基于超顺磁性四氧化三铁磁性纳米颗粒的恶性胶质瘤靶向联合基因治疗
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摘要
第一章超顺磁性Fe_3O_4纳米颗粒基因转运体系建立及体外基因转运效率评价
目的:制备能够应用于胶质瘤靶向联合基因治疗的四氧化三铁磁性纳米颗粒,建立以超顺磁性Fe_3O_4磁性纳米颗粒为基因载体的胶质瘤基因治疗基因转运体系,并评价该体系的体外基因转运效率。
方法:以2-吡咯烷酮和乙酰丙酮铁为原料,采用高温分解铁有机物法是将铁前驱体高温分解得到铁原子,再由铁原子生成铁纳米颗粒,将铁纳米颗粒控制氧化得到四氧化三铁纳米颗粒;再以偶联剂γ-氨丙基三乙氧基硅烷(NH_2C_3H_6Si(OC_2H_5)_3)对Fe_3O_4磁性纳米颗粒(Fe_3O_4magnetic nanoparticle,Fe_3O_4MNP)表面修饰;将CD基因转染U251胶质瘤细胞,得到U251-CD细胞,免疫荧光染色检测本体系的转染效率。通过Fe_3O_4MNP与质粒pCMVCD结合试验,Fe_3O_4MNP-pCMVCD复合物的DNase-Ⅰ消化,血清消化保护实验结果检测Fe_3O_4MNP结合DNA的能力及对DNA的保护效果。采用RT-PCR、Western blotting,和高效液相色谱法等检测CD基因的mRNA水平和CD蛋白水平,U251-CD细胞。MTT法测定检测U251-CD细胞的5-FC化疗效果及旁观者效应。
结果:成功地制备出稳定性好、单分散、粒径小、粒径分布范围窄(10±2nm)的磁性Fe_3O_4/氨基硅烷复合纳米颗粒,以γ-氨丙基三乙氧基硅烷作分散稳定剂,改善了磁性Fe_3O_4纳米颗粒表面的疏水环境,有效地阻止了四氧化三铁纳米颗粒团聚的发生。当Fe_3O_4MNP与质粒pCMVCD质量比为0.05:1时,结合的质粒量<20%;当二者质量比为1:1时,几乎可以结合体系中的全部质粒。Fe_3O_4MNP-pCMVCD复合物能够保护质粒pCMVCD免受Dnase-Ⅰ及血清的消化作用,保持其结构稳定。使用Fe_3O_4MNP作为载体成功将CD基因成功转染U251细胞,转染细胞有CD基因稳定表达,呈时间相关性增强表达模式(转染后5天内呈现表达持续增加模式),转染效果明显优于脂质体转染对照组。Fe_3O_4MNP的转染效果与Fe_3O_4MNP的用量呈正相关,且无明显的细胞毒性,经MTT检测Fe_3O_4MNP在50mg/L以下时不影响U251细胞的生长曲线。U251-CD细胞加入5-氟胞嘧啶(5-FC)后,细胞培养液上清中,经高效液相色谱检测出U251-CD细胞生成的高浓度5-氟尿嘧啶。能显著的抑制U251细胞生长。U251与U251-CD混合细胞中,在加入5-FC(终浓度1000uml/L)后,10%的U251-CD细胞可以杀灭20～30%的U251与U251-CD混合细胞与对照组(0%组)相比P<0.01,15%的U251-CD细胞可以杀灭约50%U251与U251-CD的混合细胞;30%的U251-CD细胞可以杀灭约80%U251与U251-CD的混合细胞;50%以上的U251-CD细胞可以完全的杀灭U251与U251-CD的混合细胞。
结论:本章制备的经γ-氨丙基三乙氧基硅烷修饰的Fe_3O_4磁性纳米颗粒粒径小,生物相容性提高,达到作为基因载体应用于胶质瘤靶向联合基因治疗要求。超顺磁性Fe_3O_4纳米颗粒基因转运体系在体外U251胶质瘤细胞系应用中转染效果显著。Fe_3O_4MNP/CD/5-FC胶质瘤基因治疗系统应用于胶瘤基因治疗体外试验抑瘤效果突出,可应用于下一步动物体内实验检测。
第二章Fe_3O_4MNP介导CD/5-FC系统联合wt-p53及wt-p16治疗恶性胶质瘤的体外实验研究
目的在第一章的Fe_3O_4MNP/CD/5-FC胶质瘤基因治疗系统成功研发的基础上,为优化该基因治疗系统,研发wt-p53、wt-p16基因联合胞嘧啶脱氨酶(Cytosine Deaminase,CD)/5-FC系统治疗神经胶质瘤的可行性,以期能提高胶质瘤化疗效果及逆转胶质瘤干细胞对化疗药物产生多药耐药性。
