),14 45 -1 44 9 .
[2] Mattson M P.Apoptosis in neurodegenerative disorders.Nat Rev Mol Cell Biol,2000 ,1:1 20-129.
[3] Rathmell JC ,Thompson C B.P thways of apoptosis in lymphocyted evelopment,homeostasis,and disease.Cell,2002,109 Suppl:97 -107
[4] Reed CJ.Apoptosis and cancer: strategies for integratingp rogrammed cell death.Semin Hematol,2000,37 :9-16.
[5] LaCasse EC,Baird S,Korneluk RG,et al .The inhibitors of apoptosis(I A-Ps)and their emerging role in cancer.Oncogene,1998,17 :3247-3259.
[6] Thompson CB.Apoptosis in the pathogenesis and treatment of disease[J]. Science,1995,267(10):1456-1462.
[7] Deveraux Q L and Reed J C.IAP family proteins-suppressors of apoptosis[J]. Genes Dev, 1999,13 (3):239-252.
[8] Miller L. An exegesis of IAPs: salvation and surprises from BIR motifs[J]. Trends Cell Biol, 1999,9 (5):323-328.
[9] Richter B W, Mir S S, Eiben L J, et al. Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family[J ]. Mol Cell Biol, 2001,21 (13): 4292-4301.
[10] Liu Z, Sun C, Olejniczak E T, et al. Structural basis for binding of Smac/ DIABLO to the XIAP BIR3 domain[J ]. Nature, 2000,408 (6815): 1004-1008.
[11] Wu G, Chai J, Suber T L, et al. Structural basis of IAP recognition by SMAC/DIABLO[J], Nature, 2000,408 (6815):1008-1012.
[12] Kasof GM,Gomes BC. Livin ,a novel inhibitor of apoptosis protein family member[J ] .Biol Chem,2001,276 (5) :3238 - 3246.
[13] Vucic D ,Stennicke HR ,Pisabarro MT ,et al . ML- IAP ,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas [J ] . Curr Biol, 2000 ,10(21) :1359-1366.
[14] Ashhab Y, Alian A , Polliack A ,et al. Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue dist ribution pattern [ J ] .FEBS Lett ,2001,495 (4) :56
[15]Lin JH,Deng G,Huang Q,et al.KIAP,a novel member of the inhibitor of apoptosis protein family[J].Biochem Biophys Res Commun,2000,279(6),820-831.
[16]Zhang M,Latham D E,Delaney M A,et al.Survivin mediates resistance to antiandrogen therapy in prostate cancer[J].Oncogene,2005,24(15),2474-2482.
[17]Vucic D,Deshayes K,Ackerly H et al SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP)[J].J Biol Chem,2002;277(14):12275-9
[18]Chai J,Du C,Wu J W,et al.Structural and biochemical basis of apoptotic activation by SMAC/DIABLO[J].Nature,2000,406(6798):855-862.
[19]Mads H A,Sine R,Jurgen C.et al.The melanoma inhibitor of apoptosis protein:a target forspontaneous cytotoxic T cell responses[J].J Invest Dermatol,2004,122(2):392
[20]Schmollinger JC,Dranoff G.Targeting melanoma inhibitor of apoptosis protein with cancer immunotherapy[J].Apoptosis,2004,9:309
[21]Vucic D,Deshayes K,Ackerly H et al.SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP)[J].J Biol Chem,2002;277(14):12275-9
[22]Hariu H,Hirohashi Y,Torigoe T,et al.Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family,Livin/ML2IAP in lung cancer[J].Clin Cancer Res,2005,11(3):1000-1009
[23]Schimmer A D,Dalili S,Batey R A,et al.Targeting XIAP for the treatment of malignancy[J].Cell Death Differ,2006,13(2)179-188.
[24]Schimmer A D,Dalili S.Targeting the IAP family of caspase inhibitors as an emerging therapeutic strategy[J].Hematology,2005,16(5),215-219.
[25]张华东,袁守军,陈惠鹏,等.Livin mRNA的反义核酸诱导MCF27乳癌细胞凋亡作用[J].中国临床药理学与治疗学,2004,9(12):1353-1156.
[26]Crnkovic M,Felix H S,Karin B.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene[J].Oncogene,2003,53(11):8330
[27]Crnkovic M,Wagener N,Semzow J,et,al.Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J].Cell Mol Life Sci,2007,64(9):1137-1144
[28]Brand S.Antisense-RNA regulation and RNA interference.Biochem Bophys Acta,2002,1575:15-25.
[29] Agrawal N,Dasaradhi PV,Mohmmed A ,et al.RNA interference: biology, mechanism, and applications. Microbiol Mol Rev,2003,67:657-685.
[30] Downward J .RNA interference[M].BMJ,2004,328:1245-1248
[31]Carete JE,Overmeer RM,Schagen FH,et al. Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells.Cancer Res,2004,64:2663-2667.
