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）凋亡的可能机制。方法TCE处理NHEK后，分光光度法测细胞上清中半胱氨酸蛋白水解酶（Caspase）-3、8和9的活性，膜联蛋白V／碘化丙啶（PI）双染后流式细胞仪（FCM）测细胞凋亡，Rhl23／PI双染后FCM测线粒体膜电位（△mm）；用Caspase-3抑制剂或Caspase-9抑制剂预处理NHEK1h，验证Caspase的活化。结果不同浓度TCE处理NHEK4h后培养不同时间，Caspase-3和9活性呈剂量和时间依赖的升高，培养12、24h，各TCE处理组Caspase-3和9活性与溶剂对照比差异均有统计学意义（均P〈0．05），而Caspase-8活性变化不明显。0．125、0．25、0．5、1．0和2．0mmol／LTCE处理NHEK4h后培养12h，膜联蛋白V＋／PI-为（20．1±4．1）％、（30．0±7．5）％、（42．1±8．2）％、（56．0±6．1）％和（79．1±4．3）％，除0．125mmol／L组外，其余组与溶剂对照组（9．4±3．0）％比较，差异均有统计学意义（均P〈0．05）。膜联蛋白V＋／PI-与Caspase-3和9活性都呈正相关（r=0．786、0．736，均P〈0．05），Caspase-3活性与Caspase-9活性也呈正相关（r=0．845，P〈0．05），膜联蛋白V＋／PI-和Caspase-3活性均与Caspase-8活性无相关。100txmol／Lz-DEVD—FMK预处理使2．0mmol／LTCE处理的NHEK中Caspase-3活性从（0．963±0．043）nmo]pNA·min-1·ml-1。下降为（0．349±0．045）nmolpNA·min-1·ml-1膜联蛋白V＋／PI～从（80．0±5．5）％下降为（16．3±3．2）％（P〈0．01），Caspase-9活性变化不大（P〉0．05）。100μmol／L的Z．LEHD-FMK预处理不仅使得2．0mmol／LTCE处理的NHEK中Caspase-3活性下降为（0．338±0．011）nmolpNA·min-1·ml-1，膜联蛋白V＋／PI-下降为（16．1±1．7）％，而且Caspase-9活性也从（0．821±0．031）nmo]pNA-rain～·ml。下降为（0．240±0．043）nmo｜pNA·rain～·ml～，差异有统计学意义（P〈0．01）。2．0mmol／L的TCE处理NHEK4h后，Rhl23荧光强度在培养4、8、12、24h时均明显低于0h（18．7±0．5，均P〈0．01）；0．125、0．5和2．0mmolZL的TCE处理NHEK4h后培养8h，Rhl23荧光强度为16．1±0．5、12．1±0．6和8．1±0．6，均低于溶剂对照组（18．1±0．5，均P〈0．01）。结论TCE诱导NHEK凋亡中，包括线粒体膜电位下降和Caspse-9依赖的Caspase-3的激活在内的内部凋亡途径可能发挥重要作用。
Objective To explore the potential mechanism of trichloroethylene （TCE） -induced apoptosis in normal human epidermis keratinocyte （NHEK） by assaying the Caspase activities, mitochondrial membrane potential （A±m） and apoptosis in vitro. Methods NHEK was exposed to TCE and Caspases-3, 8 and 9 activities were determined using a commercial assay kit. Apoptosis and A±m were detected by flow cytometry （FCM） after double-stained with annexin-V and PI, Rhl23 and PI respectively. NHEK was pretreated with inhibitor of Caspase-3 or 9 to verify the activation of Caspases by TCE treatment. Results Various dose of TCE exposure could increase the Caspases-3 and 9 activities in dose- and time- dependent way. There was marked difference between TCE-treated group and control at 12 or 24 h. But no significant influence of Caspase-8 activity was evoked. 0. 125, 0. 25, 0. 5, 1.0 and 2. 0 mmol/L TCE treated NHEK 4 h then cultured for 12 h. Annexin-V ±/PI- proportion were （20. 1 ± 4. 1 ）%, （30. 0 ± 7.5 ） % , （42. 1 _± 8.2 ） % , （ 56. 0 ± 6. 1 ） % and （ 79. 1 ± 4. 3 ） % respectively. There was marked difference between TCE-treated group except for 0. 125 mmol/L and control （9.4 ± 3.0）% （all P 〈 0. 05 ）. FITC ＋/ PI - proportion were marked positive correlation with Caspase-3 and 9 activities, r = 0. 786, 0. 736（ both P 〈 0. 05 ）. Caspase-3 activities had also a marked positive correlation with Caspase-9 activities, r = 0. 845 （P 〈 0. 05）. There was no correlation with Caspase-8 activities. Pretreatment for 1 h with 100μmol/L Z-DEVD- FMK decreased the Caspase-3 activities from （0. 963 ±0. 043） to（0. 349 ±0. 045） nmol pNA ± rain〈 ± ml -l, annexin-V ±/PI - proportion decreased from （ 80. 0 ± 5.5 ） % to （ 16. 3 ± 3.2 ） % in 2. 0 mmol/L TCE treated NHEK with a significant difference （P 〈0. 01 ）, but there was no change of Caspase-9 activities. 100 p.mol/L Z-LEHD-FMK pretreatment decreased the Caspase-3 activities to （0. 338 ± O. 011 ） nmol pNA ± min-1 ± m1-1, annexin-V ＋/PI- proportion decreased to （ 16. 1 ± 1.7 ）% in 2.0 mmol/L TCE treated NHEK. And the Caspase-9 activities decreased from （0. 821 ± 0. 031 ） to （0. 240 ± 0. 043 ） nmol pNA · min - 1 . ml - 1 with a significant difference（ P 〈 0. 01 ）. NHEK was cultured for 4, 8, 12, 24 h after a 4-hour treatment with 2. 0 mmol/L TCE. Rhod123 fluorescence intensity （FI） were respectively with a marked decrease as compared with 0 h（ 18. 7 ± O. 5, all P 〈 0. O1 ）. At O. 125, 0. 5 and 2.0 mmol/L TCE treated NHEK for 4 h then cultured 8 h, Rh123 FI were 16. 1 ± 0. 5, 12. 1 ± 0. 6 and 8. 1 ± 0. 6 with a marked decrease as compared with control（ 18. 1 ±0.5, allP 〈0. 01 ）. Conclusion TCE-induced NHEK apoptosis is mediated intrinsically through the mitochondrial pathway of the decrease of △ψm and the Caspase-9 dependent activation of Caspase-3.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2009年第44期3101-3105,共5页
National Medical Journal of China
基金
基金项目：国家自然科学基金（30671787、30872147）
安徽省教育厅自然科学重点科研项目（KJ2009A73）
关键词
三氯乙烯
线粒体膜
细胞凋亡
角质形成细胞
Trichloroethylene
Mitochondrial membranes
Apoptosis
Epidermis keratinocytes
