【摘要】 目的探讨表达hTERT蛋白的重组腺病毒rAd-hTERT感染体外原代培养的肝细胞后对其增殖情况的影响。方法将表达hTERT蛋白的重组腺病毒rAd-hTERT感染体外原代培养的肝细胞。同时设空载体对照组及空白对照组。通过定量PCR及Western-blot检测基因在细胞中的表达情况;MTT法观察其对细胞增殖的影响;采用流式细胞术检测rAd-hTERT对细胞周期和凋亡的影响。结果(1)以50MOI rAd—hTERT感染肝细胞可明显促进其生长增殖,但不会引起细胞毒性作用。(2)定量PCR在转录水平检测rAd-hTERT感染后的细胞中hTERT表达量明显增加。(3)Western-blot在蛋白水平验证rAd-hTERT感染后的细胞中hTERT蛋白的表达及其蛋白活性。(4)流式细胞DNA含量分析显示,rAdhTERT可引起肝细胞周期S+G2／M期增加,并抑制细胞的凋亡。结论rAd-hTERT在体外能有效促进原代肝细胞的生长。与阴性对照比较,同期生长的肝细胞S+G2／M期细胞增加,凋亡细胞减少(P<0．05),并未引起细胞的不典型增生表现。更多还原

【Abstract】 Objective To determine whether the induction of exogenous hTERT gene can facilitate the growth of primarily cultured liver cells. Methods The primary culture of murine liver tissue and monolayer hepatocytes was performed in a simple way of non-perfusion under the ice bath treating the liver tissues with a modified low-serum medium. The cultured cells were proved to be hepatocytes by observing morphology and structure under the light microscope and electron microscope as well as by detecting albumin level in the supernatant of the cultures. The primarily cultured liver cells were infected with rAd-hTERT. MTT assay was used to determine the infection rate and growth rate. RT-PCR and Western blot were employed to observe the expression of hTERT. Flow cytometry was used to analyze cell cycle progress and apoptosis. Results The albumin was detected in the supernatant of primary culture. Meanwhile, the rose-like hepatic granule and a great deal of cytoplasm were found under the electron microscope. RT-PCR and Western blot showed that hTERT protein was expressed in the primarily cultured cells. MTT assay revealed that rAd-hTERT could significantly facilitate the growth of primarily cultured liver cells. Flow cytometry showed that rAd-hTERT could enhance the cells of S+ G2 /M phase and prevent apoptosis. Conclusions rAd-hTERT can significantly facilitate the growth of primarily cultured liver cells.更多还原