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目的了解醋酸铅(PbAc)诱发肾上腺皮质细胞凋亡的发生与c-Akt/PKB(proteinkinase B,PKB,蛋白激酶B)活性变化的关系。方法体外分离培养豚鼠肾上腺皮质细胞,分别设立空白对照组、12.50、25.00、50.00和100.00 mol/L PbAc剂量组,染毒细胞2 h,以蛋白因子添加素V和碘化丙啶联合标记,流式细胞仪检测细胞凋亡;分别以0、6.25、12.50、25.00、50.00、100.00、200.00 mol/L PbAc与细胞孵育30 min,采用免疫沉淀-化学发光法测定c-AKT/PKB活性。结果给予不同剂量的PbAc(12.50~100.00 mol/L)染毒2 h,其中100.00 mol/L剂量组出现细胞凋亡的百分率(36.53±14.30)明显增加,与对照组(7.62±6.33)相比差异有统计学意义(P<0.01)。肾上腺皮质细胞凋亡的发生率与PbAc剂量之间具有较好的相关关系(r=0.708,P<0.01)。6.25~200.00 mol/L6个剂量PbAc处理30 min,结果表明各剂量组c-Akt/PKB条带密度呈低剂量轻微受抑制、高剂量逐渐升高的趋势,灰度值分别为7 874.67(对照组)、6 395.87、7 194.07、8 457.13、9 463.87、9 398.47和12 110.4,两者之间具有良好的相关关系(r=0.928,P<0.01)。结论高剂量PbAc作用可一定程度地诱发肾上腺皮质细胞凋亡和肾上腺皮质细胞c-Akt/PKB增加。
Objective To investigate the relationship between the occurrence of adrenocortical apoptosis induced by lead acetate (PbAc) and the changes of c-Akt / PKB protein kinase B (PKB) activity. Methods The guinea pig adrenal cortical cells were isolated and cultured in vitro. The blank control group, 12.50, 25.00, 50.00 and 100.00 mol / L PbAc dose groups were established respectively. The cells were infected with protein V and propidium iodide for 2 h. Cellular apoptosis was detected by flow cytometry. The cells were incubated with PbAc at 0, 6, 25, 12.50, 25.00, 50.00, 100.00 and 200.00 mol / L for 30 min. The activity of c-AKT / PKB was measured by immunoprecipitation-chemiluminescence. Results The percentage of apoptotic cells in the 100.00 mol / L dose group (36.53 ± 14.30) was significantly increased after exposure to different doses of PbAc (12.50-100.00 mol / L) for 2 h compared with that of the control group (7.62 ± 6.33) The difference was statistically significant (P <0.01). There was a good correlation between the incidence of adrenocortical apoptosis and PbAc dose (r = 0.708, P <0.01). The results showed that the density of c-Akt / PKB bands in each dose group was slightly inhibited at low doses and gradually increased at high doses, with the gray values of 7 874.67 (control 6 395.87, 7 194.07, 8 457.13, 9 463.87, 9 398.47 and 12 110.4, respectively. There was a good correlation between them (r = 0.928, P <0.01). Conclusion High-dose PbAc can induce apoptosis of adrenocortical cells and increase of c-Akt / PKB in adrenocortical cells.