【作者】 王颖；

【导师】 文继舫； 冯德云；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 浓度诱导细胞发生不可逆的细胞凋亡。因此,这种重组活化的Caspase-3分子在以诱导凋亡为目标的基因治疗策略中有着非常好的应用潜能。如何使导入的Caspase-3基因只在有HCV感染的肝细胞表达和激活,即治疗中的靶向性,是该策略需要解决的关键问题。有研究发现在人肝细胞系中HCV核心蛋白能特异性地与报告基因中的2’-5’寡核苷酸合成酶(OAS)基因启动子结合并激活,即使HCV核心蛋白有少数氨基酸的变异亦不会影响它与OAS启动子的结合和激活效率,而且HCV核心蛋白基因比较保守,如果我们利用OAS启动子来调控Caspase-3基因的表达,可以解决基因治疗的特异性难题。目的:根据上述研究进展,我们拟利用HCV表达的核心蛋白与OAS启动子特异性结合、启动下游基因表达的特性,使重组活化的Caspase-3基因处于OAS启动子调控下,构建HCV特异性Caspase-3治疗系统,并研究其选择性诱导HCV核心蛋白阳性肝细胞凋亡的作用。方法:首先,我们从人基因组DNA中克隆OAS启动子并构建荧光素酶报告载体。然后建立稳定表达HCV核心蛋白的肝细胞系L02/core,并分析OAS启动子在L02/core中以及正常肝细胞L02中的活性。接下来利用基因重组的方法构建OAS启动子调控下的重组型Caspase-3表达载体pGL3-OAS-re-Caspase-3,进一步将其转染入实验细胞中,测定细胞中Caspase-3酶活性。在已确定该表达载体能够特异性地诱导Caspase-3活性表达的基础上,使用荧光显微镜技术和流式细胞技术来探索OAS启动子调控下的重组Caspase-3系统在肝细胞中诱导凋亡的作用。结果:限制性酶切及基因测序证实成功构建了OAS启动子调控下的荧光素酶报告载体pGL3-OAS-Luci。RT-PCR及Western-blot证实成功建立了稳定表达HCV-core的肝细胞系L02/core。在L02/core中,OAS启动子的转录活性明显增高,表明HCV核心蛋白可以特异性激活OAS启动子。限制性酶切及基因测序证实成功构建了OAS启动子调控下的重组型Caspase-3表达载体pGL3-OAS-re-Caspase-3,通过检测Caspase-3特异性底物证实了重组型Caspase-3具有酶活性。进一步使用荧光显微镜技术和流式细胞技术证实了该表达载体具有明显的靶向性诱导HCV核心蛋白阳性肝细胞凋亡的作用。结论:1.成功克隆了OAS启动子,并证实HCV核心蛋白能特异性激活OAS启动子。2.成功构建了重组型Caspase-3基因,并首次构建OAS启动子调控下的真核表达载体pGL3-OAS-re-Caspase-3。3.首次确定了pGL3-OAS-re-Caspase-3系统能特异性地诱导HCV核心蛋白阳性肝细胞的凋亡。4.该实验为发展一种有吸引力的、特异性较强的丙型肝炎基因治疗新策略奠定了一定的基础。更多还原

【Abstract】 Introduction:Hepatitis C virus(HCV) infection is a major cause of chronic hepatitis,liver cirrhosis and hepatocellular carcinoma worldwide. The current standard therapy for chronic hepatitis C consists of a combination of pegylated IFN alpha(pegIFNα) and ribavirin.It achieves a sustained viral clearance in only 50-60%of patients.Moreover,this treatment is associated with substantial side effects,precluding its use by many individuals.Thus,current therapies are inadequate for the majority of patients,and there is an urgent need for the novel hepatitis C therapeutic strategies.It is generally thought that HCV infects only a small fraction of hepatocytes,approximately 1-20%as judged by the detection of HCV proteins or HCV RNA in liver biopsy samples.Therefore,induction of hepatic apoptosis is a potential approach for treatment of chronic hepatitis C.Activation of effector caspases is a central and ultimate step in many apoptosis pathways.Caspase-3 is the key executioner caspase,it exists as an inactive zymogen that is activated by upstream signals.Several groups have considered using the human caspase-3 gene as a novel form of anticancer gene therapy.However,overexpression of the wild-type caspase-3 in mammalian cells does not induce apoptosis,which is due to their inability to undergo autocatalytic processing without upstream caspase for activation.Recently,constitutively active recombinant caspase-3(re-caspase-3) has been was generated by making its small subunit preceding its large subunit.Unlike its wild-type counterpart that is the large subunit preceding the small subunit,the re-caspase-3 is capable of autocatalytic processing and inducing apoptosis independent of the upstream initiator caspase molecules.In addition,it could resist the effect of some apoptosis restraining genes.As caspase-3 is the most downstream executioner of apoptosis,the re-caspase-3 could be used at very low concentrations to induce apoptosis in target cells.However,if caspases-3 is transferred to normal tissues,it would be predicted to also undergo apoptosis,resulting in undesirable damage.To restrict induction of apoptosis to HCV infected cells and increase the safety of this approach,we needed to establish a HCV-specific caspase expression system.The HCV-core protein is currently considered to be a multifunctional protein that plays an important role in persistent infection and hepatocellular carcinogenesis.Naganuma et al found that HCV-core protein specifically activated the interferon(IFN)-inducible 2’-5’oligoadenylate synthetase(OAS) gene promoter in human hepatocyte cells,while the E1,E2,and NS5A proteins did not activate the OAS gene promoter.Moreover,the activation by the core protein is a general phenomenon,regardless of HCV genotype and strain.Therefore, utilization of the OAS gene promoter that is predominantly active in HCV-core positive hepatocytes would be an ideal system to restrict the cytotoxic caspase expression.Objective:Our current investigation attempts to construct an expression vector consisting of the re-caspase-3 under the OAS gene promoter(pGL3-OAS -re-caspase-3) and then investigate its effect on HCV-core positive liver cells.Methods:Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418.Expression of HCV-core was detected by RT-PCR and western blot.The OAS promoter sequences was amplified from the genomic DNA and inserted into pGL3-Basic vector.The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed.Recombinant caspase-3 gene was constructed by making its small subunit preceding its large subunit.An expression vector consisting of the re-caspase-3 under the OAS gene promoter(pGL3-OAS -re-caspase-3) was constructed and transfectd into cells to investigate its effect on HCV-core positive liver cells.Results:L02/core cell line stably expressing HCV-core protein was established.The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.The recombinant caspase-3 gene had enzymatic activity.The pGL3-OAS -re-caspase-3 construct induced apoptosis in HCV-core positive liver cells,but not in normal liver cells.Conclusion:1.We successfully cloned the 2’-5’oligoadenylate synthetase(OAS) gene promoter and demonstrated that it can be activated by HCV-core protein.2.We successfully constructed the expression vector consisting of recombinant caspase-3 gene under the OAS promoter, pGL3-OAS -re-caspase-3.3.For the first time,we demonstrated that pGL3-OAS -re-caspase-3 induced apoptosis in HCV-core positive liver cells significantly and specifically4.The present results strongly suggest that the transfer pGL3-OAS -re-caspase-3 is a novel targeting approach for the treatment of HCV infection.更多还原