慢病毒载体介导的针对端粒酶的RNA干扰技术治疗肝癌的体内外研究
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摘要
肝脏肿瘤是亚洲最常见的恶性肿瘤,初次诊断多数为晚期。传统的手术、放疗及化疗等治疗效果欠佳。因此明确其发生发展机理,探索有效的治疗方法已成为当今普通外科亟待解决的问题。
随着分子生物学的发展,人们逐渐认识到肿瘤发生的机制主要是由于癌基因的激活和抑癌基因的失活而导致的肿瘤细胞增殖失控、凋亡缺陷以及细胞正常的有限生命周期缺陷,使细胞永生化,继而形成肿瘤。目前国内外研究发现细胞的永生化主要由于在某些因素的作用下激活了细胞内部的端粒酶,进而通过端粒酶来增加端粒序列,稳定端粒长度,从而提高细胞增殖能力并使其永生化。因此,端粒酶的激活可以说是细胞永生化的关键步骤。
端粒是指真核细胞线状染色体末端的一段区域,由许多有种属特异性的简单重复的DNA序列及与其特异性结合的蛋白质构成,它给染色体末端提供一个保护性“帽”。在人和其它脊椎动物中重复序列为5’-TTAGGG-3’。1984年Greider和Blackburn在美国柏克莱的Blackburn的实验室首次从四膜虫细胞提取液中发现有端粒酶的活性。1989年Morin首次在人的癌细胞系Hela细胞中发现了端粒酶。端粒酶是一种核糖核蛋白体(ribonucleoprotein、RNP),由RNA与蛋白质构成的复合体,是一种特殊的反转录RNA依赖性DNA聚合酶,能以自身的RNA为模板,通过逆转录而合成端粒,将端粒重复片段加到染色体的末端。因此它解决了DNA半保留复制模式中末端复制问题。Counter等1994年首先在转移性卵巢癌病人中检测到端粒酶活性。近几年来各国学者报道了大量有关肿瘤和端粒酶之间关系的文献,结果发现人正常体细胞中除生殖细胞和大多数发育中胚胎细胞及有增殖能力的更新组织中有较高的端粒酶活性外,大多数端粒酶阴性,通过对5000例以上的肿瘤标本进行端粒酶活性的检测发现,其中约85%为阳性,90%以上的恶性肿瘤中端粒酶阳性,且端粒酶的活性随着肿瘤恶性程度的增高而增高。
人端粒酶包括自身RNA(human telomerase RNA, hTR)、催化亚基(human telomerase reverse transcriptase, hTERT)和相关蛋白1(telomerase associated protein 1,TP1)。hTERT属于逆转录酶(reverse transcriptase,RT)家属成员,是一个有约40kb大小的单拷贝基因,定位于5p15.33。催化亚基基因首先是Lendvay在酵母中发现。1997年编码hTERT的基因被克隆出来。hTERT启动子序列于1999年被克隆出,有1.7Kb长,其中有251bp (-211~+40)是核心部分。近年来的研究发现hTERT mRNA除在胸腺、睾丸、小肠等几种正常组织中有低水平表达外,在大多数正常组织(心、脑、乳腺、肝、骨骼肌、前列腺、胎盘、卵巢)中都没有检测到。但对各种肿瘤组织的检测发现,hTERT mRNA在大多数肿瘤组织中表达,在所有的端粒酶阳性的肿瘤组织中都可检测到hTERT mRNA。在对肝癌中hTERT mRNA的研究中发现: hTERT mRNA在肝癌细胞中存在高表达,hTERT mRNA和hTERT蛋白表达水平与肝癌的恶性程度密切相关,且随着肿瘤的恶性度增加而升高,而正常肝组织中则没有hTERT的表达。目前认为hTERT是端粒酶的催化蛋白组分,同时也是端粒酶活化和细胞癌变的限速步骤,抑制其表达可有效地抑制肿瘤细胞的生长和诱导凋亡,可作为肝癌基因治疗的理想靶标。
当前针对肝癌的基因治疗方案主要包括免疫增强治疗、基因介导的酶前体药物治疗、抑癌基因替代治疗和反义治疗等。这些方法主要是通过将外源性目的基因导入宿主靶细胞内,使之表达以纠正细胞基因缺陷,达到疾病治疗目的。随着分子生物学技术的发展,基因治疗的技术不断得到优化和改进。
近年来研究发现,一些小的双链RNA(double-stranded RNA, dsRNA)可以高效、特异地阻断体内特定基因的表达,促使其mRNA降解,诱使细胞出现特定基因缺失的表型,这种技术即RNA干扰(RNA interference, RNAi),属于转录后基因沉默(post-transcrptional gene silencing, PTGS)。RNAi现象广泛存在于大多数真核细胞中,在小鼠和人的体外培养细胞中利用RNAi技术也可以成功阻断基因的表达,实现细胞水平的基因敲除。
基因治疗成功与否的关键在于采用理想的载体将靶基因高效地转导入靶细胞并获得良好的表达,这就对基因治疗的载体提出了较高的要求。目前常用的载体分为非病毒载体和病毒载体两大类,前者虽具有较高的安全性,但转染效率较差,因此近年来人们更专注于病毒载体的开发和应用。慢病毒载体(Leniviral Vector, pLenti)是一种具有较高转染效率和低细胞毒性的病毒载体。不仅能感染增殖期细胞,还能感染非增殖期细胞及静止期细胞如神经元细胞,无任何毒副作用。
基于以上几点,本试验首先以pLenti-GFP为基础,构建pLenti-hTERT-siRNA,在体外转染人肝癌细胞HepG2细胞,通过RNAi的机制抑制肝癌细胞高表达的hTERT,探讨其对肝癌细胞生长的抑制作用。
