以腺相关病毒为载体的肿瘤特异启动子驱动的TRAIL靶向杀伤作用研究

【作者】 张颖；

【导师】 刘彦信； 郑德先；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 基因治疗,尤其是肿瘤的基因治疗目前是人们关注的热点。重组AAV病毒载体由于其独有的优势而在近些年来被认为是最理想的病毒载体之一,本实验室利用重组AAV病毒进行肝癌及肺癌的基因治疗研究也取得了一些比较理想的结果。在基因治疗中,靶向性是非常重要的问题。在我们既往利用的重组AAV病毒载体中,外源基因的表达是通过CAG启动子来驱动的,CAG启动子是一个组成型启动子,在几乎所有细胞中都能驱动外源基因的表达,缺乏良好的选择性,因此,我们希望对CAG启动子进行改造,来更好的实现以重组AAV病毒为载体的靶向基因治疗,以避免外源基因对正常组织器官的伤害。本研究中,我们构建了两种肿瘤特异启动子,即人端粒酶逆转录酶单位（hTERT）启动子和存活因子（survivin）启动子,比较研究了它们在体内、体外驱动外源基因表达的情况及对细胞生命活动的影响。（一）含有肿瘤特异启动子的重组AAV载体的构建及其活性研究我们构建了hTERT启动子或survivin启动子与AAV2的重组质粒载体。通过报告基因EGFP的表达比较了两种肿瘤特异启动子在不同肝细胞癌（HCC）细胞株及人原代正常肝细胞（PHH）中的活性,结果表明hTERT启动子与survivin启动子均能驱动EGFP基因在HCC细胞中的表达,hTERT启动子驱动的EGFP表达在SMMC-7721、BEL-7402、HepG2和Hep3B肝癌细胞中的平均荧光强度分别为44.4%、32.7%、35.2%和12.9%,survivin启动子驱动的EGFP表达在四株细胞中的平均荧光强度为25.4%、21.8%、13.5%和11.7%,虽然这两种启动子之间的活性有所差别,但它们在PHH中均没有活性,平均荧光强度在5%以下。此外,两启动子驱动的TRAIL基因还可引起不同程度的HCC细胞存活率降低,而对PHH并无影响。进一步,我们又对hTERT启动子在包括肺癌、结肠癌、乳腺癌、宫颈癌、肝癌、白血病等不同种类的肿瘤细胞株及正常细胞中的活性进行检测。结果表明,hTERT启动子在多种肿瘤细胞中均有活性,而在正常细胞中则没有,说明它具有良好的肿瘤特异选择性。（二）含有肿瘤特异启动子的重组病毒的制备及其功能研究我们还通过包装纯化重组AAV病毒颗粒,进一步研究了hTERT启动子驱动的AAV.TRAIL重组病毒单独作用或与化疗药物5-Fu联合作用的情况。经对重组病毒侵染后的肿瘤细胞及正常细胞DNA、mRNA及蛋白分析的结果表明,hTERT启动子可驱动TRAIL基因在肿瘤细胞中特异表达。抗肿瘤活性分析表明,重组病毒侵染72小时后,肝细胞癌SMMC-7721细胞、肺癌A549细胞、宫颈癌Hela细胞和正常人肝细胞PHH的凋亡率分别为:SMMC-7721 57.4%、A549 22.9%、Hela 33.3%、PHH 4.9%。应用裸鼠移植瘤模型进行的体内研究结果表明,hTERT启动子驱动的TRAIL基因表达明显抑制SMMC-7721人肝细胞癌在小鼠体内的生长,5×10¹¹)Gps重组病毒可使肿瘤增长抑制率达77%,而且,hTERT启动子驱动的TRAIL基因表达也能明显抑制A549人肺腺癌细胞在小鼠体内的生长。应用化疗药物5-Fu与重组病毒联合给药结果表明,40mg/kg的5-Fu能增强重组病毒抗肿瘤作用,肿瘤增长的抑制率达到90%。免疫组织化学分析显示,5-Fu能增强肿瘤组织中TRAIL受体DR5的表达。此外,我们还观察到hTERT启动子驱动的TRAIL表达具有给药途径的选择性,即局部瘤内注射有效而系统给药（尾静脉注射和肌肉注射）则无效。我们还对hTERT驱动的AAV.TRAIL进行了安全性检测。病理分析及转氨酶测定表明,无论是瘤内注射还是系统给药,重组病毒对动物正常组织器官尤其是肝脏没有影响,说明在hTERT启动子驱动下,重组病毒是安全的。综上,我们的研究表明肿瘤特异启动子能特异性驱动外源基因在肿瘤细胞中的表达及效应。在肿瘤特异启动子驱动下,重组AAV病毒能使外源基因TRAIL安全有效的发挥作用,这为实现以重组AAV病毒为载体的肿瘤靶向性基因治疗提供了新的思路。更多还原

