醋酸铅诱发肾上腺皮质细胞凋亡及与c—Akt／PKB相关性研究

目的了解醋酸铅（PbAc）诱发肾上腺皮质细胞凋亡的发生与c—AkCPKB（protein kinase 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100．00moL／LPbAc剂量组，染毒细胞2h，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、200．00mol／LPbAc与细胞孵育30min，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（12．50～100．00mol/L）染毒2h，其中100．00mol／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）。6．25～200．00moL／L6个剂量PbAe处理30min，结果表明各剂量组c—AkCPKB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7874．67（对照组）、6395．87、7194．07、8457．13、9463．87、9398．47和12110．4，两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。

Apoptosis of adrenocorticol cells induced by lead acetate and its relativity with c -Akt/PKB

Objective To study apoptosis induced by lead acetate （PbAc） and its relativity with c - Akt/PKB in adrenocortical cells. Methods Fascieulata - glomerulosa （FG） cells of male guinea pigs were dispersed and primarily cultured in vitro. Dose - effect study： PbAc was incubated with FG cells at the levels of 0, 12.50, 25.00, 50.00 and 100.00 mol/L respectively for 2 hours, features of apoptotie cells were observed using combined labeling of annexin - V and propidium iodide （PI） and flow cytometry. The FG cells were incubated with 0, 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00 mol/L PbAc for 30 minutes, the activity of c - Akt/PKB was determined with immunoprecipitation and the chemilumines- cence assay. Results It was showed that 12.50 - 100.00 mol/L PbAc could induced apoptosis of FG cells. Compared with the control （7.62 - 6.33 ）, the significant difference was shown in the 100 mol/L group （36.53 ± 14.30） （ P 〈 O. 01 ）. Regression analysis showed that occurrence of apoptosis correlated with the dose of PbAc in a dose -response pattern （r = 0. 708 ,P 〈 0.01 ）. The gray scales of c -Akt/PKB appeared the characteristic of slightly decrease in low doses and gradually increase in high doses. The correspondent c- Akt/PKB values were 7 874.67 （control）, 6 395.87, 7 194.07,8 457. 13, 9 463.87, 9 398.47 and 12 110.4, respectively. PbAc could increase c- Akt/PKB in the FG cells, with significant correlation （r =0.928,P 〈 0.01 ）. Conclusion PbAc with concentrations 12.50 to 100.00 moL/L might induce apoptosis. Since c - Akt/PKB appeared the characteristic of slightly decrease in low doses and gradually increase in high doses, it was suggested that high dosage of lead might possibly elicit the stress response.