由于肝癌的常规治疗方法效果不佳,探求新的治疗策略已成当务之急。肝癌的基因治疗近年来发展迅速,并取得一定进展。但由于基因治疗本身的一些制约条件,比如载体的安全性及转染效率较低等,使得基因治疗的发展受到一定的限制。因此,探讨应用一些高转染效率,低细胞毒性的新型靶向载体来介导基因产物进行治疗已成为肝癌基因治疗研究的新热点。
选择合适的基因转运载体将目的基因安全高效地导入细胞,是基因治疗成功的关键。从目前发展趋势来看,病毒载体在基因转运方面呈现显著优势,其中慢病毒载体以其优越的理化性能和较理想的基因转运能力而日益受到重视。研究证明,pLenti-GFP可以高效介导基因转染多种肿瘤细胞株和原代培养细胞,并且具有缓释、安全无毒等优点。
本研究利用GFP荧光标记的hTERT-siRNA作为治疗基因,选择pLenti-GFP作为基因转运载体,在体外转染人肝癌细胞HepG-2,利用荧光显微镜分析转染效率,并以空载体为对照,对慢病毒载体pLenti-GFP的细胞毒性进行了连续观察和分析。此部分研究旨在探索利用pLenti-GFP慢病毒载体转基因治疗肝癌的可行性,进一步为肝癌基因治疗寻求一种较理想的基因转运手段。
研究结果表明:通过荧光显微镜检测慢病毒载体pLenti-GFP对人肝癌细胞HepG-2的转染效率,得到pLenti-GFP的转染效率为90%以上,完全可用于肝癌基因治疗的体外试验。此外,空载体组细胞生长状态良好,细胞凋亡率与不加任何干预的空白对照组相近,这就证明了pLenti-GFP是一种低细胞毒性的载体,可以用于肝癌基因治疗的研究。
hTERT是端粒酶催化亚基,常常只在增殖的细胞中表达,是端粒酶活化和细胞癌变的限速步骤,抑制其表达可有效地抑制肿瘤细胞的生长和诱导凋亡,已成为目前肿瘤基因治疗的理想靶标。RNAi通过双链RNA产生序列特异性的转录后基因沉默,在哺乳动物细胞内可高效特异地阻断特定基因的表达。它是将导入生物体内的或内源性转录生成的dsRNA被一种RNaseⅢ类的核酸酶Dicer切割成21~25nt的干扰性小RNA,即siRNA,siRNA进一步与其他多种蛋白成分结合形成RNA诱导的沉默复合体(RNA-induced silencing complex, RISC),最后由RISC介导siRNA反义链与靶mRNA分子互补结合并引起同源性靶mRNA分子的切割效应。
我们在第一部分中已经成功制备了具有高转染效率的基因治疗载体pLenti-GFP,在这一部分,我们设计了针对hTERT的siRNA,对人肝癌细胞系HepG-2细胞进行基因干扰治疗的研究,结果分别用MTT法和RT-PCR法分析转染后细胞增殖率、hTERT mRNA的表达,探讨其对肝癌细胞生长的抑制作用及其机制。结果显示:hTERT-siRNA在体外可明显抑制HepG-2细胞系的生长,凋亡增多,随着时间的延长,抑制作用增强,第7天时,其细胞抑制率为57.5%,和对照组差异有统计学意义。hTERT-siRNA转染HepG-2细胞后,hTERT mRNA的表达明显减少,端粒酶活性水平显著下降;空白组和空载组转染后hTERT mRNA及端粒酶活性无明显改变。由上述结果可以看出:hTERT-siRNA能抑制肿瘤细胞hTERT mRNA的表达,使肿瘤细胞的端粒酶活性水平下降,从而抑制肿瘤细胞生长、增殖,促进肿瘤细胞的凋亡。
在这一部分,我们设计了针对hTERT的siRNA,对人肝癌细胞系HepG-2细胞进行基因干扰治疗的体内研究。我们采用在裸鼠皮下分别接种转染和未转染目的基因的HepG2细胞。每3天用游标卡尺测量肿瘤的长径和短径,计算肿瘤体积(V = 1/ 2×长径×短径×短径)。30天后处死裸鼠,取出瘤体,多聚甲醛固定后包埋制作石蜡切片。连续切片,常规HE染色,光镜检查肿瘤组织学,TUNEL法检测细胞凋亡情况。结果显示:治疗组肿瘤生长缓慢,随着时间的延长,差异越来越明显。常规病理HE染色显示坏死增多,周围炎性细胞浸润。TUNEL法检测显示细胞凋亡增多。
综合以上三部分的实验结果,我们认为,hTERT是肝癌基因治疗的理想靶点,RNAi和pLenti-GFP基因转运技术有机结合治疗肝癌是一种切实可行的新策略,可望为日后肝癌的基因治疗开辟一个全新的技术平台,具有广阔的应用前景。但与此同时,许多重要的问题包括hTERT表达调控网络及其表达沉默诱导细胞凋亡机理等仍有待更深入的研究。
Hepatocellular carcinoma (HCC) is the most common malignant disease in Asia. Most HCC have developed to the advanced stage when patients get diagnosed at the first time. The conventional treatments of HCC, including surgery, radiation therapy and chemotherapy, have not yet getten the satisfied efficacy in patients. Therefore, clear-cut its etiopathogenesis and developing effective therapeutic approach would be a urgent project in treatment of HCC.