方法分离培养并鉴定U251细胞系及30例临床胶质瘤病理标本(WHOⅢ级、Ⅳ级)的胶质胶干细胞。利用Fe_3O_4MNP为基因载体分别将质粒pCDNA3.1-p16、pYD5-p53,及pCMVCD转染至U251细胞、各组病理组织分离的胶质瘤细胞及其胶质瘤干细胞(Glioma stemcells,GSC)。采用RT-PCR、Western blotting检测目标基因转染后在相应的细胞中的表达情况。MTT法检测不同基因治疗方案的U251细胞及U251GSC细胞的对CD/5-FC的治疗敏感性;并检测四种临床常用化疗药物VM-26,VP-16,TMZ,5-FU对不同基因治疗方案的U251及U251GSC的生长抑制率。使用AnnexinV-PI双染法对10例病理标本分离出来的并经转染后的各组Ⅲ～Ⅳ级胶质瘤细胞实施不同的基因治疗方案后的细胞凋亡情况进行检测。
结果成功以Fe_3O_4MNP为载体将wt-p53、wt-p16,及CD基因转染U251细胞、30例病理标本分离出来胶质瘤细胞,及其胶质瘤干细胞,并稳定表达。RT-PCR、Western blotting检测结果显示胶质瘤细胞和胶质瘤干细胞的目标基因表达情况无差别(p<0.01)。体外转染wt-p53、wt-p16基因都能明显提高CD/5-FC人对U251细胞系的化疗效果。以VM-26、VP16、TMZ,5-FU的1倍血浆峰浓度值的细胞增殖抑制率,各化疗药物组对U251胶质瘤细胞的体外增殖均有不同程度的抑制,以TMZ效果最好;但U251GCS对各种化疗敏感度均较U251细胞明显下降(p<0.01),其中TMZ效果最好;P53/P16转染组U251GCS对VM-26、VP16、TMZ,5-FU的敏感性较U251GCS均明显提高(p<0.01),TMZ效果仍是最好。对10例病理标本分离出来的并经转染后的各组Ⅲ~Ⅳ级胶质干瘤细胞实施不同的基因治疗方案后的细胞凋亡情况进行检测结果显示CD/P53/P16联合组胶质干瘤细胞凋亡率(46.32±12.78%)高于对照组(1.53±1.22%)及P53(30.1±10.45%)、P16(33.10±9.89%)单独转染组(p<0.01)。CD/P53/P16/5-FC组(89.87±9.02%)明显高于CD/P53/P16/5-FU组(60.18±6.23%)(p<0.01)。
结论基于Fe_3O_4磁性纳米基因载体的p53、p16联合CD/5-FC系统基因治疗体系可作为临床上胶质瘤治疗方案的一种可能选择,可在体外有效的杀灭胶质瘤细胞及胶质瘤干细胞,该基因治疗系统对肿瘤细胞杀伤作用的机理与凋亡相关。另外,该体系能逆转胶质瘤干细胞对多种临床常用的化疗药物的耐药性,明显提高化疗效果。
第三章Fe_3O_4 MNP介导CD/5-FC系统联合wt-p53及wt-p16治疗恶性胶质瘤的动物实验研究
目的Fe_3O_4磁性纳米基因载体在体内的转染效果及静脉注射时体内分布情况。在胶质瘤干细胞裸鼠皮下成瘤动物模型中验证本课题研发的“基于Fe_3O_4磁性纳米基因载体的p53、p16联合CD/5-FC系统基因治疗体系”的治疗效果。实施体内磁靶向定位基因治疗,并观察该基因治疗体系的全身副作用。
方法建立胶质瘤干细胞皮下荷瘤裸鼠模型。原子吸收分光光度计检测Fe_3O_4MNP在裸鼠体内的磁靶向定位效果及体内分布情况。免疫组化检测各目标基因的蛋白在动物模型胶质瘤组织中的表达,并采用TUNEL原位凋亡试剂盒检测各组肿瘤组织细胞凋亡情况。效液相色谱法检测5-FC体内转化为5-FU情况。体内抑瘤实验检测不同基因治疗方案下各组胶质瘤干细胞在裸鼠皮下成瘤体积,计算抑瘤率,评定治疗效果,并观察裸鼠体重的变化。对比CD/P53/P16转染组5-FC与5-FU的治疗效果及全向副作用。
