论文部分内容阅读
目的研究热休克蛋白27磷酸化在高糖诱导的人脐静脉内皮细胞凋亡中的作用。方法取健康剖腹产孕妇的脐静脉内皮细胞(HUVEC)进行培养,选择2～3代细胞进行实验。①检测高糖对内皮细胞中热休克蛋白27(HSP-27)蛋白活性的影响:实验分正常细胞组与高糖组。正常细胞组不做处理直接提取细胞总蛋白,高糖组为30.5mmol/L高糖分别干预细胞24、48、72h后提取总蛋白。②检测槲皮素对高糖诱导的内皮细胞中HSP-27活性的影响:实验分为正常细胞组,高糖组和高糖+槲皮素组。正常细胞组不做处理,高糖组为30.5mmol/L高糖干预细胞;高糖+槲皮素组为槲皮素(10μmol/L)先与细胞孵育1h后再加入刺激物高糖(30.5mmol/L),各细胞组培养48h后提取总蛋白。①与②中细胞总蛋白均采用特异性Phospho-HSP27抗体的蛋白免疫印迹法检测HSP-27的活性;采用Annexin-Ⅴ-PI染色流式细胞技术检测人脐静脉内皮细胞凋亡。分组及干预情况同②,比较各组间细胞的凋亡率。结果高糖呈时间依赖性诱导人脐静脉内皮细胞中HSP-27磷酸化,30.5mmol/L高糖作用24、48、72h,HSP-27磷酸化水平较正常对照组分别提高了100%(P<0.05),182.5%(P<0.01),117%(P<0.05)。而HSP-27选择性阻断剂槲皮素(10μmol/L)作用48h可以抑制高糖诱导的HSP-27磷酸化,与高糖组相比槲皮素组HSP-27磷酸化水平降低了52.6%(P<0.01),与此同时,可使凋亡增加,内皮细胞凋亡率较高糖组增高了37.6%(P<0.01)。结论 HSP-27磷酸化可能在高糖诱导的人脐静脉内皮细胞凋亡中起保护作用。
Objective To study the role of heat shock protein 27 phosphorylation in apoptosis induced by high glucose in human umbilical vein endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) from healthy caesarean section were cultured and selected for 2-3 passages. (1) To detect the effect of high glucose on the activity of heat shock protein 27 (HSP-27) in endothelial cells: The experiment was divided into normal cell group and high glucose group. The total cell protein was extracted directly from the normal cell group without any treatment. The total protein was extracted after 24 h, 48 h and 72 h, respectively. ② detection of quercetin on high glucose-induced endothelial cell HSP-27 activity: The experiment is divided into normal cell group, high glucose group and high glucose + quercetin group. The cells in normal group were treated with high glucose (30.5mmol / L), and the cells in high glucose + quercetin group were treated with quercetin (10μmol / L) mmol / L). The total protein was extracted from each cell group after 48 hours. The total cellular proteins in ① and ② were detected by Western blotting with specific Phospho-HSP27 antibody. The apoptosis of human umbilical vein endothelial cells was detected by Annexin-Ⅴ-PI staining. Grouping and intervention with the situation ②, comparing the apoptosis rate of cells between groups. Results High glucose induced HSP-27 phosphorylation in human umbilical vein endothelial cells in a time-dependent manner. The phosphorylation of HSP-27 at 30.5mmol / L and 24,48,72h increased by 100% compared with the control group <0.05), 182.5% (P <0.01), 117% (P <0.05). Quercetin (10 μmol / L), a selective blocker of HSP-27, inhibited the phosphorylation of HSP-27 induced by high glucose, and the phosphorylation level of HSP-27 in quercetin group decreased by 52.6 % (P <0.01). At the same time, apoptosis increased and the rate of apoptosis in endothelial cells increased by 37.6% (P <0.01). Conclusion HSP-27 phosphorylation may play a protective role in high glucose-induced apoptosis of human umbilical vein endothelial cells.