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目的 探讨bcl 2基因转染对热应激心肌细胞的保护作用。方法 用胰酶消化法分离培养Wistar大鼠乳鼠心肌细胞 ,使其暴露于 39℃、41℃和 43℃水浴进行热应激。用脂质体转染法将bcl 2基因转染入心肌细胞 ,用免疫组织化学法检测转染结果。用生物化学法测定bcl 2基因转染对热应激前、后心肌细胞培养液中乳酸脱氢酶 (LDH)活力的影响 ;用流式细胞仪 (FCM)测定bcl 2基因转染对热应激前、后心肌细胞凋亡率的影响 ;用台盼蓝染色法测定bcl 2基因转染对热应激前、后心肌细胞坏死率的影响。结果 经 39℃、41℃和 43℃热应激后 ,心肌细胞培养液中LDH活力明显增高 ,心肌细胞的凋亡率由 4.74%分别增加到 5 .12 %、16 .31%和 2 8.97% ,坏死率分别由 3.5 2 %、3.5 5 %、3.5 3%增加到 3.6 1%、5 .81%和 10 .80 %。bcl 2基因转染后 ,41℃和 43℃热应激引起的心肌细胞凋亡率明显降低 ,43℃热应激引起的心肌细胞培养液中LDH活力和心肌细胞坏死率也明显降低。结论 热应激可以引起心肌细胞损伤 ,细胞凋亡是热应激心肌细胞死亡的重要途径 ,bcl 2基因转染对热应激心肌细胞有保护作用。
Objective To investigate the protective effect of bcl 2 gene transfection on heat-stressed cardiomyocytes. Methods Wistar rat neonatal rat cardiomyocytes were isolated and cultured by trypsin digestion and exposed to 39 ℃, 41 ℃ and 43 ℃ water bath for heat stress. The bcl 2 gene was transfected into cardiomyocytes by lipofection and the transfection results were detected by immunohistochemistry. The effect of bcl 2 gene transfection on the activity of lactate dehydrogenase (LDH) in the culture medium of myocardial cells before and after heat stress was determined by biochemical method. The effect of bcl 2 gene transfection on the activity of LDH was determined by flow cytometry (FCM) The effects of bcl 2 gene transfection on cardiomyocyte apoptosis rate before and after heat stress were determined by trypan blue staining. Results LDH activity in cardiomyocyte culture medium was significantly increased after heat stress at 39 ℃, 41 ℃ and 43 ℃. The apoptosis rate of cardiomyocytes increased from 4.74% to 5.12%, 16.31% and 2.897% respectively. , The necrosis rates increased from 3.5 2%, 3.5 5% and 3.5 3% to 3.6 1%, 5.81% and 10 .80% respectively. After transfection with bcl 2 gene, the apoptotic rate of myocardial cells induced by heat stress at 41 ℃ and 43 ℃ decreased significantly, and the LDH activity and cardiomyocyte necrosis rate induced by heat stress at 43 ℃ were also significantly decreased. Conclusion Heat stress can cause injury of cardiomyocytes. Apoptosis is an important pathway of heat-induced cardiomyocyte death. Transfection of bcl 2 gene has protective effect on heat-stressed cardiomyocytes.