结果成功建立胶质瘤干细胞皮下荷瘤裸鼠模型,一般在接种后14d可见较明显皮下结节的形成。在体外肿瘤表面施加4000高斯磁场时,鼠尾静脉注射磁性纳米颗粒溶液后45min,实验组的肿瘤组织铁元素含量(μg/g)为91.46±19.94,远高于对照组30.23±6.34,(p<0.01)。说明在磁场作用下Fe_3O_4MNP定向向肿瘤组织移动,大量聚集在肿瘤组织内造成铁元素含量明显升高,另外脑组织铁含量也较正常对照组明显升高(p<0.01),说明Fe_3O_4MNP透过血脑屏障能力显著。wt-p53、wt-p16能够显著的裸鼠荷瘤的体积,并减少荷瘤裸鼠体重的下降。荷瘤裸鼠BALB/c-CD/p53/p16腹腔注射5-FC后5d起皮下肿瘤体积明显缩小,经高效液相色谱对肿瘤组织均浆检测出5-FU。而荷瘤裸鼠BALB/c-CD/p53/p16腹腔注射5-FU后1M内肿瘤体积末见缩小,反而进行性恶化。5-FC治疗组对裸鼠骨髓抑制、血常规、肝功能影响明显小于5-FU治疗组(p<0.01)。各组荷瘤裸鼠TUNEL检测凋亡情况结果与第二章体外检测结果一致。对12例不同的胶质瘤病理组织分离培养的胶质瘤干细胞裸鼠皮下形成的胶质瘤行体内Fe_3O_4MNP介导的CD/p53/p16基因治疗。12例中原病理组织P53,P16免疫组化阳性率均为25%,基因治疗总有效率为33%(4/12),微效率为25%(3/12),无效率为25%(3/12),恶化率为17%(1/12)。
结论基于Fe_3O_4磁性纳米基因载体的p53、p16联合CD/5-FC系统基因治疗体系可作为临床上胶质瘤治疗方案的一种可能选择,可在胶质瘤干细胞皮下荷瘤裸鼠模型体内有效抑制胶质瘤生长,在5-FC治疗时能有效的缩小已生成胶质瘤体积,该基因治疗系统对肿瘤细胞杀伤作用的机理与凋亡相关,体内外实验结果相符。
Objective:To synthesize Fe_O_4 magnetite nanoparticles(Fe_3O_4MNP) wich could be used in targeted glioma multi-gene therapy of malignant glioma.In this study,the newly nanotechnology and molecular biology were combined to develope a new glioma suicide gene therapy system base on superparamagnetic Fe_3O_4 magnetic nanoparticle gene vehicle and CD/5-FC system.
Methods:Fe_3O_4MNP were prepared by thermal decomposition of Fe(acac)3 in 2-pyrrolidone,and 3-aminopropyltriethoxy-silane(APTTS) was used to modify the surface of the nanoparticles.Base on XRD,TEM, analysis,the nanoparticle size were tested.The Fe_3O_4MNP was evaluated as a kind of plasmid pCMVCD carrier and transfected into human glioma cell line U251 in vitro.The mRNA and protein expression of intracellular CD gene were tested by RT-PCR,Western blotting,and Immunofluorescent staining,respectively.Methods Methyl thiazolyl tetrazolium(MTT) method was used to detect the toxicity of Fe_O_4MNP the proliferation of U251 cells in the presence of CD/5-FC system,and "bystand effect"of U251-CD.