[32] Scherr M,Morgen MA,Eder M. Gene silencing mediated by small interfering RNAs inmammalian cells. Curr Med Chem,2003,10:245-256.
[33] Caplen NJ.RNAi as a gene therapy approach.Expert Opin Biol Ther, 2003,3:575-586.
[1]Fischer U,Schulze O K.New approaches and therapeutics targeting apoptosis in disease[J].Pharmacol Rev,2005,57(2),187-215.
[2]Yang Y,Yu X.Regulation of apoptosis:the ubiquitous way[J].FASEB J,2003,17(8),790-799.
[3]Carter BZ,Gronda M,Wang Z,et al.Small molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells[J].Blood,2005,105(10),4043-4050.
[4] Zhang M, Latham D E, Delaney M A,et al. Survivin mediates resistance to antiandrogen therapy in prostate cancer[J]. Oncogene,2005,24 (15), 2474-2482.
[5] Kim E H, Kim S U, Choi K S. Rottlerin sensitizes glioma cells to TRAIL-induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP[J]. Oncogene, 2005, 24 (46),838-849.
[6] Demarchi F ,Brancolini C. Altering protein turnover in tumor cells: new opportunities for anti-cancer therapies[J]. Drug Resist Updat, 2005,8 (6),359-368.
[7] Watson R , Fitzpatrick J M. Targeting apoptosis in prostate cancer: focus on caspases and inhibitors of apoptosis proteins[J]. BJU Int 2005,96 (2),30-34.
[8] Roy N, Mahadevan M S, McLean M,et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy[J]. Cell, 1995,80(1):167-178.
[9] Rothe M, Pan M G, Henzel W J, Ayres T M ,et al. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins[J ]. Cell, 1995,83(7):1243-1252.
[10] Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes[J ]. Nature ,1996,379 (6563):349-353.
[11] Ambrosini G, Adida C , Altieri D C. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J ]. Nat Med, 1997,3 (8) : 917-921.
[12] Chen Z, Naito M, Hori S,et al. A human IAP-family gene, apollon, expressed in human brain cancer cells[J]. Biochem. Biophys Res Commun, 1999,264 (3):847-854.
[13] Richter B W, Mir S S, Eiben L J, et al. Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family [J ]. Mol Cell Biol, 2001,21 (13): 4292-4301.
[14] Kasof GM,Gomes BC. Livin ,a novel inhibitor of apoptosis protein family member[J ] .Biol Chem,2001 ,276 (5) :3238 - 3246.
[15] Lacass EC ,Baird S , Komeluk RG, et al . The inhibitors of apoptosis (IAPs) and their emerging role in cancer [J ]. Oncogene ,1998 ,17 (25) :3247 - 3259.
[16] Vucic D ,Stennicke HR ,Pisabarro MT ,et al . ML- IAP ,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas [J ] . Curr Biol, 2000 ,10(21) :1359 -1366.
[17]Boaz N,Yaqoub A,Vered B,et al.caspase-mediated cleavage convert s livin f rom an antiapoptotic to a proapoptotic factor:implications for drug-resistant melanoma[J].Cancer Res,2003,63(10):6340
[18]Ashhab Y,Alian A,Polliack A,et al.Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue dist ribution pattern[J].FEBS Lett,2001,495(4):56
[19]Lin JH,Deng G,Huang Q,et al.KIAP,a novel member of the inhibitor of apoptosis protein family[J].Biochem Biophys Res Commun,2000,279(6),820-831.
[20]Schmollinger JC,Vonderheide RH,Hoar KM,et al.Melanoma inhibitor of apoptosis protein(ML2IAP)is a target for immune-mediated tumor destruction [J].Proc Natl Acad SciUSA,2003,100(6):3398-3403.
[21]Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of livin,survivin and other apoptosis related genes in the progression of superficial bladder cancer[J].Ann Oncol,2003,14(1):85-90.
[22]李显文;杨罗艳;王红珊,等.凋亡抑制蛋白Livin在膀胱移行细胞癌中的表达及临床意义[J].临床泌尿外科杂志,2006,21(3):216-220..
[23]Kitamura H,Honma I,Torigoe T,et al.Expression of livin in renal cell carci noma and detection of anti-livin autoantibody in patients[J].Urology,2007,70(1):38-42
[24]宋希双;张志伟;车翔宇,等.凋亡抑制基因Livin在膀胱癌中的表达及意义[J].中华泌尿外科杂志,2006,27:37-39.
[25]何远桥;曾甫清;鞠文.凋亡抑制因子Livin在膀胱移行细胞癌组织中的表达及意义[J].临床泌尿外科杂志,2006,21(6):458-460.
[26]Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of livin,survivin and other apoptosis-related genes in the progression of superficial bladder cancer[J].Ann Oncol,2003,14(1):85-90.