With the development and application of molecular biology in tumorrigenesis, it has been demonstrated that the development of malignant tumors is due to the activation of protooncogenes and inactivation of tumor suppressor genes, which lead a out of control on cell proliferation and apoptosis, and a deficit of normal life span of cells resulting in cellular immortality and tumorigenesis. Recently, the studies showed that cellular immortality was caused by the activated telomerase. The role of telomerase is to maintain telomeric length and promotes cell proliferation potential. The activated telomerase is believed to be a critical event in cellular immortility.
Telomere is the component of chromosome ends in eukaryotic cells. Just as the hat it consists of species specific tandem repeats of simple sequences and binding proteins Human and other mammals contain 5'-TTAGGG-3' repeats. In 1984, Greider and Blackburn found the activity of telomerase in Blackburn laboratory. In 1989, Morin found the telomerase in human breast cancer cell“Hela”. Telomere length is progressively shortened in normal cells due to "end replicative problem" untill senescence, while it is short and stable in tumor cells. Telomerase is a special reverse transcriptase which contains both essential proteins and RNA component. The RNA component is used as the template for synthesizing the telomeric repeats to lengthen telomere. Telomerase expression has been detected in more then 90% of tumors, but it is absent in most normal somatic tissues. The higher the degree of malignancy of tumors is, the higher the telomerase activity is.
Human telomerase contains three major subunits, RNA (hTR), human telomerase catalytic subunits (hTERT), and telomerase-associated protein (TP1), have been identified recently. hTERT was cloned in 1997, its template contains 11 nucleotides. In 1999, the hTERT gene promoter was cloned, its template contains 1.7bp nucleotides, with its core is -211~+40. Recently researches found that hTERT mRNA had no expressed in most normal tissue (heart, brain, breast, liver, skeletal muscle, prostate and placenta) except thymus, testis and small intestine et al. But some researches also found that hTERT mRNA was expressed in nearly all Hepatocellular carcinoma (HCC) as well as other cancers. And the level of hTERT mRNA was higher in malignant tumors than in normal liver tissues, and correlated positively with the degree of malignancy of tumors. hTERT is similar with elementary eukaryotic telomerase reverse transcriptase in sequence similarity. The hTERT is the catalytic subunit of telomerase. Its expression is usually parallel with telomerase activity. The hTERT is the telomerase proteinum catalytic subunit, which is expressed in proliferative cells, also is the rate-limiting step of telomerase activation and cell cancerization. To restrain the expression of hTERT could effectively shutdown the growth of HCC cells and deduce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the HCC cell.
Currently treatment prescriptions in gene therapy for HCC include immunopotentiation therapy; gene mediated pre-enzyme drug therapy; gene replacement and anti-sense gene therapy. These methods mainly use exogenous gene which was imported into target cells to rectification cell gene deviancy in order to achieve goals of treatment.
Some researches found that a few dsRNA (double-stranded RNA) can specifically inhibit the expression of target gene efficiently and degraded mRNA. It is named as RNAi (RNA interference), belonging to PTGS (post-transcriptional gene silencing). This phenomenon generally emerges in eukaryotic cells and vitro cells, for successful target validation in therapeutic approaches in gene silencing.
The key of gene therapy is to have gene highly express in target cell, which is mediated by ideal carrier efficiently. So there is a high requirement for gene carrier. The current carriers include non viral vector and viral vector. However, the former one has a high safety advantage, with a low efficiently transfect rate. Leniviral Vector (pLenti-GFP) is a high rate of transduction virus vector with low cytotoxicity. The vectors can not only transduce proliferating cells, but also transduce quiescent cell, such as neuron cells, without any cytotoxicity.