Results:APTTS-modified Fe3O4 magnetite nanoparticles prepared in this part,had a narrow particle size distribution(10±2nm),good crystallinity.DNA binding assay and co-sedimentation assay showed Fe3O4MNP could absorb most plasmid pCMVCD,when the m/m ratio of pCMVCD to Fe3O4 MNP was 4:1 at pH=7.4,the binding interaction also protected DNA against the digestion of DNase-Ⅰand blood serum.The result of RT-PCR,Western blotting,and immunofluorescent staining revealed that intracellular CD gene levels continuously increased in a time-dependent manner after transfection of U251 cells with Fe3O4MNP-pCMVCD complex.MTT result showed Fe3O4MNP was safe to use in U251 gene tansfection in vitro,Fe3O4MNP/ CD/5-FC system could killed U251 cells effectively,the "bystand effect"show 10% U251-CD cells could killed 20-30%U251 and U251-CD mixed cells(p＜0.01 vs negtive control group),15%killed 50%mixed cells,35% killed 80%mixed cells,50%killed almost all cells in vitro.
Conclusion:Fe_3O_4MNP could be used as one of the ideal gene carriers for CD gene delivery.U251-CD cells could transform 5-FC into 5-FU.Fe_3O_4MNP/CD/5-FC system can become a new method of brain glioma adjunctive therapy. malignant glioma.Try to increase chemotherapy effect of glioma by reversing the multi-drug resistance of glioma stem cells.
Methods:pCDNA3.1-P16,pYD5-P53 and pCMVCD were transfected into U251 glioma cells by Fe3O4 magnetic nanoparticles. Methyl thiazolyl tetrazolium(MTT) method was used to detect the cell growth inhibition rate of U251 cells in the presence of 5-fluorocytosine, and growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251,U251-GCS cells.RT-PCR,Western blotting were used to detect target gene expression.G lioma stem cells was isolated from 30 different glioma tissue(WHO GradeⅢorⅣ),and their apoptosis after different gene therapy was test by AnnexinⅤ-FITC/PI Apoptosis Detection Kits.
Results:Glioma stem cells was isolated from 30 different glioma tissue sucessfully.wt-p53,wt-pl6 and CD gene were transfcted into U251 cells induced by Fe_3O_4 magnetic nanoparticles,and wt-p53,wt-p16 could increase the antitumor effect of CD/5-FC gene therapy system(p＜0.01), and the growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251 (p＜0.01),U251-GCS cells.Chemotherapy effect of TMZ was best. Apoptosis detection showed apoptotic rates increase in different gene therapy group,p＜0.01:CD/P53/P16 vs P53 and P16 group,CD /P53/P16/5-FC group(89.87±9.02%) better than other groups(p＜0.01).
Conclusion:The combination of wt-p53,wt-p16 gene with CD/5-FC system had extremely good antitumor effect in vivo,reversed the multi-drug resistance of glioma stem cells and increased growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251 cells.Its antitumor
Objective:To investigate the establishment of nude mice-subcutaneous glioma model induced by glioma stem cells,and its application feasibility in detecting the transfected effect of wt-p53,p16 and CD/5-FC gene therapy system base on Fe_3O_4MNP gene carrier in vivo,distribution of Fe_3O_4MNP in nude mice,and magnetic targeted localization in tumor.
Methods:Human glioma stem cell- suspension isolated from different human glioma tissues,was inolulated to the 30 male nude mice subcutaneously.Distribution of Fe_3O_4MNP in nude mice was tested by atomic absorption Spectrometry.Pathological and immunohistochemical features of tumor were studied.TUNEL(TdT-mediated dUTP nick end labeling) was used to test cells apoptosis in tumor tissue after gene therapy.
Results:Success rates of this methods were 100%,tumor grew in good condition,and the histological and immunohistochemical examination results of tumor were consistent with human glioma cell morphology.Glioma nodus could be found 14d after glioma stem cells were inolulated to nude mice subcutaneously.Under 4000 gauss magnetic field near the tumor,tumor Fer content(91.46±19.94μg/g) higher than control gruop(30.23±6.34μg/g)(p＜0.01),showed that Fe_3O_4MNP could lateralizaed to tumor under magnetic field guiding.Otherwise,Fer content of brain increased higher than control group(p＜0.01),showed that Fe_3O_4MNP could pass blood brain barrier without magnetic field guiding.Wt-p53、wt-p16 could decrease tumor volum and nude mice body weight lose.After intraperitoneal injection of 5-FC in glioma Bearing Nude Mice- BALB/c-CD/p53/p16,the tumor volume deflated grudually with 7d.TUNEL result showed glioma cells apoptosis increased in vivo after gene therapy.
Conclusion:The combination of wt-p53,wt-p16 gene with CD/5-FC system may be more suitable for clinical therapeutic trials of suicide gene therapy for malignant gliomas.Its antitumor effect was correlation with increasing cell apoptosis just as the in vitro result.
