三氯乙烯诱导人角质形成细胞凋亡过程中caspase-8及caspase-9活力的变化

目的 观察三氯乙烯(TCE)致人原代角质形成细胞(KC)损伤过程中caspase-8、caspase-9活力及细胞凋亡率的变化,探讨TCE引起KC凋亡的可能机制.方法 体外培养的KC分别给予0.125、0.250、0.500、1.000和2.000mmol/L的TCE染毒4、8、12、24 h,以100μmol/L的caspase-9特异抑制剂Z-LEHD-FMK预处理细胞1 h,再用2.000mmol/LTCE染毒12 h.MTT法检测细胞活力变化,分光光度法检测caspase-8及caspase-9活力变化.流式细胞仪检测细胞凋亡率变化.结果 与空白对照相比,2.000和1.000 mmol/LTCE染毒组作用8 h后细胞活力降低.染毒超过12 h,各剂量组细胞活力均降低,差异有统计学意义(P<0.05或P<0.01).在不同的时间段,各剂量组的caspase-8活力与对照组比较,差异无统计学意义(P>0.05).染毒8 h时,2.000mmol/LTCE组caspase-9活力明显高于对照组,差异有统计学意义(P<0.05);12和24h时,各剂量组caspase-9活力与对照组比较,差异均有统计学意义(P<0.05或P<0.01).不同剂量的TCE染毒12 h后,2.000 mmol/L TCE染毒组细胞凋亡率升高至(80.43±4.21)%,空白对照组为(9.40±2.98)%,除0.125 mmol/LTCE组外,其他各染毒组的细胞凋亡率与对照组相比,差异均有统计学意义(P<0.05),剂量-效应关系明显.Caspase-9抑制剂预处理组caspase-9活力及细胞凋亡率较2.000mmol/L TCE染毒组明显降低,差异有统计学意义(P<0.01).结论 Caspase-9的活化在TCE诱导的体外培养的人KC的凋亡过程中发挥重要作用.

Changes of caspase-8 and caspase-9 activity during apoptosis of keratinocytes induced by trichloroethylene

Objective To observe the change of caspase-8, caspase-9 activity and apoptosis rates in the process of trichloroethylene-induced damage in keratinoeytes,and explore the tentative mechanism of apoptosis. Methods Human keratinocytes were exposed to 0.125,0.250,0.500, 1.000 and 2.000 mmol/L trichloroethylene for 4,8, 12 and 24 h. The inhibitive groups were pretreated with 100 μmol/L Z-LEHI)-FMK (a specific inhibitor of caspase-9) for 1 h,and were stimulated with 2.000 mmol/I TCE for 12 h. MTT assay was used to detect the viability of different cells;The activity of caspase were calculated according to spec-trophotometry;Change of the apoptotie rates was assessed by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide(PI). Results (1)The minimum effective concentration for cell viabil-ity reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a change was 4 h at a con-centration of 2.000 mmol/L (compared with control group,P<0.01). Cell viability in all the groups markedly decreased from 12 h to 24 h (P<0.05). (2)The activity of caspase-8 in the various dosage groups at different times had no statistical difference compared with the control group, P>0.01. (3)At 8 h, 1.000 and 2.000mmol/ L TCE groups could significantly enhance caspase-9 activity(P<0.05). The caspase-9 activity in all the groups showed differences and was significantly higher than those of control cells when time was over 12 h (P<0.05).(4)After exposing to different dosages of TCE for 12 h, the rate of apoptosis rose to (80.43±4.21)% with the increase of dosage ,compared with the control group, (9.40±2.98)% ,which showed a dose-effect relationship.(5)The cells pre-treated with caspase-9 inhibitor resulted in a decrease in the caspase-9 activity and apoptosis rates (compared with 2.000 mmol/L TCE exposed group,P<0.01). However,there was no statistical signifi-cance in comparison with the control group (P>0.05). Conclusion Caspase-9 may be an important mediator of apoptosis in ker-atinocytes induced by trichloroethylene.