In this study, we constructed the gene vector pLenti-GFP containing the hTERT gene and transduced it into HepG-2 cell in vitro. We have studied the inhibitory effects of hTERT-siRNA to HepG-2 cells (one kind of human HCC cells) by using RNAi to inhabit the expression of hTERT.
Because of the unfavorable outcome of conventional therapy for malignant HCC, it is top priority to develop new therapeutic strategy for HCC. Gene therapy has been studied for the treatment of HCC. The application the hTERT-siRNA of gene therapy has been limited due to selection of suitable gene transfer vector and transfective efficiency of it. So, approach some high efficiency and hypotoxic vector to mediate gene therapy is another hot spot.
Since the effectiveness of cancer gene therapy is mainly determined by selective vector, the majority of the research in this area has been focused on designing an efficient and safe vector system. At present, viral vector, especially pLenti-GFP (Leniviral vectors) appears to be a promising solution for cancer gene therapy.
In our study, the hTERT-siRNA was used as therapeutic gene, which is linked to the GFP reporter gene in plenti-GFP vector, then was transduced into HepG-2 cells. No-load group and blank group are determined as control. The transduce rate was detected by fluorescent microscope. The results showed that the rate of transduction was more than 90% in plenti-GFP gene transduced group. In addition, the growth of cells in No-load group and blank group is similar, which indicated that the plenti-GFP is a vector with lower toxicity. It was demonstrated that pLenti-GFP can be used as a promising vector in gene therapy.
The hTERT (human telomerase reverse transcriptase) is the telomerase proteinum catalytic subunit, which is expressed in proliferative cells, also is the rate-limiting step of telomerase activation and cell cancerization. To restrain the expression of hTERT could effectively shutdown the growth of HCC cells and induce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the HCC cell. RNAi (RNA interference), for successful target validation in therapeutic approaches in cancer and many other diseases, it has emerged as a useful tool to specifically inhibit the expression of a selected factor and study its function in the disease process. Gene silencing by RNA interference requires the application of double-stranded short inhibitory RNA (siRNA) that is designed to bind a selected mRNA sequence after intracellular complexation with the RNA-induced silencing complex (RISC). Upon binding of the specific sequence the RISC complex cleaves the targeted mRNA which is subsequently degraded and ultimately results in highly efficient and selective protein synthesis inhibition. Only very few siRNA copies per cell are required to mediate efficient gene silencing which has raised this technique as a widely approached attractive therapeutic strategy for various diseases including cancer. Use 21nt siRNA can silence the expression of hTERT efficiently.
In part I, we have constructed the vector pLenti-GFP containing with hTERT, which had a high efficient transduction. Telomerase activity and hTERT were highly expressed in HCC after transduced by this vector. It suggests that hTERT maybe used as a target for RNAi gene therapy. In this part, hTERT-siRNA was transduced into human HepG-2 HCC cells. The telomerase activity and the expression of gene were examined using the same method mentioned above; the cell proliferation and apoptosis were detected by MTT assay and RT-PCR assay. The results were as follows: Proliferation activity of HepG-2 cells transduced with hTERT-siRNA was inhibited significantly and the inhibition rate was time dependent. The inhibitory rate was 57.5% at 7 day after transduction with hTERT-siRNA. Compared with control and no-load group, the telomerase activity and the expression of hTERT mRNA was lowered significantly in HepG2 cells transduced with hTERT-siRNA. There was no change in no-load group and control groups.
These results indicated that with hTERT-siRNA transduction, expression of hTERT mRNA was decreased, telomerase activity was inhibited, and proliferation of tumor cells was suppressed, moreover apoptosis of tumor cells was induced.
PARTⅢ: study on inhibitory effects of hTERT-siRNA to HepG2 cells mediated by pLenti-GFP in vivo
We have constructed the vector pLenti-GFP with a high efficient transduction. In this part, we have performed the study on inhibitory effects of hTERT-siRNA to HepG-2 cells mediated by pLenti-GFP in vivo. HepG-2 cells transduced with hTERT-siRNA and without hTERT-siRNA were implanted into subcutaneous of nude mice respectively. During the course of the study, primary tumor sizes were estimated by each other 3 days with measuring the perpendicular minor dimension (W) and major dimension (L) using sliding calipers. Approximate tumor volume was calculated by the formula (W2x L) x 1/2. The study was ended at day 30; all animals in each group were sacrificed. The primary tumors were excised, collected and fixed in paraformaldehyde and embedded in paraffin blocks for histology analysis. The apoptosis of cells were detected by TUNEL assay. As the results: Proliferation activity of HepG2 cells transduced with hTERT-siRNA was inhibited significantly and the inhibition rate was time dependent. HE staining indicated that hTERT siRNA induced cell necrosis, and inflammatory cell infiltrate to the tumor cells was displayed. TUNEL assay also demonstrated that hTERT siRNA induced cell apoptosis.
In summary, our study demonstrated that the hTERT is a promising molecular target for HCC gene therapy. The combination of the efficient transduction of pLenti-GFP and the effective targeting of siRNA for hTERT will provide a potential therapeutic approach to treating HCC and also extend the application of siRNA to nano-based therapies and to basic research. Meanwhile, there are many questions need to be deeply understood such as regulation of hTERT gene expression and induction of apoptosis in tumor cells.