目的:制备能够应用于胶质瘤靶向联合基因治疗的四氧化三铁磁性纳米颗粒,建立以超顺磁性Fe_3O_4磁性纳米颗粒为基因载体的胶质瘤基因治疗基因转运体系,并评价该体系的体外基因转运效率。
方法:以2-吡咯烷酮和乙酰丙酮铁为原料,采用高温分解铁有机物法是将铁前驱体高温分解得到铁原子,再由铁原子生成铁纳米颗粒,将铁纳米颗粒控制氧化得到四氧化三铁纳米颗粒;再以偶联剂γ-氨丙基三乙氧基硅烷(NH_2C_3H_6Si(OC_2H_5)_3)对Fe_3O_4磁性纳米颗粒(Fe_3O_4magnetic nanoparticle,Fe_3O_4MNP)表面修饰;将CD基因转染U251胶质瘤细胞,得到U251-CD细胞,免疫荧光染色检测本体系的转染效率。通过Fe_3O_4MNP与质粒pCMVCD结合试验,Fe_3O_4MNP-pCMVCD复合物的DNase-Ⅰ消化,血清消化保护实验结果检测Fe_3O_4MNP结合DNA的能力及对DNA的保护效果。采用RT-PCR、Western blotting,和高效液相色谱法等检测CD基因的mRNA水平和CD蛋白水平,U251-CD细胞。MTT法测定检测U251-CD细胞的5-FC化疗效果及旁观者效应。
结果:成功地制备出稳定性好、单分散、粒径小、粒径分布范围窄(10±2nm)的磁性Fe_3O_4/氨基硅烷复合纳米颗粒,以γ-氨丙基三乙氧基硅烷作分散稳定剂,改善了磁性Fe_3O_4纳米颗粒表面的疏水环境,有效地阻止了四氧化三铁纳米颗粒团聚的发生。当Fe_3O_4MNP与质粒pCMVCD质量比为0.05:1时,结合的质粒量<20%;当二者质量比为1:1时,几乎可以结合体系中的全部质粒。Fe_3O_4MNP-pCMVCD复合物能够保护质粒pCMVCD免受Dnase-Ⅰ及血清的消化作用,保持其结构稳定。使用Fe_3O_4MNP作为载体成功将CD基因成功转染U251细胞,转染细胞有CD基因稳定表达,呈时间相关性增强表达模式(转染后5天内呈现表达持续增加模式),转染效果明显优于脂质体转染对照组。Fe_3O_4MNP的转染效果与Fe_3O_4MNP的用量呈正相关,且无明显的细胞毒性,经MTT检测Fe_3O_4MNP在50mg/L以下时不影响U251细胞的生长曲线。U251-CD细胞加入5-氟胞嘧啶(5-FC)后,细胞培养液上清中,经高效液相色谱检测出U251-CD细胞生成的高浓度5-氟尿嘧啶。能显著的抑制U251细胞生长。U251与U251-CD混合细胞中,在加入5-FC(终浓度1000uml/L)后,10%的U251-CD细胞可以杀灭20～30%的U251与U251-CD混合细胞与对照组(0%组)相比P<0.01,15%的U251-CD细胞可以杀灭约50%U251与U251-CD的混合细胞;30%的U251-CD细胞可以杀灭约80%U251与U251-CD的混合细胞;50%以上的U251-CD细胞可以完全的杀灭U251与U251-CD的混合细胞。
结论:本章制备的经γ-氨丙基三乙氧基硅烷修饰的Fe_3O_4磁性纳米颗粒粒径小,生物相容性提高,达到作为基因载体应用于胶质瘤靶向联合基因治疗要求。超顺磁性Fe_3O_4纳米颗粒基因转运体系在体外U251胶质瘤细胞系应用中转染效果显著。Fe_3O_4MNP/CD/5-FC胶质瘤基因治疗系统应用于胶瘤基因治疗体外试验抑瘤效果突出,可应用于下一步动物体内实验检测。
第二章Fe_3O_4MNP介导CD/5-FC系统联合wt-p53及wt-p16治疗恶性胶质瘤的体外实验研究
目的在第一章的Fe_3O_4MNP/CD/5-FC胶质瘤基因治疗系统成功研发的基础上,为优化该基因治疗系统,研发wt-p53、wt-p16基因联合胞嘧啶脱氨酶(Cytosine Deaminase,CD)/5-FC系统治疗神经胶质瘤的可行性,以期能提高胶质瘤化疗效果及逆转胶质瘤干细胞对化疗药物产生多药耐药性。
方法分离培养并鉴定U251细胞系及30例临床胶质瘤病理标本(WHOⅢ级、Ⅳ级)的胶质胶干细胞。利用Fe_3O_4MNP为基因载体分别将质粒pCDNA3.1-p16、pYD5-p53,及pCMVCD转染至U251细胞、各组病理组织分离的胶质瘤细胞及其胶质瘤干细胞(Glioma stemcells,GSC)。采用RT-PCR、Western blotting检测目标基因转染后在相应的细胞中的表达情况。MTT法检测不同基因治疗方案的U251细胞及U251GSC细胞的对CD/5-FC的治疗敏感性;并检测四种临床常用化疗药物VM-26,VP-16,TMZ,5-FU对不同基因治疗方案的U251及U251GSC的生长抑制率。使用AnnexinV-PI双染法对10例病理标本分离出来的并经转染后的各组Ⅲ～Ⅳ级胶质瘤细胞实施不同的基因治疗方案后的细胞凋亡情况进行检测。
