摘要
目的探讨有机溶剂三氯乙烯(TCE)诱导正常人表皮角质形成细胞(NHEK)凋亡过程中可能的信号通路。方法用0.125、0.25、0.5、1.0、2.0mmol/L的TCE处理NHEK4h,并培养4、8、12、24h,分光光度法测上清中Caspase-9和Caspase-3活性,Annexin-V/PI双染后流式细胞仪(FCM)测细胞凋亡;Caspase-3抑制剂Z-DEVD-FMK和Caspase-9抑制剂Z-LEHD-FMK预处理组,用100μmol/LZ-DEVD-FMK或Z-LEHD-FMK预处理NHEK1h,再用2.0mmol/LTCE染毒4h后培养至12h。结果不同浓度TCE处理NHEK4h后培养不同时间,可诱导Caspase-3和Caspase-9活性升高,培养12、24h时,各TCE处理组Caspase-3和Caspase-9活性与溶剂对照比差异有统计学意义(P<0.05)。不同浓度TCE处理NHEK4h后培养12h,凋亡细胞(Annexin-V+/PI-)所占百分比随TCE剂量增加而升高,除0.125mmol/LTCE组外,其它各处理组Annexin-V+/PI-细胞所占百分比与对照组比差异均有统计学意义(P<0.05)。相关分析显示Annexin-V+/PI-细胞所占百分比与Caspase-3和Caspase-9活性都呈正相关(P<0.05)。100μmol/LZ-DEVD-FMK预处理明显抑制2.0mmol/LTCE诱导的Caspase-3活性上升和Annexin-V+/PI-细胞所占百分比的增加(P<0.05),对Caspase-9活性影响不明显(P>0.05)。100μmol/LZ-LEHD-FMK预处理不仅抑制2.0mmol/LTCE诱导的Caspase-3活性上升和Annexin-V+/PI-细胞所占百分比的增加,还抑制Caspase-9活性上升(P<0.01)。结论TCE诱导NHEK凋亡中,内部凋亡途径中上游关键酶Caspase-9和下游效应分子Caspase-3均激活,且Caspase-3的激活依赖Casapase-9的活化。
Objective To explore the potential mechanism of triehloroethylene (TCE)-induced apoptosis in normal human epidermis keratinoeytes (NHEK)by assayed Caspase-3 and Caspase-9 activities as well as apoptosis in vitro. Methods NHEK were exposed to 0. 125,0. 25,0.5,1.0,2.0 mmol/L TCE for 4 h, then cultured for 4,8,12 or 24 h,Caspase-3 and Caspase-9 activities in NHEK were determined using a commercial assay kit, and apoptosis was detected by flow eytometry ( FCM ) after double-stained with Annexin-V and PI. For inhibition group, NHEK were pretreated with 100 μmol/L Z-DEVD-FMK or Z-LEHD-FMK for 1 h and exposure to 2. 0 mmol/L TCE for 4 h then cultured for 12 h. Results Various dose of rICE exposure could cause Caspase-3 and Caspase-9 activities with NHEK increased,there were significant difference between TCE treated group and vehicle control at 12 or 24 h. Annexin-V^+/PI^- proportion were increased with the elevation of TCE concentration, there were significant difference between TCE treated group except for 0. 125 mmoL/L and vehicle control. Correlation analysis indicated FITC +/PI- proportion were significant positive correlation with Caspase-3 and Caspase-9 activities. In additional, Caspase-3 activities were also significant positive correlation with Caspase-9 activities. 100μmol/L Z-DEVD-FMK pretreated NHEK for 1 h, could significantly supress the elevation of Caspase-3 activities and Annexin-V + PI- proportion could induce by 2.0 mmol/L TCE, but not elevation of Caspase-9 activities. 100 μmol/L Z-LEHD-FMK pretreated NHEK for 1 h, could significantly supress not only the elevation of Caspase-3 activities and Annexin-V + PI proportion induced by 2.0 mmol/L TCE,but also the elevation of Caspase-9 activities. Conclusion These results suggest that Caspase-9,a key initiating molecular in intrinsic apotosis pathway, and Caspase-3 can activate in TCE-induced NHEK apoptosis,and activation of Caspase-3 is dependent upon activation of Caspase-9.
出处
《安徽医科大学学报》
CAS
北大核心
2009年第6期699-703,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金项目(编号:30671787
30872147)
安徽省教育厅自然科学重点科研项目(编号:KJ2009A73)