【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(gè)公眾問題。目前治療PCa的方法包括外科手術(shù)、放射治療和內(nèi)分泌治療等。但是這些方法對(duì)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(shí)驗(yàn)構(gòu)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(guān)病毒（adeno-associated virus, AAV），AAV感染PCa細(xì)胞后持續(xù)地表達(dá)、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(gòu)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(guān)病毒rAAV-TK、rAAV-Tum5、rAAV-TK-IRES-Tum5、rAAV-Tum5-IRES-TK的體外功能實(shí)驗(yàn)。方法： 1.通過基因重組技術(shù)將Tum5和HSV-TK基因克隆入pIRES-MCS的不同位點(diǎn)，構(gòu)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(xì)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(xì)胞（PC3）和人臍靜脈內(nèi)皮細(xì)胞（HUVEC），通過熒光顯微鏡、實(shí)時(shí)定量PCR、Western blot檢測(cè)病毒感染效率，，通過倒置顯微鏡觀察細(xì)胞生長狀況，通過流式細(xì)胞儀和MTT法觀察細(xì)胞的凋亡情況和細(xì)胞周期變化。 結(jié)果： 1.成功構(gòu)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(guān)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(xì)胞，并能夠表達(dá)目的蛋白，Tum5蛋白能夠抑制HUVEC的成管能力，促進(jìn)HUVEC凋亡，轉(zhuǎn)染TK基因的PC3細(xì)胞凋亡細(xì)胞比例要遠(yuǎn)大于對(duì)照組。 結(jié)論： 所構(gòu)建的AAV-TK-IRES-Tum5重組腺相關(guān)病毒可以感染PC3及HUVEC細(xì)胞并能夠表達(dá)目的蛋白。Tum5蛋白抑制了HUVEC的成管能力并促進(jìn)凋亡，TK基因促進(jìn)了PC3細(xì)胞的凋亡，為進(jìn)一步的體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。
[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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 趙麗琴;席斌;彭華松;;腺相關(guān)病毒(AAV)載體研究進(jìn)展[J];生物技術(shù)進(jìn)展;2012年02期

2 周利群;;前列腺癌的病因、診斷與治療進(jìn)展[J];繼續(xù)醫(yī)學(xué)教育;2006年08期

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