结果成功以Fe_3O_4MNP为载体将wt-p53、wt-p16,及CD基因转染U251细胞、30例病理标本分离出来胶质瘤细胞,及其胶质瘤干细胞,并稳定表达。RT-PCR、Western blotting检测结果显示胶质瘤细胞和胶质瘤干细胞的目标基因表达情况无差别(p<0.01)。体外转染wt-p53、wt-p16基因都能明显提高CD/5-FC人对U251细胞系的化疗效果。以VM-26、VP16、TMZ,5-FU的1倍血浆峰浓度值的细胞增殖抑制率,各化疗药物组对U251胶质瘤细胞的体外增殖均有不同程度的抑制,以TMZ效果最好;但U251GCS对各种化疗敏感度均较U251细胞明显下降(p<0.01),其中TMZ效果最好;P53/P16转染组U251GCS对VM-26、VP16、TMZ,5-FU的敏感性较U251GCS均明显提高(p<0.01),TMZ效果仍是最好。对10例病理标本分离出来的并经转染后的各组Ⅲ~Ⅳ级胶质干瘤细胞实施不同的基因治疗方案后的细胞凋亡情况进行检测结果显示CD/P53/P16联合组胶质干瘤细胞凋亡率(46.32±12.78%)高于对照组(1.53±1.22%)及P53(30.1±10.45%)、P16(33.10±9.89%)单独转染组(p<0.01)。CD/P53/P16/5-FC组(89.87±9.02%)明显高于CD/P53/P16/5-FU组(60.18±6.23%)(p<0.01)。
结论基于Fe_3O_4磁性纳米基因载体的p53、p16联合CD/5-FC系统基因治疗体系可作为临床上胶质瘤治疗方案的一种可能选择,可在体外有效的杀灭胶质瘤细胞及胶质瘤干细胞,该基因治疗系统对肿瘤细胞杀伤作用的机理与凋亡相关。另外,该体系能逆转胶质瘤干细胞对多种临床常用的化疗药物的耐药性,明显提高化疗效果。
第三章Fe_3O_4 MNP介导CD/5-FC系统联合wt-p53及wt-p16治疗恶性胶质瘤的动物实验研究
目的Fe_3O_4磁性纳米基因载体在体内的转染效果及静脉注射时体内分布情况。在胶质瘤干细胞裸鼠皮下成瘤动物模型中验证本课题研发的“基于Fe_3O_4磁性纳米基因载体的p53、p16联合CD/5-FC系统基因治疗体系”的治疗效果。实施体内磁靶向定位基因治疗,并观察该基因治疗体系的全身副作用。
方法建立胶质瘤干细胞皮下荷瘤裸鼠模型。原子吸收分光光度计检测Fe_3O_4MNP在裸鼠体内的磁靶向定位效果及体内分布情况。免疫组化检测各目标基因的蛋白在动物模型胶质瘤组织中的表达,并采用TUNEL原位凋亡试剂盒检测各组肿瘤组织细胞凋亡情况。效液相色谱法检测5-FC体内转化为5-FU情况。体内抑瘤实验检测不同基因治疗方案下各组胶质瘤干细胞在裸鼠皮下成瘤体积,计算抑瘤率,评定治疗效果,并观察裸鼠体重的变化。对比CD/P53/P16转染组5-FC与5-FU的治疗效果及全向副作用。
结果成功建立胶质瘤干细胞皮下荷瘤裸鼠模型,一般在接种后14d可见较明显皮下结节的形成。在体外肿瘤表面施加4000高斯磁场时,鼠尾静脉注射磁性纳米颗粒溶液后45min,实验组的肿瘤组织铁元素含量(μg/g)为91.46±19.94,远高于对照组30.23±6.34,(p<0.01)。说明在磁场作用下Fe_3O_4MNP定向向肿瘤组织移动,大量聚集在肿瘤组织内造成铁元素含量明显升高,另外脑组织铁含量也较正常对照组明显升高(p<0.01),说明Fe_3O_4MNP透过血脑屏障能力显著。wt-p53、wt-p16能够显著的裸鼠荷瘤的体积,并减少荷瘤裸鼠体重的下降。荷瘤裸鼠BALB/c-CD/p53/p16腹腔注射5-FC后5d起皮下肿瘤体积明显缩小,经高效液相色谱对肿瘤组织均浆检测出5-FU。而荷瘤裸鼠BALB/c-CD/p53/p16腹腔注射5-FU后1M内肿瘤体积末见缩小,反而进行性恶化。5-FC治疗组对裸鼠骨髓抑制、血常规、肝功能影响明显小于5-FU治疗组(p<0.01)。各组荷瘤裸鼠TUNEL检测凋亡情况结果与第二章体外检测结果一致。对12例不同的胶质瘤病理组织分离培养的胶质瘤干细胞裸鼠皮下形成的胶质瘤行体内Fe_3O_4MNP介导的CD/p53/p16基因治疗。12例中原病理组织P53,P16免疫组化阳性率均为25%,基因治疗总有效率为33%(4/12),微效率为25%(3/12),无效率为25%(3/12),恶化率为17%(1/12)。
结论基于Fe_3O_4磁性纳米基因载体的p53、p16联合CD/5-FC系统基因治疗体系可作为临床上胶质瘤治疗方案的一种可能选择,可在胶质瘤干细胞皮下荷瘤裸鼠模型体内有效抑制胶质瘤生长,在5-FC治疗时能有效的缩小已生成胶质瘤体积,该基因治疗系统对肿瘤细胞杀伤作用的机理与凋亡相关,体内外实验结果相符。