随着分子生物学的发展,人们逐渐认识到肿瘤发生的机制主要是由于癌基因的激活和抑癌基因的失活而导致的肿瘤细胞增殖失控、凋亡缺陷以及细胞正常的有限生命周期缺陷,使细胞永生化,继而形成肿瘤。目前国内外研究发现细胞的永生化主要由于在某些因素的作用下激活了细胞内部的端粒酶,进而通过端粒酶来增加端粒序列,稳定端粒长度,从而提高细胞增殖能力并使其永生化。因此,端粒酶的激活可以说是细胞永生化的关键步骤。
端粒是指真核细胞线状染色体末端的一段区域,由许多有种属特异性的简单重复的DNA序列及与其特异性结合的蛋白质构成,它给染色体末端提供一个保护性“帽”。在人和其它脊椎动物中重复序列为5’-TTAGGG-3’。1984年Greider和Blackburn在美国柏克莱的Blackburn的实验室首次从四膜虫细胞提取液中发现有端粒酶的活性。1989年Morin首次在人的癌细胞系Hela细胞中发现了端粒酶。端粒酶是一种核糖核蛋白体(ribonucleoprotein、RNP),由RNA与蛋白质构成的复合体,是一种特殊的反转录RNA依赖性DNA聚合酶,能以自身的RNA为模板,通过逆转录而合成端粒,将端粒重复片段加到染色体的末端。因此它解决了DNA半保留复制模式中末端复制问题。Counter等1994年首先在转移性卵巢癌病人中检测到端粒酶活性。近几年来各国学者报道了大量有关肿瘤和端粒酶之间关系的文献,结果发现人正常体细胞中除生殖细胞和大多数发育中胚胎细胞及有增殖能力的更新组织中有较高的端粒酶活性外,大多数端粒酶阴性,通过对5000例以上的肿瘤标本进行端粒酶活性的检测发现,其中约85%为阳性,90%以上的恶性肿瘤中端粒酶阳性,且端粒酶的活性随着肿瘤恶性程度的增高而增高。
人端粒酶包括自身RNA(human telomerase RNA, hTR)、催化亚基(human telomerase reverse transcriptase, hTERT)和相关蛋白1(telomerase associated protein 1,TP1)。hTERT属于逆转录酶(reverse transcriptase,RT)家属成员,是一个有约40kb大小的单拷贝基因,定位于5p15.33。催化亚基基因首先是Lendvay在酵母中发现。1997年编码hTERT的基因被克隆出来。hTERT启动子序列于1999年被克隆出,有1.7Kb长,其中有251bp (-211~+40)是核心部分。近年来的研究发现hTERT mRNA除在胸腺、睾丸、小肠等几种正常组织中有低水平表达外,在大多数正常组织(心、脑、乳腺、肝、骨骼肌、前列腺、胎盘、卵巢)中都没有检测到。但对各种肿瘤组织的检测发现,hTERT mRNA在大多数肿瘤组织中表达,在所有的端粒酶阳性的肿瘤组织中都可检测到hTERT mRNA。在对肝癌中hTERT mRNA的研究中发现: hTERT mRNA在肝癌细胞中存在高表达,hTERT mRNA和hTERT蛋白表达水平与肝癌的恶性程度密切相关,且随着肿瘤的恶性度增加而升高,而正常肝组织中则没有hTERT的表达。目前认为hTERT是端粒酶的催化蛋白组分,同时也是端粒酶活化和细胞癌变的限速步骤,抑制其表达可有效地抑制肿瘤细胞的生长和诱导凋亡,可作为肝癌基因治疗的理想靶标。
当前针对肝癌的基因治疗方案主要包括免疫增强治疗、基因介导的酶前体药物治疗、抑癌基因替代治疗和反义治疗等。这些方法主要是通过将外源性目的基因导入宿主靶细胞内,使之表达以纠正细胞基因缺陷,达到疾病治疗目的。随着分子生物学技术的发展,基因治疗的技术不断得到优化和改进。
近年来研究发现,一些小的双链RNA(double-stranded RNA, dsRNA)可以高效、特异地阻断体内特定基因的表达,促使其mRNA降解,诱使细胞出现特定基因缺失的表型,这种技术即RNA干扰(RNA interference, RNAi),属于转录后基因沉默(post-transcrptional gene silencing, PTGS)。RNAi现象广泛存在于大多数真核细胞中,在小鼠和人的体外培养细胞中利用RNAi技术也可以成功阻断基因的表达,实现细胞水平的基因敲除。
基因治疗成功与否的关键在于采用理想的载体将靶基因高效地转导入靶细胞并获得良好的表达,这就对基因治疗的载体提出了较高的要求。目前常用的载体分为非病毒载体和病毒载体两大类,前者虽具有较高的安全性,但转染效率较差,因此近年来人们更专注于病毒载体的开发和应用。慢病毒载体(Leniviral Vector, pLenti)是一种具有较高转染效率和低细胞毒性的病毒载体。不仅能感染增殖期细胞,还能感染非增殖期细胞及静止期细胞如神经元细胞,无任何毒副作用。
基于以上几点,本试验首先以pLenti-GFP为基础,构建pLenti-hTERT-siRNA,在体外转染人肝癌细胞HepG2细胞,通过RNAi的机制抑制肝癌细胞高表达的hTERT,探讨其对肝癌细胞生长的抑制作用。
由于肝癌的常规治疗方法效果不佳,探求新的治疗策略已成当务之急。肝癌的基因治疗近年来发展迅速,并取得一定进展。但由于基因治疗本身的一些制约条件,比如载体的安全性及转染效率较低等,使得基因治疗的发展受到一定的限制。因此,探讨应用一些高转染效率,低细胞毒性的新型靶向载体来介导基因产物进行治疗已成为肝癌基因治疗研究的新热点。
