热应激诱导奶牛乳腺上皮细胞凋亡及其对HSP70基因表达的影响

Effects of Heat Stress on Apoptosis and Expression of HSP70 mRNA in Bovine Mammary Epithelial Cells

为探讨热应激对乳腺上皮细胞凋亡的影响,以体外培养的奶牛乳腺上皮细胞为模型,取对数生长期的乳腺上皮细胞分别置于39℃和41℃热处理1 h(以37℃正常培养的细胞为对照),在37℃恢复培养0、6、12 h后收集细胞,采用MTT法检测细胞生长抑制率,Annexin V/PI双染法检测细胞凋亡,比色法分析Caspase-3活性,荧光定量PCR检测HSP70基因的表达水平。结果表明,39℃热应激组细胞生长抑制率和死亡率高峰期均发生在热修复6 h,细胞死亡率为15.42%,而41℃热应激组发生在热修复12 h,细胞死亡率高达23.18%;高温处理后,Caspase-3活性呈上升趋势;39℃热应激组在热恢复6 h,HSP70基因表达量显著高于对照组,41℃热应激组在热恢复0、6、12 h,HSP70基因的表达量分别为对照组的3.53倍、5.61倍和2.93倍。综上可知,高温应激激活了Caspase-3活性,从而诱导乳腺上皮细胞凋亡,并随高温强度的增加而作用增强,同时高温诱导了HSP70基因过表达,协助细胞获得热耐受性。

To explore the effects of heat stress on the apoptosis of the bovine mammary epithelial cells and the expression of HSP70 mRNA, the logarithmic growth phase mammary epithelial cells were exposed to 39℃ and 41℃ for 1 h（the control group was cultured at 37℃） ,and cells were collected in the recovery times at 0,6,12 h. Cell growth inhibition rate was detected by MTT method,cell apoptosis was measured by Annexin V/PI double staining method, Caspase-3 activity was detected by colorimetric assay and the expression level of HSP70 was tested by RQ-PCR. The results showed that the highest growth inhibition rate and maximum death rate of mammary epithelial cells occurred in the recovery 6 h against 39℃ and the cell mortality rate was 15. 42% , while they occurred in the recovery 12 h against 41℃ and the largest cell mortality rate was 23. 18% . After high temperature treatment, the activity of Caspase-3 increased. The expression level of HSP70 mRNA in the recovery 6 h against 39℃ was significantly higher than that in control. And it was 3. 53 times,5. 61 times and 2. 93 times significantly higher than that of control in the recovery 0,6, 12 h respectively against 41℃ . This suggested that heat stress activated the activity of Caspase-3 in the mammary epithelial cells and induced cell apoptosis with the increase of temperature. At the same time,the cells over-expressed HSP70 by heat stress,and could acquire tolerance.