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目的检测三氯乙烯(TCE)对人表皮角质形成细胞(NHEK)的凋亡诱导作用,探讨TCE皮肤毒性的作用靶。方法采用中性红吸附试验测定TCE对体外无血清培养的NHEK的中性红吸附减少50%的浓度(NR50值),确定TCE染毒剂量;测定丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力反映细胞脂质过氧化作用和氧化状态;用透射电子显微镜(TEM)和流式细胞仪(FCM)观察细胞凋亡形态学改变和测定细胞DNA含量,计算凋亡发生率及增殖指数(PI)。结果TCE对NHEK的NR50值为4.53(3.92～5.13)mmol/L;TCE处理NHEK4h后,MDA含量的增加和SOD活力的抑制均具有剂量-效应关系(r=0.98,r=0.93,P<0.01);TEM观察显示,与对照相比,TCE处理组细胞可见明显凋亡改变;FCM测定显示,DNA含量直方图中G1期前可见明显的凋亡峰,与对照组相比,TCE处理组细胞凋亡率明显增高(空白对照及TCE0.125、0.500、2.000mmol/L组分别为18.42%、31.83%、38.63%、44.35%),而PI则明显降低(空白对照及TCE0.125、0.500、2.000mmol/L组分别为4.99%、3.26%、2.48%、2.07%)。结论在体外培养条件下,TCE可通过脂质过氧化和氧化应激作用诱导NHEK凋亡,抑制其增殖。
Objective To investigate the apoptosis-inducing effect of trichlorethylene (TCE) on human epidermal keratinocytes (NHEK) and to explore the target of TCE on skin toxicity. Methods The concentration of TCE was determined by Neutral Red Adsorption Test (RCE). The concentration of TCE on NRCE decreased by 50% in NHEK without serum and the dose of TCE was determined. The contents of malondialdehyde (MDA) and superoxide dismutase The activity of enzyme (SOD) reflected lipid peroxidation and oxidative status of cells. The morphological changes of apoptotic cells and the content of DNA were observed by transmission electron microscopy (TEM) and flow cytometry (FCM) Proliferation index (PI). Results The NR50 of TCE against NHEK was 4.53 (3.92 ~ 5.13) mmol / L. After treatment with NHEK for 4 hours, the increase of MDA content and the inhibition of SOD activity were dose-effect relationship (r = 0.98, r = 0.93, P <0.01) ); TEM observation showed that compared with the control, TCE treated group of cells showed significant changes in apoptosis; FCM assay showed DNA content in the histogram obvious G1 peak before apoptosis peak compared with the control group, TCE treated cells (P <0.05). The apoptotic rate was significantly higher in the control group (18.42%, 31.83%, 38.63%, 44.35% in the blank control and TCE 0.125, 2.000 mmol / L group were 4.99%, 3.26%, 2.48%, 2.07%, respectively). Conclusion TCE can induce NHEK apoptosis and inhibit its proliferation through lipid peroxidation and oxidative stress under in vitro culture conditions.