选择合适的基因转运载体将目的基因安全高效地导入细胞,是基因治疗成功的关键。从目前发展趋势来看,病毒载体在基因转运方面呈现显著优势,其中慢病毒载体以其优越的理化性能和较理想的基因转运能力而日益受到重视。研究证明,pLenti-GFP可以高效介导基因转染多种肿瘤细胞株和原代培养细胞,并且具有缓释、安全无毒等优点。
本研究利用GFP荧光标记的hTERT-siRNA作为治疗基因,选择pLenti-GFP作为基因转运载体,在体外转染人肝癌细胞HepG-2,利用荧光显微镜分析转染效率,并以空载体为对照,对慢病毒载体pLenti-GFP的细胞毒性进行了连续观察和分析。此部分研究旨在探索利用pLenti-GFP慢病毒载体转基因治疗肝癌的可行性,进一步为肝癌基因治疗寻求一种较理想的基因转运手段。
研究结果表明:通过荧光显微镜检测慢病毒载体pLenti-GFP对人肝癌细胞HepG-2的转染效率,得到pLenti-GFP的转染效率为90%以上,完全可用于肝癌基因治疗的体外试验。此外,空载体组细胞生长状态良好,细胞凋亡率与不加任何干预的空白对照组相近,这就证明了pLenti-GFP是一种低细胞毒性的载体,可以用于肝癌基因治疗的研究。
hTERT是端粒酶催化亚基,常常只在增殖的细胞中表达,是端粒酶活化和细胞癌变的限速步骤,抑制其表达可有效地抑制肿瘤细胞的生长和诱导凋亡,已成为目前肿瘤基因治疗的理想靶标。RNAi通过双链RNA产生序列特异性的转录后基因沉默,在哺乳动物细胞内可高效特异地阻断特定基因的表达。它是将导入生物体内的或内源性转录生成的dsRNA被一种RNaseⅢ类的核酸酶Dicer切割成21~25nt的干扰性小RNA,即siRNA,siRNA进一步与其他多种蛋白成分结合形成RNA诱导的沉默复合体(RNA-induced silencing complex, RISC),最后由RISC介导siRNA反义链与靶mRNA分子互补结合并引起同源性靶mRNA分子的切割效应。
我们在第一部分中已经成功制备了具有高转染效率的基因治疗载体pLenti-GFP,在这一部分,我们设计了针对hTERT的siRNA,对人肝癌细胞系HepG-2细胞进行基因干扰治疗的研究,结果分别用MTT法和RT-PCR法分析转染后细胞增殖率、hTERT mRNA的表达,探讨其对肝癌细胞生长的抑制作用及其机制。结果显示:hTERT-siRNA在体外可明显抑制HepG-2细胞系的生长,凋亡增多,随着时间的延长,抑制作用增强,第7天时,其细胞抑制率为57.5%,和对照组差异有统计学意义。hTERT-siRNA转染HepG-2细胞后,hTERT mRNA的表达明显减少,端粒酶活性水平显著下降;空白组和空载组转染后hTERT mRNA及端粒酶活性无明显改变。由上述结果可以看出:hTERT-siRNA能抑制肿瘤细胞hTERT mRNA的表达,使肿瘤细胞的端粒酶活性水平下降,从而抑制肿瘤细胞生长、增殖,促进肿瘤细胞的凋亡。
在这一部分,我们设计了针对hTERT的siRNA,对人肝癌细胞系HepG-2细胞进行基因干扰治疗的体内研究。我们采用在裸鼠皮下分别接种转染和未转染目的基因的HepG2细胞。每3天用游标卡尺测量肿瘤的长径和短径,计算肿瘤体积(V = 1/ 2×长径×短径×短径)。30天后处死裸鼠,取出瘤体,多聚甲醛固定后包埋制作石蜡切片。连续切片,常规HE染色,光镜检查肿瘤组织学,TUNEL法检测细胞凋亡情况。结果显示:治疗组肿瘤生长缓慢,随着时间的延长,差异越来越明显。常规病理HE染色显示坏死增多,周围炎性细胞浸润。TUNEL法检测显示细胞凋亡增多。
综合以上三部分的实验结果,我们认为,hTERT是肝癌基因治疗的理想靶点,RNAi和pLenti-GFP基因转运技术有机结合治疗肝癌是一种切实可行的新策略,可望为日后肝癌的基因治疗开辟一个全新的技术平台,具有广阔的应用前景。但与此同时,许多重要的问题包括hTERT表达调控网络及其表达沉默诱导细胞凋亡机理等仍有待更深入的研究。
Hepatocellular carcinoma (HCC) is the most common malignant disease in Asia. Most HCC have developed to the advanced stage when patients get diagnosed at the first time. The conventional treatments of HCC, including surgery, radiation therapy and chemotherapy, have not yet getten the satisfied efficacy in patients. Therefore, clear-cut its etiopathogenesis and developing effective therapeutic approach would be a urgent project in treatment of HCC.
