热应激对乳鼠心肌细胞损伤作用的研究

INJURY OF HEAT-STRESS TO RAT CARDIOMYOCYTES

目的 :探讨热应激对心肌细胞的损伤作用及其机制。方法 :用胰酶消化法分离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。用生物化学法测定培养液中乳酸脱氢酶 (LDH)活性的变化 ,用流式细胞仪 (FCM )测定心肌细胞凋亡率的变化 ,用台盼蓝 (trypanblue)染色法测定细胞坏死率的变化 ,用荧光分光光度法测定细胞内和培养液中活性氧 (ROS)含量的变化。结果 :39℃、41℃和 43℃热应激后 ,心肌细胞培养液中LDH的活性显著增高 ,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 .8% ,应激后心肌细胞凋亡率随时间变化 ,6h时达最高峰 ,应激后细胞坏死率随时间延长逐渐降低。同时 ,应激后心肌细胞内和培养液中ROS含量明显增加。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主要途径 ,热应激所致细胞ROS升高是导致细胞损伤的重要机制之一。

Aim:To observe the injury and its mechanism of heat-stress to rat cardiomyocytes. Methods:Rat cardiomyocytes were cultured with Typsin digestion method and stressed in water at 39℃、41℃and 43℃ repectively. The variation of lactate dehydrogenase (LDH) in medium was biochemistry method, the apoptosis rate of cardiomyocytes with Flow Cytometry (FCM) mthod,the necrosis rate of cardiomyocytes with trypan blue dying method and the intensity of reactive oxygene species(ROS) in cardiomyocytes and medium with fluorescence spectrophotometer method. Results:After heat stress at 39℃、41℃ and 43℃, the activation of LDH in medium increasd significantly , and the apoptosis rate (%) of cardiomyocytes increased from 4.74(37℃) to 5.12、16.31 and 28.97 respectively. The necrosis rate(%) of cardiomyocytes increased from 3.52 to 3.61 after heat stress at 39℃,from 3.55 to 5.81 at 41℃,from 3.53 to 10.80 at 43℃.The apoptosis rate of cardiomyocytes changed with cultured time after stress and it was highest at the sixth hour after stress.The necrosis rate of cardiomyocytes after heat stress decreased with time. The intensity of ROS in heat stressed cardiomyocytes and medium increased significantly than that in normal cardiomyocytes and medium. Conclution: Cardiomyocytes can be injured by heat stress and apoptosis is the main way of the death of cardiomyocytes caused by heat stress. Increased ROS in cardiomyocytes is one of important mechanisms leading to injury of cardiomyocytes caused by heat-stress.