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  本文選題：293A細(xì)胞　切入點：噬斑試驗　出處：《福建醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:將攜帶HGF目的基因(Ad-HGF)的腺病毒載體轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)并進(jìn)行缺氧培養(yǎng),通過檢測缺氧不同時間下凋亡相關(guān)蛋白的表達(dá),探索Ad-HGF BMSCs最佳缺氧培養(yǎng)時間。方法:(1)采用239A細(xì)胞進(jìn)行體外擴(kuò)增Ad-HGF、Ad-Lac Z腺病毒,噬斑法檢測病毒滴度。(2)分離、培養(yǎng)、病毒感染、鑒定大鼠BMSCs:采用貼壁法分離大鼠BMSCs,常氧及缺氧培養(yǎng)(5%CO2+95%N2)大鼠BMSCs。采用x-gal染色法檢測腺病毒感染率。采用流式細(xì)胞法鑒定細(xì)胞表型。(3)檢測BMSCs表達(dá)HGF:BMSCs感染Ad-HGF、Ad-Lac Z后采用ELISA法檢測上清液中HGF濃度。(4)RT PCR及Western blot法檢測不同缺氧時間下Ad-HGF轉(zhuǎn)染對BMSCs表達(dá)Bax、Bcl-2、Caspase-3的影響。結(jié)果:(1)噬斑法檢測病毒滴度:Ad-Lac Z為2.5×109pfu/ml,Ad-HGF為1.2×109pfu/ml。(2)當(dāng)感染復(fù)數(shù)(MOI)=150,Ad-Lac Z對BMSCs感染率達(dá)高峰95.6%。(3)Ad-HGF感染BMSCs后隨著細(xì)胞培養(yǎng)時間增加,HGF含量顯著增加,第7d時HGF濃度達(dá)高峰。(4)缺氧6 h時HGF基因轉(zhuǎn)染BMSCs表達(dá)Bax/Bcl-2比值及Caspase-3蛋白表達(dá)最小(P0.05)。結(jié)論:缺氧培養(yǎng)HGF基因轉(zhuǎn)染BMSCs發(fā)現(xiàn),缺氧6 h時Bax/Bcl-2比值及caspase-3蛋白表達(dá)最小,該時間點可能是缺氧聯(lián)合HGF基因轉(zhuǎn)染BMSCs移植治療的最佳時間點。
[Abstract]:Objective: to carry the HGF gene (Ad-HGF) adenovirus vector transfection of rat bone marrow mesenchymal stem cells (BMSCs) and hypoxia, hypoxia by detecting the expression of proteins associated with apoptosis at different time, explore the time Ad-HGF BMSCs best hypoxia. Methods: (1) were amplified by Ad-HGF using 239A cells. Ad-Lac Z adenovirus, the titer of virus plaque assay. (2) the separation, culture, identification of virus infection, BMSCs: rats were isolated by adherent BMSCs rats, normoxia and hypoxia (5%CO2+95%N2) rat BMSCs. X-gal assay was used to detect adenovirus infection. Cell phenotype by flow cytometry method. (3) to detect the expression of BMSCs HGF:BMSCs Ad-HGF infection, HGF concentration in the supernatants were determined by ELISA method. Ad-Lac Z (4) RT PCR detection of different hypoxia time and Western blot Ad-HGF Bax on BMSCs Bcl-2 expression, and Caspase-3 effect. Results: (1) The titer of virus plaque assay: Ad-Lac Z 2.5 * 109pfu/ml, 1.2 * Ad-HGF 109pfu/ml. (2) when the multiplicity of infection (MOI) =150, Ad-Lac Z on the infection rate of BMSCs reached the peak at 95.6%. (3) Ad-HGF BMSCs after infection with the cell culture time increased, HGF content increased significantly, the concentration of HGF reached the peak at 7d. (4) 6 h hypoxia HGF gene transfection and expression of BMSCs Bax/Bcl-2 and Caspase-3 protein expression ratio of the minimum (P0.05). Conclusion: hypoxia HGF gene transfection BMSCs found that hypoxia 6 h ratio of Bax/Bcl-2 and caspase-3 protein expression in the minimum time point may be the best time of hypoxia and HGF gene transfection of BMSCs transplantation.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R54

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