With the development and application of molecular biology in tumorrigenesis, it has been demonstrated that the development of malignant tumors is due to the activation of protooncogenes and inactivation of tumor suppressor genes, which lead a out of control on cell proliferation and apoptosis, and a deficit of normal life span of cells resulting in cellular immortality and tumorigenesis. Recently, the studies showed that cellular immortality was caused by the activated telomerase. The role of telomerase is to maintain telomeric length and promotes cell proliferation potential. The activated telomerase is believed to be a critical event in cellular immortility.
Telomere is the component of chromosome ends in eukaryotic cells. Just as the hat it consists of species specific tandem repeats of simple sequences and binding proteins Human and other mammals contain 5'-TTAGGG-3' repeats. In 1984, Greider and Blackburn found the activity of telomerase in Blackburn laboratory. In 1989, Morin found the telomerase in human breast cancer cell“Hela”. Telomere length is progressively shortened in normal cells due to "end replicative problem" untill senescence, while it is short and stable in tumor cells. Telomerase is a special reverse transcriptase which contains both essential proteins and RNA component. The RNA component is used as the template for synthesizing the telomeric repeats to lengthen telomere. Telomerase expression has been detected in more then 90% of tumors, but it is absent in most normal somatic tissues. The higher the degree of malignancy of tumors is, the higher the telomerase activity is.
Human telomerase contains three major subunits, RNA (hTR), human telomerase catalytic subunits (hTERT), and telomerase-associated protein (TP1), have been identified recently. hTERT was cloned in 1997, its template contains 11 nucleotides. In 1999, the hTERT gene promoter was cloned, its template contains 1.7bp nucleotides, with its core is -211~+40. Recently researches found that hTERT mRNA had no expressed in most normal tissue (heart, brain, breast, liver, skeletal muscle, prostate and placenta) except thymus, testis and small intestine et al. But some researches also found that hTERT mRNA was expressed in nearly all Hepatocellular carcinoma (HCC) as well as other cancers. And the level of hTERT mRNA was higher in malignant tumors than in normal liver tissues, and correlated positively with the degree of malignancy of tumors. hTERT is similar with elementary eukaryotic telomerase reverse transcriptase in sequence similarity. The hTERT is the catalytic subunit of telomerase. Its expression is usually parallel with telomerase activity. The hTERT is the telomerase proteinum catalytic subunit, which is expressed in proliferative cells, also is the rate-limiting step of telomerase activation and cell cancerization. To restrain the expression of hTERT could effectively shutdown the growth of HCC cells and deduce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the HCC cell.
Currently treatment prescriptions in gene therapy for HCC include immunopotentiation therapy; gene mediated pre-enzyme drug therapy; gene replacement and anti-sense gene therapy. These methods mainly use exogenous gene which was imported into target cells to rectification cell gene deviancy in order to achieve goals of treatment.
Some researches found that a few dsRNA (double-stranded RNA) can specifically inhibit the expression of target gene efficiently and degraded mRNA. It is named as RNAi (RNA interference), belonging to PTGS (post-transcriptional gene silencing). This phenomenon generally emerges in eukaryotic cells and vitro cells, for successful target validation in therapeutic approaches in gene silencing.
The key of gene therapy is to have gene highly express in target cell, which is mediated by ideal carrier efficiently. So there is a high requirement for gene carrier. The current carriers include non viral vector and viral vector. However, the former one has a high safety advantage, with a low efficiently transfect rate. Leniviral Vector (pLenti-GFP) is a high rate of transduction virus vector with low cytotoxicity. The vectors can not only transduce proliferating cells, but also transduce quiescent cell, such as neuron cells, without any cytotoxicity.
In this study, we constructed the gene vector pLenti-GFP containing the hTERT gene and transduced it into HepG-2 cell in vitro. We have studied the inhibitory effects of hTERT-siRNA to HepG-2 cells (one kind of human HCC cells) by using RNAi to inhabit the expression of hTERT.
