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目的:利用体外培养心肌细胞缺氧模型,观察不同缺氧时间心肌细胞凋亡的发生及与Fas表达的关系。方法:原代培养乳鼠心肌细胞,随机分为对照组、缺氧1h、2h、3h、4h组,构建心肌细胞缺氧模型,应用膜联蛋白-V与溴化丙啶双染法与Hoechst33258染色法检测缺氧心肌细胞凋亡,应用免疫组化法检测Fas在心肌细胞上表达,应用West-ernblot检测Fas在心肌细胞表达。结果:膜联蛋白-V与溴化丙啶双染法检测发现对照组、缺氧1h、2h、3h和4h组细胞凋亡率分别为(0.20±0.15)%,(4.32±0.56)%,(6.32±1.22)%,(8.32±1.19)%,(5.01±1.56)%(P<0.05);Hoechst33258染色显示,与对照组比较,缺氧1h、2h、3h和4h组细胞的凋亡率明显升高;免疫组化与对照组比较,缺氧1h、2h、3h、4h组阳性细胞数明显升高(P<0.05),缺氧3h组最高,缺氧4h组略下降;Westernblot检测结果提示对照组与4h组Fas表达减弱,缺氧1h、2h和3h组Fas表达上调。结论:缺氧可诱导心肌细胞的凋亡,缺氧时间延长引起凋亡率逐渐上升,至4h时凋亡率开始下降,Fas表达量的变化趋势与凋亡率的变化趋势一致,Fas可能介导了缺氧诱导的心肌细胞凋亡。
OBJECTIVE: To study the relationship between the occurrence of cardiomyocyte apoptosis and the expression of Fas in different time of hypoxia by culturing cardiomyocyte hypoxia in vitro. Methods: Primary cultured neonatal rat cardiomyocytes were randomly divided into control group, hypoxia 1h, 2h, 3h, 4h group, the model of cardiomyocyte hypoxia was established. Using Annexin-V and propofol double staining method, Hoechst33258 The apoptosis of hypoxic cardiomyocytes was detected by staining. The expression of Fas in cardiomyocytes was detected by immunohistochemistry. The expression of Fas in cardiomyocytes was detected by West-ernblot. Results: The apoptosis rates of control group were (0.20 ± 0.15)% and (4.32 ± 0.56)% respectively at 1h, 2h, 3h and 4h after hypoxia, (6.32 ± 1.22)%, (8.32 ± 1.19)% and (5.01 ± 1.56)%, respectively (P <0.05). Hoechst33258 staining showed that compared with the control group, the apoptotic rates of HUVECs at 1h, 2h, 3h and 4h (P <0.05). Compared with the control group, the number of positive cells in hypoxia group was significantly increased at 1h, 2h, 3h, 4h (P <0.05) Suggesting that the expression of Fas decreased in the control group and the 4h group, and was up-regulated in the 1h, 2h and 3h hypoxia groups. CONCLUSION: Hypoxia can induce cardiomyocyte apoptosis. The apoptosis rate gradually increases after prolonged hypoxia, and the apoptosis rate begins to decline at 4h. The change trend of Fas expression is consistent with that of apoptosis rate. Fas may mediate Hypoxia-induced cardiomyocyte apoptosis.