缺氧-复氧诱导人肥大心肌细胞凋亡信号的变化

Hypoxia-reoxygenation induced apoptosis and its signal regulation in human hypertrophic cardiomyocytes

目的:研究缺氧-复氧诱导人肥大心肌细胞凋亡信号的变化。方法:通过胶原酶/胰酶联合消化法获取成人肥大心肌细胞,采用缺氧-复氧诱导细胞凋亡,改良TUNEL法检测细胞凋亡率,并用免疫细胞化学法检测bcl-2、bax、caspase3、细胞色素C和磷酸酪氨酸蛋白的变化,用原位杂交法检测bcl-2 mRNA、caspase3 mRNA和Akt1 mRNA表达的变化。结果:缺氧24 h-复氧4 h后细胞凋亡率为(17.50±0.67)%,对照组为(2.88±0.27)%;凋亡信号的平均吸光度(A)值:bcl-2为0.189±0.006,对照组为0.238±0.004;bax为0.240±0.002,对照组为0.218±0.004;caspase-3为0.230±0.002,对照组为0.202±0.004;细胞色素C为0.225±0.003,对照组为0.191±0.009;磷酸酪氨酸蛋白为0.186±0.005,对照组为0.154±0.003;bcl-2 mRNA为0.300±0.003,对照组为0.360±0.001;caspase 3 mRNA为0.307±0.004,对照组为0.271±0.002;Akt1 mRNA为0.274±0.007,对照组为0.301±0.008,差异均有统计学意义(P<0.01)。结论:缺氧-复氧可造成人肥大心肌细胞凋亡,bcl-2蛋白及Bcl-2 mRNA和Akt1 mRNA表达降低,bax、caspase3、细胞色素C和磷酸酪氨酸蛋白及caspase3 mRNA表达增加。

Objective ： To investigate the changes of apoptosis and its signal regulation in human hypertrophic cardiomyocytes induced by hypoxia and reoxygenation. Methods： Adult human hypertrophic cardiomyocytes were cultured and digested in trypsin and collagenase. The apoptotic cells were induced by hypoxia-reoxygenation and their rate was evaluated by a modified TUNEL method. The changes in the Bcl-2, Bax, Caspase 3, cytochrome C and Phosphotyrosine proteins were detected by immunocytochemistry （ICC）, those in Bcl-2 mRNA, Caspase 3 mRNA, and Aktl mRNA by ISH Detection Kit, and the resuits were statistically analyzed by SPSS 10.0. Results ： The apoptosis rate of cardiomyocytes induced by 24 h hypoxia-4 h reoxygenation （H/R） was （17.50±0.67）% vs （2.88±0.27）% in the control group; the average A values of the apoptotic signals were （0. 189 ±0. 006） in Bcl-2, （0. 240± 0. 002） in Bax, （0. 230 ±0. 002） in Caspase 3, （0.225 ±0. 003） in Cytochrome C, and （0. 186 ± 0.005） in Phosphotyrosine proteins, as compared with （0. 238 ± 0. 004）, （0. 218± 0. 004）, （0. 202 ±0. 004）, （0. 191 ±0.009） and （0. 154 ±0. 003） respectively in the control group; and the expression of bcl-2 mRNA was （0. 300 ±0. 003 ）, Caspase 3 mRNA （0. 307 ± 0. 004） and Aktl mRNA （0.274 ±0.007） in comparison with （0. 360 ±0. 001）, （0. 271±0. 002） and （0. 301 ±0. 008） in the controls, all with statistically significant differences between the two groups （ P 〈 0. 01 ）. Conclitsion： Hypoxiaeoxygenation can significantly increase the apoptosis rate of hypertrophic cardiomyocytes, down-regulate the expressions of the Bcl-2 protein, Bcl-2 mRNA and Aktl mRNA, and up-regulate the expressions of Bax, Caspase 3, Cytochrome C and Phosphotyrosine proteins and Caspase 3 mRNA in cardiomyocytes.