Zhu Qi-xing, Ye Liang-ping, Wang Li-jie, Shen Tong
Department of Occupational and Environmental Health, School of Public Health, Anhui Medical University, Hefei, 230032, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2009 Apr;27(4):193-7.
To observe the change of caspase-8, caspase-9 activity and apoptosis rates in the process of trichloroethylene-induced damage in keratinocytes, and explore the tentative mechanism of apoptosis.
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 micromol/L Z-LEHD-FMK (a specific inhibitor of caspase-9) for 1 h, and were stimulated with 2.000 mmol/l TCE for 12 h. MTT assay was used to detect the viability of different cells; The activity of caspase were calculated according to spectrophotometry; Change of the apoptotic rates was assessed by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide (PI).
(1) The minimum effective concentration for cell viability reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a change was 4 h at a concentration 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 2.000 mmol/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 significance in comparison with the control group (P > 0.05).
Caspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.
观察三氯乙烯诱导角质形成细胞损伤过程中半胱天冬酶-8（caspase-8）、半胱天冬酶-9（caspase-9）活性及凋亡率的变化，探讨凋亡的初步机制。
将人角质形成细胞分别暴露于0.125、0.250、0.500、1.000和2.000 mmol/L三氯乙烯中4、8、12和24小时。抑制组先用100 μmol/L Z-LEHD-FMK（caspase-9的特异性抑制剂）预处理1小时，再用2.000 mmol/L三氯乙烯刺激12小时。采用MTT法检测不同细胞的活力；根据分光光度法计算半胱天冬酶的活性；用膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）和碘化丙啶（PI）双染后，通过流式细胞仪（FCM）评估凋亡率的变化。
（1）细胞活力降低的最小有效浓度在12小时时为0.125 mmol/L，产生变化所需的最短时间在浓度为2.000 mmol/L时为4小时（与对照组相比，P < 0.01）。从12小时到24小时，所有组的细胞活力均显著下降（P < 0.05）。（2）不同剂量组在不同时间的caspase-8活性与对照组相比无统计学差异，P > 0.01。（3）在8小时时，1.000和2.