【Abstract】 Gene therapy represents an attractive approach to treating cancers.For a good cancer gene therapy delivery system,an ideal vector is absolutely necessary for effective and safe therapeutic.Among quite a few viral vectors which have been tested in recent years,Adeno-associated virus(AAV) is considered to be the most promising vector due to its nonpathogenic property,wide tropisms,non immunogenecity to host and long-term transgene expression.Experimental and clinical trials using recombinant AAV(rAAV) as the delivery vehicles have been proved to be an effective way in gene therapy.However, in gene therapy,especially in treating tumors,the most concerned problem is how to limit the cytotoxic exogenous gene expression in the tumors without harm to normal cells.Since the wide host range infectivity of rAAV may become a disadvantage for its applications in cancer gene therapy,due to its nonselective tissue transduction,safety issue of rAAV as the vector of carrying exogenous genes,which are often toxic to both cancer and normal cells in treating tumors,must be considered.Using tumor-specific promoters has been proved to be a potential way to control the exogenous genes in limited sites.Human telomerase reverse transcriptase(hTERT) is considered to be the most crucial unit in telomerase activity,which is present in a majority of human cancers.The hTERT promoter has been proved to be able to controlling the expression and effect of exogenous genes in tumors without implication of normal tissues.Survivin is the member of the inhibitors of apoptosis(IAP),which is expressed in various human cancers but not in normal adult human tissues.As its expression is activated transcriptionally,the promoter of survivin was used as a cancer-specific promoter in cancer gene therapy.In recent years,TNF-related apoptosis-inducing ligand(TRAIL),as an inducer of tumor cell apoptosis,has been proven to be a promising agent for cancer treatment. However,the potential utility and safety of TRAIL has been questioned because its potential toxicity on normal human cells(e.g.hepatocytes) and the mechanism of the related toxicity still hangs in doubt.From a point of clinical view,safety is the most critical problem in developing a drug.Thus,how to control the effect of TRAIL specifically on tumor cells appears to be imperative.Several studies have proved the tumor-specific efficiency of TRAIL mediated by hTERT promoter using adenovirus or plasmids.However,the capability of rAAV-mediated TRAIL expression driven by hTERT promoter to lead tumor-specific killing effect is unknown.Therefore,in the present study,by using rAAV2 as the vector,we tested whether hTERT promoter and survivin promoter are promising candidates in rAAV-mediated tumor-specific therapy.First all,we constructed the EGFP report gene and soluble TRAIL gene controlled by hTERT promoter(about 284bp) or survivin promoter(about 980bp) in rAAV2 vector, repectively.The specificity of EGFP or TRAIL expression driven by the two promoters was tested in human hepatocellular carcinoma(HCC) cells and primary human hepatocyte(PHH).It showed that hTERT and survivin promoter were both efficient in driving tumor-specific expression of EGFP gene in HCC cells and the activity of hTERT promoter seemed higher compared with that of survivin,but both the two promoters had no acitivity in PHH.The apoptosis of HCC induced by TRAIL expression under the control of tumor-specific promoters was proved that hTERT promoter was more efficient in driving the exogenous gene expression than that of survivin promoter.The second,we tested the activity of hTERT promoter in immortal cell line HEK293 and various tumor cell lines,including human carcinoma of lung,breast,colon, cervix,and lymphoma etc.and human kidney proximal tubular cells(HKC).The result demonstrated that hTERT promoter was active in all the tumor cells although they were not same.Moreover,the AAV.hTERT.TRAIL leaded to apoptosis of various tumor cells, including A549,Hela,SMMC-7721,but not in PHH.Thirdly,in vivo study was carried out and proved that hTERT promoter mediated the surppression of tumor formation and growth by apoptosis without toxic to normal tissues,suggesting it is a promising construct for cancer gene therapy.Fatherly,we studied the effect of combination of AAV.hTERT.TRAIL and chemotherapeutic drugs 5-Fu in vivo.The result showed that the combination of the AAV.hTERT.TRAIL and 5-Fu suppressed the tumor growth more efficiently than either one agent,indicating a novel strategy of AAV-mediated gene threpay combined with chemotherapeutic drugs for cancer treatment.更多还原