非融合雙靶點重組TK-IRES-Tum5腺相關病毒構建及體外實驗研究
發(fā)布時間：2018-11-19 20:41
【摘要】：研究背景： 前列腺癌（prostate cancer, PCa）持續(xù)增長的發(fā)病率和持續(xù)增加的死亡率是目前非常重要的一個公眾問題。目前治療PCa的方法包括外科手術、放射治療和內(nèi)分泌治療等。但是這些方法對激素非依賴PCa治療效果不夠理想�；蛑委熓悄壳坝锌赡苤斡[瘤的一種方法。本實驗構建腫瘤血管內(nèi)皮抑素功能片段（Tum5）和自殺基因（TK）非融合重組腺病毒相關病毒（adeno-associated virus, AAV），AAV感染PCa細胞后持續(xù)地表達、分泌Tum5蛋白，阻止腫瘤內(nèi)部新生血管形成，雙靶位治療PCa。 目的： 1.構建pAAV-TK、pAAV-Tum5、pAAV-TK-IRES-Tum5、pAAV-Tum5-IRES-TK質(zhì)粒。 2.rAAV-TK、rAAV-Tum5、rAAV-TK-IRES-Tum5、rAAV-Tum5-IRES-TK病毒顆粒的包裝、濃縮、純化與鑒定。 3.基因重組腺相關病毒rAAV-TK、rAAV-Tum5、rAAV-TK-IRES-Tum5、rAAV-Tum5-IRES-TK的體外功能實驗。方法： 1.通過基因重組技術將Tum5和HSV-TK基因克隆入pIRES-MCS的不同位點，構建pAAV-TK、pAAV-Tum5、pAAV-TK-IRES-Tum5、pAAV-Tum5-IRES-TK質(zhì)粒，通過酶切鑒定載體質(zhì)粒正確。 2.采用HEK293細胞包裝病毒AAV Help free系統(tǒng)轉(zhuǎn)染；采用氯仿-PEG/NaCl-氯仿抽提分離、濃縮和純化病毒。 3.培養(yǎng)人前列腺癌細胞（PC3）和人臍靜脈內(nèi)皮細胞（HUVEC），通過熒光顯微鏡、實時定量PCR、Western blot檢測病毒感染效率，，通過倒置顯微鏡觀察細胞生長狀況，通過流式細胞儀和MTT法觀察細胞的凋亡情況和細胞周期變化。 結果： 1.成功構建pAAV-TK、pAAV-Tum5、pAAV-TK-IRES-Tum5、pAAV-Tum5-IRES-TK質(zhì)粒。成功得到濃縮的重組腺相關病毒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均能夠順利感染PC3及HUVEC細胞，并能夠表達目的蛋白，Tum5蛋白能夠抑制HUVEC的成管能力，促進HUVEC凋亡，轉(zhuǎn)染TK基因的PC3細胞凋亡細胞比例要遠大于對照組。 結論： 所構建的AAV-TK-IRES-Tum5重組腺相關病毒可以感染PC3及HUVEC細胞并能夠表達目的蛋白。Tum5蛋白抑制了HUVEC的成管能力并促進凋亡，TK基因促進了PC3細胞的凋亡，為進一步的體內(nèi)實驗奠定基礎。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25
【參考文獻】
相關期刊論文 前2條
1 趙麗琴;席斌;彭華松;;腺相關病毒(AAV)載體研究進展[J];生物技術進展;2012年02期
2 周利群;;前列腺癌的病因、診斷與治療進展[J];繼續(xù)醫(yī)學教育;2006年08期
本文編號：2343371
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