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目的应用氯化钴(CoCl2)诱导乳鼠心肌细胞缺氧凋亡,探讨缺氧条件下大鼠心肌细胞survivin mRNA和蛋白的改变以及与心肌细胞凋亡的相关性。方法(1)不同浓度(0,10,100,200,500,1000μmol/L)CoCl2干预心肌细胞;(2)流式细胞仪(Annexin VFITC/PI)分析细胞凋亡率(AR)和死亡率(DR);(3)免疫细胞化学、Rt-PCR和Western blot方法明确survivin的表达。结果(1)与常氧(CoCl20μmol/L)比较,除10μmol/LCoCl2(P=0.069)外,其余不同浓度CoCl2干预均可显著增加心肌细胞AR(P<0.01)和DR(P<0.01);(2)与常氧比较,不同浓度CoCl2干预均可显著增加心肌细胞survivin mRNA(0.127±0.004、0.186±0.011、0.154±0.008、0.146±0.003、0.141±0.005和0.137±0.004,P<0.01)和蛋白(0.353±0.012、1.588±0.021、1.016±0.019、0.899±0.042、0.823±0.018和0.549±0.011,P<0.01)的表达,均以10μmol/LCoCl2时表达最高,且随CoCl2干预浓度增加而呈下降趋势(P<0.05);(3)心肌细胞AR和survivin mRNA(r=-0.617,P=0.014)及蛋白(r=-0.549,P=0.009)显著负相关。结论缺氧可致心肌细胞发生凋亡并表达survivin mRNA和蛋白,在其凋亡进程中,survivin基因可能减轻心肌细胞的凋亡。
Objective To investigate the effects of cobalt chloride (CoCl 2) on hypoxia and apoptosis in neonatal rat cardiomyocytes, and to explore the relationship between the changes of survivin mRNA and protein in cardiomyocytes and the apoptosis of cardiomyocytes under hypoxia. Methods (1) CoCl2 (0, 10, 100, 200, 500, 1000μmol / L) was used to interfere the cardiomyocytes. (2) The apoptosis rate (AR) and mortality (DR) were analyzed by flow cytometry (Annexin VFITC / PI) ) Immunocytochemistry, Rt-PCR and Western blot confirm the expression of survivin. Results Compared with normal saline (CoCl20μmol / L), the addition of CoCl2 at 10μmol / L CoCl2 (P = 0.069) significantly increased AR (P <0.01) and DR (P <0.01) (2) Compared with normoxia, the intervention of CoCl2 at different concentrations could significantly increase the mRNA expression of survivin (0.127 ± 0.004,0.186 ± 0.011,0.154 ± 0.008,0.146 ± 0.003,0.141 ± 0.005 and 0.137 ± 0.004, P <0.01) and The expressions of proteins (0.353 ± 0.012, 1.588 ± 0.021, 1.016 ± 0.019, 0.899 ± 0.042, 0.823 ± 0.018 and 0.549 ± 0.011, P <0.01) were all highest expressed at 10μmol / L CoCl2 and increased with the concentration of CoCl2 (P <0.05). (3) There was a significant negative correlation between AR and survivin mRNA (r = -0.617, P = 0.014) and protein (r = -0.549, P = 0.009) in cardiomyocytes. Conclusion Hypoxia can induce apoptosis of cardiomyocytes and express survivin mRNA and protein. Survivin gene may reduce the apoptosis of cardiomyocytes in the process of apoptosis.