[27]Yagihashi A,Asanuma K,Kobayashi D,et al.Detection of autoantibodies to Livin and survivin in Sera from lung cancer patients[J].Lung Cancer,2005,48(2):217-221.
[28]Tanabe H,Yagihashi A,Ysuji N,et al.Expression of Survivin mRNA and Livin mRNA in non-small-cell lung cancer[J].Lung Cancer,2004,46(3):299-304.
[29]孙建国,廖荣霞,陈正堂,等.一种新的凋亡抑制蛋白Livin在非小细胞肺癌组 织中的表达[J].重庆医学,2004,33(7):982
[30]Kim DK,Alvarado CS,Abramowsky CR,et al.Expression of inhibitor of apoptosis protein(IAP)Livin by neuroblastoma cells:correlation with prognostic factors and outcome[J].PediatrDev Pathol,2005,8(6):621-629.
[31]向欣,黄正松,石忠松,等.脑星形细胞瘤L ivin基因表达与细胞增殖的关系[J].中华实验外科杂志,2005,22(9):1141-1146.
[32]Qiuping Z,Jei X,Youxin J,et al.CC chemokine ligand 25 enhances resistance to apoptosis in CD4+T cells from patients with T cell lineage acute and chronic lymphocytic leukemia by means of Livin activation[J].Cancer Res,2004,64(20):7579-7587.
[33]Choi J,Hwang Y K,Sung,Ki W,et al.Expression of Livin,an antiapoptotic protein,is an independent favorable prognostic factor in childhood acute lymphoblastic leukemia[J].Blood,2007,109(2):471-477
[34]Yagihashi A,Ohmura T,Asanuma K,et al.Detection of autoantibodies to survivin and Livin in sera from patients with breast cancer[J].Clin Chim Acta,2005,362(1-2):125-130.
[35]Yagihashi A,Asanuma K,Tsuji N,et al.Detection of anti-Livin antibody in gastrointestinal cancer patients[J].Clin Chem,2003,49(7):1206-1208.
[36]Yagihashi A,Asanuma K,Nakamura M,et al.Detection of anti-Survivin antibody in gastrointestinal cancer patients[J].Clin Chem,2001,47(7):1729-1736
[37]Deveraux Q L and Reed J C.IAP family proteins-suppressors of apoptosis[J].Genes Dev,1999,13(3):239-252.
[38]Miller L.An exegesis of IAPs:salvation and surprises from BIR motifs[J].Trends Cell Biol,1999,9(5):323-328.
[39]Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biol Chem,1998,273(14):7787-7790.
[40]Sun C,Cai M,Gunasekera A H,et al.NMR structure and mutagenesis of the inhibitor of apoptosis protein XIAP[J].Nature,1999,401(6755):818-822.
[41]甄海宁,章翔.新的凋亡抑制因子Livin[J].第四军医大学学报,2004,25(19):1822-1823.
[42]Sanna MG,da Silva Correia J,DucreyO,et al.IAP suppression of apoptosis involves distinct mechanisms:the TAK1/JNK1 signaling cascade and Caspase inhibition[J].Mol Cell Biol,2002,22(6):1754-1766.
[43] Vucic D, Franklin MC,Wallweber HJ, et al. Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: imp lications for Smac-dependent anti-apop totic activity of ML-IAP [ J ]. Biochem J, 2005, 385 (Pt 1): 11 -20.
[44] Vucic D , Deshayes K, Ackerly H et al . SMAC negatively regulatesthe anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP) [J] . J Biol Chem, 2002 ;277 (14): 12275-9
[45] Crnkovic M I, Semzow J , Hoppe S F, et al. Isoform specific silencing of the Livin gene by RNA interference defines Livin beta as key mediator of apoptosis inhibition in HeLa cells [ J ]. J Mol Med. 2006, 84 (3): 232 -240.
[46] Eckelman B P , Salvesen G S.The human anti apoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases[J ]. J Biol Chem, 2006,281(12): 3254-3260.
[47] Samuel T, Welsh K, Lober T,et al.Distinct BIR domains of cIAP1 mediate binding to and ubiquitination of tumor necrosis factor receptor-associated factor 2 and second mitochondrial activator of caspases[J]. J Biol Chem., 2006,281 (2): 1080-1090.
[48] Riedl S J, Renatus M, Schwarzenbacher R,et al.Structural basis for the inhibition of caspase-3 by XIAP[J].Cell, 2001,104 (5):791-800.
[49] Liu Z, Sun C, Olejniczak E T, et al. Structural basis for binding of Smac/ DIABLO to the XIAP BIR3 domain[J ]. Nature, 2000,408 (6815): 1004-1008.
[50] Riedl S J, Renatus M, Schwarzenbacher R,et al.Structural basis for the inhibition of caspase-3 by XIAP[J].Cell, 2001,104 (5):791-800.
