非融合双靶点重组TK-IRES-Tum5腺相关病毒构建及体外实验研究
发布时间：2018-11-19 20:41
【摘要】：研究背景： 前列腺癌（prostate cancer, PCa）持续增长的发病率和持续增加的死亡率是目前非常重要的一个公众问题。目前治疗PCa的方法包括外科手术、放射治疗和内分泌治疗等。但是这些方法对激素非依赖PCa治疗效果不够理想。基因治疗是目前有可能治愈肿瘤的一种方法。本实验构建肿瘤血管内皮抑素功能片段（Tum5）和自杀基因（TK）非融合重组腺病毒相关病毒（adeno-associated virus, AAV），AAV感染PCa细胞后持续地表达、分泌Tum5蛋白，阻止肿瘤内部新生血管形成，双靶位治疗PCa。 目的： 1.构建pAAV-TK、pAAV-Tum5、pAAV-TK-IRES-Tum5、pAAV-Tum5-IRES-TK质粒。 2.rAAV-TK、rAAV-Tum5、rAAV-TK-IRES-Tum5、rAAV-Tum5-IRES-TK病毒颗粒的包装、浓缩、纯化与鉴定。 3.基因重组腺相关病毒rAAV-TK、rAAV-Tum5、rAAV-TK-IRES-Tum5、rAAV-Tum5-IRES-TK的体外功能实验。方法： 1.通过基因重组技术将Tum5和HSV-TK基因克隆入pIRES-MCS的不同位点，构建pAAV-TK、pAAV-Tum5、pAAV-TK-IRES-Tum5、pAAV-Tum5-IRES-TK质粒，通过酶切鉴定载体质粒正确。 2.采用HEK293细胞包装病毒AAV Help free系统转染；采用氯仿-PEG/NaCl-氯仿抽提分离、浓缩和纯化病毒。 3.培养人前列腺癌细胞（PC3）和人脐静脉内皮细胞（HUVEC），通过荧光显微镜、实时定量PCR、Western blot检测病毒感染效率，，通过倒置显微镜观察细胞生长状况，通过流式细胞仪和MTT法观察细胞的凋亡情况和细胞周期变化。 结果： 1.成功构建pAAV-TK、pAAV-Tum5、pAAV-TK-IRES-Tum5、pAAV-Tum5-IRES-TK质粒。成功得到浓缩的重组腺相关病毒rAAV-Tum5、 rAAV-TK、rAAV-Tum5-IRES-TK、rAAV-TK-IRES-Tum5。 2.rAAV-Tum5、rAAV-TK、rAAV-Tum5-IRES-TK、rAAV-TK-IRES-Tum5均能够顺利感染PC3及HUVEC细胞，并能够表达目的蛋白，Tum5蛋白能够抑制HUVEC的成管能力，促进HUVEC凋亡，转染TK基因的PC3细胞凋亡细胞比例要远大于对照组。 结论： 所构建的AAV-TK-IRES-Tum5重组腺相关病毒可以感染PC3及HUVEC细胞并能够表达目的蛋白。Tum5蛋白抑制了HUVEC的成管能力并促进凋亡，TK基因促进了PC3细胞的凋亡，为进一步的体内实验奠定基础。
[Abstract]:Background: the increasing incidence and mortality of prostate cancer (prostate cancer, PCa) is a very important public issue at present. Current treatments for PCa include surgery, radiotherapy and endocrine therapy. But these methods are not ideal for hormone-independent PCa treatment. Gene therapy is currently a possible cure for cancer. In this study, we constructed tumor vascular endostatin functional fragment (Tum5) and suicide gene (TK) non-fusion recombinant adenovirus-associated virus (adeno-associated virus, AAV), AAV) after infection with PCa cells to continuously express and secrete Tum5 protein. Prevention of Neovascularization in tumor and treatment of PCa. with double targets Objective: 1. PAAV-TK,pAAV-Tum5,pAAV-TK-IRES-Tum5,pAAV-Tum5-IRES-TK plasmid was constructed. 2. Packaging, concentration, purification and identification of rAAV-TK-IRES-Tum5 rAAV-Tum5-IRES-TK virus particles. 3. Functional experiment of recombinant adeno-associated virus rAAV-TK,rAAV-Tum5,rAAV-TK-IRES-Tum5,rAAV-Tum5-IRES-TK in vitro. Methods: 1. Tum5 and HSV-TK genes were cloned into different sites of pIRES-MCS by gene recombination technique, and pAAV-TK,pAAV-Tum5,pAAV-TK-IRES-Tum5,pAAV-Tum5-IRES-TK plasmid was constructed. The plasmid was confirmed by restriction endonuclease digestion. 2. HEK293 cell package virus AAV Help free system was used to transfect the virus, chloroform-PEG/NaCl- was used to extract and purify the virus. 3. Cultured human prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC),) were used to detect the viral infection efficiency by fluorescence microscope and real-time quantitative PCR,Western blot, and the growth status of the cells was observed by inverted microscope. Apoptosis and cell cycle were observed by flow cytometry and MTT. Results: 1. PAAV-TK,pAAV-Tum5,pAAV-TK-IRES-Tum5,pAAV-Tum5-IRES-TK plasmid was successfully constructed. Successful production of concentrated recombinant adeno-associated virus rAAV-Tum5, rAAV-TK,rAAV-Tum5-IRES-TK,rAAV-TK-IRES-Tum5. 