Because of the unfavorable outcome of conventional therapy for malignant HCC, it is top priority to develop new therapeutic strategy for HCC. Gene therapy has been studied for the treatment of HCC. The application the hTERT-siRNA of gene therapy has been limited due to selection of suitable gene transfer vector and transfective efficiency of it. So, approach some high efficiency and hypotoxic vector to mediate gene therapy is another hot spot.
Since the effectiveness of cancer gene therapy is mainly determined by selective vector, the majority of the research in this area has been focused on designing an efficient and safe vector system. At present, viral vector, especially pLenti-GFP (Leniviral vectors) appears to be a promising solution for cancer gene therapy.
In our study, the hTERT-siRNA was used as therapeutic gene, which is linked to the GFP reporter gene in plenti-GFP vector, then was transduced into HepG-2 cells. No-load group and blank group are determined as control. The transduce rate was detected by fluorescent microscope. The results showed that the rate of transduction was more than 90% in plenti-GFP gene transduced group. In addition, the growth of cells in No-load group and blank group is similar, which indicated that the plenti-GFP is a vector with lower toxicity. It was demonstrated that pLenti-GFP can be used as a promising vector in gene therapy.
The hTERT (human telomerase reverse transcriptase) is the telomerase proteinum catalytic subunit, which is expressed in proliferative cells, also is the rate-limiting step of telomerase activation and cell cancerization. To restrain the expression of hTERT could effectively shutdown the growth of HCC cells and induce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the HCC cell. RNAi (RNA interference), for successful target validation in therapeutic approaches in cancer and many other diseases, it has emerged as a useful tool to specifically inhibit the expression of a selected factor and study its function in the disease process. Gene silencing by RNA interference requires the application of double-stranded short inhibitory RNA (siRNA) that is designed to bind a selected mRNA sequence after intracellular complexation with the RNA-induced silencing complex (RISC). Upon binding of the specific sequence the RISC complex cleaves the targeted mRNA which is subsequently degraded and ultimately results in highly efficient and selective protein synthesis inhibition. Only very few siRNA copies per cell are required to mediate efficient gene silencing which has raised this technique as a widely approached attractive therapeutic strategy for various diseases including cancer. Use 21nt siRNA can silence the expression of hTERT efficiently.
In part I, we have constructed the vector pLenti-GFP containing with hTERT, which had a high efficient transduction. Telomerase activity and hTERT were highly expressed in HCC after transduced by this vector. It suggests that hTERT maybe used as a target for RNAi gene therapy. In this part, hTERT-siRNA was transduced into human HepG-2 HCC cells. The telomerase activity and the expression of gene were examined using the same method mentioned above; the cell proliferation and apoptosis were detected by MTT assay and RT-PCR assay. The results were as follows: Proliferation activity of HepG-2 cells transduced with hTERT-siRNA was inhibited significantly and the inhibition rate was time dependent. The inhibitory rate was 57.5% at 7 day after transduction with hTERT-siRNA. Compared with control and no-load group, the telomerase activity and the expression of hTERT mRNA was lowered significantly in HepG2 cells transduced with hTERT-siRNA. There was no change in no-load group and control groups.
These results indicated that with hTERT-siRNA transduction, expression of hTERT mRNA was decreased, telomerase activity was inhibited, and proliferation of tumor cells was suppressed, moreover apoptosis of tumor cells was induced.
PARTⅢ: study on inhibitory effects of hTERT-siRNA to HepG2 cells mediated by pLenti-GFP in vivo
We have constructed the vector pLenti-GFP with a high efficient transduction. In this part, we have performed the study on inhibitory effects of hTERT-siRNA to HepG-2 cells mediated by pLenti-GFP in vivo. HepG-2 cells transduced with hTERT-siRNA and without hTERT-siRNA were implanted into subcutaneous of nude mice respectively. During the course of the study, primary tumor sizes were estimated by each other 3 days with measuring the perpendicular minor dimension (W) and major dimension (L) using sliding calipers. Approximate tumor volume was calculated by the formula (W2x L) x 1/2. The study was ended at day 30; all animals in each group were sacrificed. The primary tumors were excised, collected and fixed in paraformaldehyde and embedded in paraffin blocks for histology analysis. The apoptosis of cells were detected by TUNEL assay. As the results: Proliferation activity of HepG2 cells transduced with hTERT-siRNA was inhibited significantly and the inhibition rate was time dependent. HE staining indicated that hTERT siRNA induced cell necrosis, and inflammatory cell infiltrate to the tumor cells was displayed. TUNEL assay also demonstrated that hTERT siRNA induced cell apoptosis.
In summary, our study demonstrated that the hTERT is a promising molecular target for HCC gene therapy. The combination of the efficient transduction of pLenti-GFP and the effective targeting of siRNA for hTERT will provide a potential therapeutic approach to treating HCC and also extend the application of siRNA to nano-based therapies and to basic research. Meanwhile, there are many questions need to be deeply understood such as regulation of hTERT gene expression and induction of apoptosis in tumor cells.
引文
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