慢病毒介导的可诱导Caspase9细胞凋亡模型的建立

发布时间：2018-04-22 01:34

本文选题：二聚化 + 自杀基因　；参考：《新乡医学院》2015年硕士论文

【摘要】：背景：基因修饰T细胞免疫治疗越来越显示出其优越性,在癌症患者的治疗上起到了积极的作用。然而,这种“超自然”T淋巴细胞的调控是临床基因和细胞治疗需要解决的关键问题,利用自杀基因系统调控基因修饰的细胞是基因治疗的安全措施之一。研究显示,iCasp9自杀基因系统能够高效快速的诱导细胞凋亡,并且iCasp9几乎全部是人源性的,所以转导细胞引起免疫反应的风险大大降低,因此,该系统受到了人们的广泛关注。目的：建立可诱导的Caspase9自杀基因系统诱导Jurkat T细胞,利用阳性化合物诱导细胞凋亡,获得T细胞条件消融的相关数据,为基因修饰T细胞的免疫治疗提供安全保障。方法：1、利用分子克隆技术将目的基因(FKBP12/Caspase9)克隆入慢病毒载体pCDH-CMV-MCS-EFl-puro中,构建piCasp9表达质粒,三质粒(piCasp9、pVSVG、 pDe18.9)磷酸钙法共转染293T细胞制备慢病毒(LV-KLC9),并进行滴度测定。2、用病毒液感染Jurkat T细胞,经puromycin筛选扩增。Western blot检测目的基因在细胞中的表达；利用Annexin V染色法对AP20187诱导二聚体介导的细胞凋亡情况进行分析。结果：1、成功构建piCasp9表达质粒,三质粒共转染293T细胞制备的慢病毒LV-KLC9具有高效性,72h后95%的293T细胞可以观察到绿色荧光。采用逐孔稀释滴度测定法测定病毒滴度,病毒滴度可达3.0×107 IU/ml。2、病毒感染Jurkat T细胞的效率达65%；Western blot检测发现约在49kD处出现明显的特异条带,而对照组无此条带,说明慢病毒将融合基因导入Jurkat细胞,并在细胞中成功表达。给予10 nM AP20187对细胞进行凋亡诱导,并于不同时间点检测细胞凋亡,流式细胞术结果显示：细胞凋亡率随着时间的延长逐渐增加,表现为时间依赖性的细胞死亡。结论：成功建立了iCasp9自杀基因系统可诱导细胞凋亡生物效应的细胞模型,体外给予AP20187后,携带目的基因的Jurkat细胞呈现时问依赖性的凋亡,这有望为基因修饰T细胞的免疫治疗提供安全保障。
[Abstract]:Background: gene-modified T-cell immunotherapy (GMT) has shown its advantages and played an active role in the treatment of cancer patients. However, the regulation of supernatural T lymphocytes is a key problem in clinical gene and cell therapy. It is one of the safe measures for gene therapy to regulate the genetically modified cells by using suicide gene system. Studies have shown that iCasp9 suicide gene system can induce apoptosis efficiently and rapidly, and iCasp9 is almost all human origin, so the risk of immune response caused by transduction cells is greatly reduced. Therefore, the system has attracted wide attention. Aim: to establish an inducible Caspase9 suicide gene system to induce Jurkat T cells, to induce apoptosis by using positive compounds, and to obtain the relevant data of T cell conditioned ablation, so as to provide a safe guarantee for the immunotherapy of genetically modified T cells. Methods the target gene (FKBP12 / Caspase9) was cloned into lentivirus vector pCDH-CMV-MCS-EFl-puro by molecular cloning technique, and the piCasp9 expression plasmid was constructed. Three plasmids (piCasp9 pVSVG, pDe18.9) calcium phosphate were co-transfected into 293T cells to prepare lentivirus LV-KLC9. The expression of the target gene in the cells was detected by puromycin screening and amplification, and the apoptosis induced by AP20187 was analyzed by Annexin V staining. Results the piCasp9 expression plasmid was constructed successfully. The lentivirus LV-KLC9 prepared by co-transfection of the three plasmids into 293T cells had high efficiency. After 72 hours, 95% of the 293T cells could be observed green fluorescence. The virus titer was determined by one-hole dilution titer assay. The virus titer reached 3.0 脳 10 ~ 7 IU / ml 路2. The efficiency of virus infection in Jurkat T cells was 65%. Western blot detection showed that there were obvious specific bands in 49kD, but there was no specific band in the control group. The results showed that lentivirus could transfer the fusion gene into Jurkat cells and express it successfully. Apoptosis was induced by 10 nm AP20187, and apoptosis was detected at different time points. The results of flow cytometry showed that the rate of apoptosis increased with time, and showed time dependent cell death. Conclusion: the iCasp9 suicide gene system was successfully established to induce the biological effect of apoptosis. After AP20187 was given in vitro, the Jurkat cells carrying the target gene showed time-dependent apoptosis. This is expected to provide a safe guarantee for the immunotherapy of genetically modified T cells.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.51

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