Objective:To synthesize Fe_O_4 magnetite nanoparticles(Fe_3O_4MNP) wich could be used in targeted glioma multi-gene therapy of malignant glioma.In this study,the newly nanotechnology and molecular biology were combined to develope a new glioma suicide gene therapy system base on superparamagnetic Fe_3O_4 magnetic nanoparticle gene vehicle and CD/5-FC system.
Methods:Fe_3O_4MNP were prepared by thermal decomposition of Fe(acac)3 in 2-pyrrolidone,and 3-aminopropyltriethoxy-silane(APTTS) was used to modify the surface of the nanoparticles.Base on XRD,TEM, analysis,the nanoparticle size were tested.The Fe_3O_4MNP was evaluated as a kind of plasmid pCMVCD carrier and transfected into human glioma cell line U251 in vitro.The mRNA and protein expression of intracellular CD gene were tested by RT-PCR,Western blotting,and Immunofluorescent staining,respectively.Methods Methyl thiazolyl tetrazolium(MTT) method was used to detect the toxicity of Fe_O_4MNP the proliferation of U251 cells in the presence of CD/5-FC system,and "bystand effect"of U251-CD.
Results:APTTS-modified Fe3O4 magnetite nanoparticles prepared in this part,had a narrow particle size distribution(10±2nm),good crystallinity.DNA binding assay and co-sedimentation assay showed Fe3O4MNP could absorb most plasmid pCMVCD,when the m/m ratio of pCMVCD to Fe3O4 MNP was 4:1 at pH=7.4,the binding interaction also protected DNA against the digestion of DNase-Ⅰand blood serum.The result of RT-PCR,Western blotting,and immunofluorescent staining revealed that intracellular CD gene levels continuously increased in a time-dependent manner after transfection of U251 cells with Fe3O4MNP-pCMVCD complex.MTT result showed Fe3O4MNP was safe to use in U251 gene tansfection in vitro,Fe3O4MNP/ CD/5-FC system could killed U251 cells effectively,the "bystand effect"show 10% U251-CD cells could killed 20-30%U251 and U251-CD mixed cells(p＜0.01 vs negtive control group),15%killed 50%mixed cells,35% killed 80%mixed cells,50%killed almost all cells in vitro.
