胰岛素对烧伤血清诱导血管内皮细胞凋亡的影响及机制

Effect of insulin on the apoptosis of vascular endothelial cells induced by burn serum

目的探讨胰岛素对烧伤血清诱导血管内皮细胞凋亡的影响及相关机制。方法体外常规培养内皮细胞系ECV304，随机分为正常对照组（n=6），体积分数15％（V/V）正常大鼠血清培养；烧伤血清刺激组（n=6），体积分数15％（V/V）自制烧伤大鼠血清培养，血清采自背部30％TBSAⅢ°烫伤大鼠模型；胰岛素处理组（n=6），体积分数15％（V/V）自制烧伤大鼠血清中加入10^-7M／L胰岛素培养；刺激6h后采用原位末端标记法（TUNEL）观察内皮细胞凋亡、免疫组织化学染色和蛋白印迹法检测抗凋亡蛋白bel-2及eNOS的蛋白表达变化，数据以表示，均数比较采用单因素方差分析，以P〈0．05为差异具有统计学意义。结果与正常对照组比较，烧伤血清促进内皮细胞凋亡（18．5％±3．1％），免疫组化染色bcl-2平均吸光度低（0．14±0．02），蛋白印迹实验显示bel-2（0．36±0．12）和p-eNOS（0．55±O．28）表达降低（P〈0．01）。相对于烧伤血清刺激组，胰岛素处理组细胞凋亡显著减少（9．6±2．8％），免疫组化染色bel-2平均吸光度升高（0．21±0．03），蛋白印迹实验显示bcl-2（0．94±0．25）和p-eNOS（O．89±0．16）表达明显恢复（P〈0．01）。eNOS在各组中无明显变化。结论胰岛素明显抑制烧伤血清诱导下的内皮细胞凋亡并增加抗凋亡蛋白bel-2的表达，此作用可能与eNOS的磷酸化有关。

Objective To investigate the effects of insulin on the apoptosis of vascular endothelial cells cuinduced by bum serum in order to explore its possible mechanism. Method Cultured human ECV304 cells were randomly divided into 3 groups： control group,the ECV304 cells hured by 15% （V/V） rat normal serum （ n = 6）; burn serum group,the ECV304 cells simulated by 15% （V/V） self-made bum serum collected from rats with 30% TBSA full-thickness burns on the back （ n = 6） ; and bum serum ＋ insulin group, the ECV304 ceils cuhared by insulin （10-7mol/L） and 15% （V/V） self-made rat burn serum （n = 6）. The transferase mediated nick end labeling （TUNEL） method was employed to measure the apoptosis of endothelial ceils at 6 hours after Meanwhile, immunohistochemical technique and Western blotting were used to determine the protein expressions of bcl-2 and eNOS. Data are expressed as mean ±SEM. Statistical comparison was made using oneway analysis of variance. Significance was accepted at P 〈 0.05. Results Compared with the control group, bum serum induced the apoptosis （ 18.5 ± 3.1% ） and down-regulated bcl-2 （0.36 ± 0.12） and p-eNOS （0.55 ± 0.28） protein expressions of HUVECs （ P 〈 0.01 ）. Bum serum ＋ insulin significantly decreased the apoptosis （9.6 ± 2.8 % ） and up-regulated bcl-2 （0.94± 0.25） and p-eNOS （0.89 ± 0.16） protein expressions of HU- VECs in comparison with the bum serum group （P 〈 0.01）. eNOS showed no significant differences in three groups. Conclusions Insulin could markedly inhibit the apoptosis and up-regulate bcl-2 protein expression of HUVECs induced by burn sertun, and its mechanism might involve the protein expression of phosphorylated eNOS.