2. RAAV-Tum5-IRES-Tum5 can successfully infect PC3 and HUVEC cells and express target protein. Tum5 protein can inhibit the ability of HUVEC tube formation and promote HUVEC apoptosis. The percentage of apoptotic cells in PC3 cells transfected with TK gene was much higher than that in control group. Conclusion: the constructed AAV-TK-IRES-Tum5 recombinant adeno-associated virus can infect PC3 and HUVEC cells and express the target protein. Tum5 protein inhibits the tubular ability of HUVEC and promotes apoptosis. TK gene can promote the apoptosis of PC3 cells. It lays the foundation for further in vivo experiment.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2343371
[Abstract]:Background: the increasing incidence and mortality of prostate cancer (prostate cancer, PCa) is a very important public issue at present. Current treatments for PCa include surgery, radiotherapy and endocrine therapy. But these methods are not ideal for hormone-independent PCa treatment. Gene therapy is currently a possible cure for cancer. In this study, we constructed tumor vascular endostatin functional fragment (Tum5) and suicide gene (TK) non-fusion recombinant adenovirus-associated virus (adeno-associated virus, AAV), AAV) after infection with PCa cells to continuously express and secrete Tum5 protein. Prevention of Neovascularization in tumor and treatment of PCa. with double targets Objective: 1. PAAV-TK,pAAV-Tum5,pAAV-TK-IRES-Tum5,pAAV-Tum5-IRES-TK plasmid was constructed. 2. Packaging, concentration, purification and identification of rAAV-TK-IRES-Tum5 rAAV-Tum5-IRES-TK virus particles. 3. Functional experiment of recombinant adeno-associated virus rAAV-TK,rAAV-Tum5,rAAV-TK-IRES-Tum5,rAAV-Tum5-IRES-TK in vitro. Methods: 1. Tum5 and HSV-TK genes were cloned into different sites of pIRES-MCS by gene recombination technique, and pAAV-TK,pAAV-Tum5,pAAV-TK-IRES-Tum5,pAAV-Tum5-IRES-TK plasmid was constructed. The plasmid was confirmed by restriction endonuclease digestion. 2. HEK293 cell package virus AAV Help free system was used to transfect the virus, chloroform-PEG/NaCl- was used to extract and purify the virus. 3. Cultured human prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC),) were used to detect the viral infection efficiency by fluorescence microscope and real-time quantitative PCR,Western blot, and the growth status of the cells was observed by inverted microscope. Apoptosis and cell cycle were observed by flow cytometry and MTT. Results: 1. PAAV-TK,pAAV-Tum5,pAAV-TK-IRES-Tum5,pAAV-Tum5-IRES-TK plasmid was successfully constructed. Successful production of concentrated recombinant adeno-associated virus rAAV-Tum5, rAAV-TK,rAAV-Tum5-IRES-TK,rAAV-TK-IRES-Tum5. 2. RAAV-Tum5-IRES-Tum5 can successfully infect PC3 and HUVEC cells and express target protein. Tum5 protein can inhibit the ability of HUVEC tube formation and promote HUVEC apoptosis. The percentage of apoptotic cells in PC3 cells transfected with TK gene was much higher than that in control group. Conclusion: the constructed AAV-TK-IRES-Tum5 recombinant adeno-associated virus can infect PC3 and HUVEC cells and express the target protein. Tum5 protein inhibits the tubular ability of HUVEC and promotes apoptosis. TK gene can promote the apoptosis of PC3 cells. It lays the foundation for further in vivo experiment.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
【参考文献】
相关期刊论文 前2条
1 赵丽琴;席斌;彭华松;;腺相关病毒(AAV)载体研究进展[J];生物技术进展;2012年02期
2 周利群;;前列腺癌的病因、诊断与治疗进展[J];继续医学教育;2006年08期
本文编号：2343371
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