Conclusion:Fe_3O_4MNP could be used as one of the ideal gene carriers for CD gene delivery.U251-CD cells could transform 5-FC into 5-FU.Fe_3O_4MNP/CD/5-FC system can become a new method of brain glioma adjunctive therapy. malignant glioma.Try to increase chemotherapy effect of glioma by reversing the multi-drug resistance of glioma stem cells.
Methods:pCDNA3.1-P16,pYD5-P53 and pCMVCD were transfected into U251 glioma cells by Fe3O4 magnetic nanoparticles. Methyl thiazolyl tetrazolium(MTT) method was used to detect the cell growth inhibition rate of U251 cells in the presence of 5-fluorocytosine, and growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251,U251-GCS cells.RT-PCR,Western blotting were used to detect target gene expression.G lioma stem cells was isolated from 30 different glioma tissue(WHO GradeⅢorⅣ),and their apoptosis after different gene therapy was test by AnnexinⅤ-FITC/PI Apoptosis Detection Kits.
Results:Glioma stem cells was isolated from 30 different glioma tissue sucessfully.wt-p53,wt-pl6 and CD gene were transfcted into U251 cells induced by Fe_3O_4 magnetic nanoparticles,and wt-p53,wt-p16 could increase the antitumor effect of CD/5-FC gene therapy system(p＜0.01), and the growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251 (p＜0.01),U251-GCS cells.Chemotherapy effect of TMZ was best. Apoptosis detection showed apoptotic rates increase in different gene therapy group,p＜0.01:CD/P53/P16 vs P53 and P16 group,CD /P53/P16/5-FC group(89.87±9.02%) better than other groups(p＜0.01).
Conclusion:The combination of wt-p53,wt-p16 gene with CD/5-FC system had extremely good antitumor effect in vivo,reversed the multi-drug resistance of glioma stem cells and increased growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251 cells.Its antitumor
Objective:To investigate the establishment of nude mice-subcutaneous glioma model induced by glioma stem cells,and its application feasibility in detecting the transfected effect of wt-p53,p16 and CD/5-FC gene therapy system base on Fe_3O_4MNP gene carrier in vivo,distribution of Fe_3O_4MNP in nude mice,and magnetic targeted localization in tumor.
Methods:Human glioma stem cell- suspension isolated from different human glioma tissues,was inolulated to the 30 male nude mice subcutaneously.Distribution of Fe_3O_4MNP in nude mice was tested by atomic absorption Spectrometry.Pathological and immunohistochemical features of tumor were studied.TUNEL(TdT-mediated dUTP nick end labeling) was used to test cells apoptosis in tumor tissue after gene therapy.
Results:Success rates of this methods were 100%,tumor grew in good condition,and the histological and immunohistochemical examination results of tumor were consistent with human glioma cell morphology.Glioma nodus could be found 14d after glioma stem cells were inolulated to nude mice subcutaneously.Under 4000 gauss magnetic field near the tumor,tumor Fer content(91.46±19.94μg/g) higher than control gruop(30.23±6.34μg/g)(p＜0.01),showed that Fe_3O_4MNP could lateralizaed to tumor under magnetic field guiding.Otherwise,Fer content of brain increased higher than control group(p＜0.01),showed that Fe_3O_4MNP could pass blood brain barrier without magnetic field guiding.Wt-p53、wt-p16 could decrease tumor volum and nude mice body weight lose.After intraperitoneal injection of 5-FC in glioma Bearing Nude Mice- BALB/c-CD/p53/p16,the tumor volume deflated grudually with 7d.TUNEL result showed glioma cells apoptosis increased in vivo after gene therapy.
Conclusion:The combination of wt-p53,wt-p16 gene with CD/5-FC system may be more suitable for clinical therapeutic trials of suicide gene therapy for malignant gliomas.Its antitumor effect was correlation with increasing cell apoptosis just as the in vitro result.
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