[51] Liu Z, Sun C, Olejniczak E T, et al. Structural basis for binding of Smac/ DIABLO to the XIAP BIR3 domain[J ]. Nature, 2000,408 (6815): 1004-1008.
[52] Wu G, Chai J, Suber T L, et al. Structural basis of IAP recognition by SMAC/DIABLO[J], Nature, 2000,408 (6815): 1008-1012.
[53] Chai J, Du C, Wu J W, et al. Structural and biochemical basis of apoptotic activation by SMAC/DIABLO[J].Nature, 2000,406 (6798):855-862.
[54] Yang Q H ,Du C. Smac/ DIABLO selectively reduces the levels of c-IAP1 and c-IAP2 but not that of XIAP and livin in HeLa cells [ J ] . J Biol Chem , 2004 , 279 (17): 16963
[55] Morita T,Yoshida K.RNAi provides a new tool for functional analyses of the mammalian genes.Tanpakushitsu Kakusan Koso,2002;47:1839-1845
[56] Franklin M C, Kadkhodayan S,Ackerly H, et al. Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis(ML-IAP)[J].Biochem-istry,2003,42(27):8223-8231.
[57]Stucki J W,Simon H U,Mathematical modeling of the regulation of caspase-3activation and degradation[J].J Theor Biol,2005,234(1):123-131.
[58]Crnkovic M,Felix H S,Karin B.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene[J].Oncogene,2003,53(11):8330
[59]Mads H A,Sine R,Jurgen C.et al.The melanoma inhibitor of apoptosis protein:a target forspontaneous cytotoxic T cell responses[J].J Invest Dermatol,2004,122(2):392
[60]Schmollinger JC,Dranoff G.Targeting melanoma inhibitor of apoptosis protein with cancer immunotherapy[J].Apoptosis,2004,9:309
[61]Takeuchi H;Morton,DL;Elashoff D;et al.Survivin expression by metastatic melanoma predicts poor disease outcome in patients receiving adjuvant polyvalent vaccine.Int J Cancer,2005,117(6):1032-1038
[62]Hariu H,Hirohashi Y,Torigoe Y,et al.Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family,Livin/ML2IAP in lung cancer[J].Clin Cancer Res,2005,11(3):1000-1009
[63]Schmolinger JC,Vonderheide RH,Hoar KM,et al.Melanoma inhibitor of apoptosis protein(ML-IAP)is a target for immune-mediated tumor destruction[J].Proc Natl Acad Sci USA,2003,100(6):3398-3403.
[64]Schimmer A D,Dalili S,Batey R A,et al.Targeting XIAP for the treatment of malignancy[J].Cell Death Differ,2006,13(2)179-188.
[65]Schimmer A D,Dalili S.Targeting the IAP family of caspase inhibitors as an emerging therapeutic strategy[J].Hematology,2005,16(5),215-219.
[66]张华东,袁守军,陈惠鹏,等.Livin mRNA的反义核酸诱导MCF27乳癌细胞凋亡作用[J].中国临床药理学与治疗学,2004,9(12):1353-1156.
[67]Chi K N,Gleave M E.Antisense approaches in prostate cancer[J].Expert Opin Biol Ther,2004,4(6),927-936.
[68]Puri N,Eller M S,Byers H R,et al.Telomere-based DNA damage responses:a new approach to melanoma.FASEB J,2004,18(12),1373-1381.
[69]Izquierdo M.Short interfering RNAs as a tool for cancer gene therapy[J].Cancer Gene Ther,2005,12(8),217-227.
[70]Hatano M,Mizuno M,Yoshida J.Enhancement of C2-ceramide antitumor activity by small interfering RNA on X chromosome-linked inhibitor of apoptosis protein in resistant human glioma cells[J]. Neurosurg J, 2004,101 (1),119-127.
[71] Zhang Q , Zhang Z F, Rao JY, et al.Treatment with siRNA and antisense oligonucleotides targeted to HIF-1 alpha induced apoptosis in human tongue squamous cell carcinomas[J]. Int J Cancer, 2004,111 (6), 849-857.
[72] Yamaguchi Y, Shiraki K, Fuke H, et al.Targeting of X-linked inhibitor of apoptosis protein or survivin by short interfering RNAs sensitize hepatoma cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic agent-induced cell death[J]. Oncol. Rep,2005,14 (5), 1311-1316.
[73] Yonesaka K, Tamura K, Kurata T,et al. Small interfering RNA targeting surviv in sensitizes lung cancer cell with mutant p53 to adriamycin[J].Int J Cancer,2006 ,118 (4), 812-820.
[74] Crnkovic M, Wagener N, Semzow J,et,al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J].Cell Mol Life Sci, 2007,64 (9): 1137-1144