SET缺陷对三氯乙烯诱导L-02细胞增殖和凋亡及组蛋白去乙酰化酶的影响

Effect of SET deficiency on the trichloroethylene-induced alteration of cell proliferation and cell apoptosis and DNA methylation in human hepatic L-02 cells

目的比较三氯乙烯（trichloroethylene，TCE）诱导人正常肝细胞（L-02细胞）和SET基因缺陷L02细胞（SET缺陷细胞）中细胞增殖、凋亡、组蛋白去乙酰化酶活力及表达水平的变化，探讨TCE染毒对组蛋白修饰的影响及SET在表观遗传调控中的作用。方法选用前期建立的SET缺陷细胞为研究对象，以未经TCE处理的L-02细胞和SET缺陷细胞作为各自的对照组，用2．00和8．00mmol／LTCE染毒两种细胞24h，用CCK．8法和Hoechst 33342染色法检测细胞的增殖水平和凋亡率。用0．25、0．50、1．00、2．00、4．00、8．00mmol／LTCE染毒两种细胞，检测细胞的组蛋白去乙酰化酶活力和蛋白去乙酰化酶的蛋白表达水平。结果TCE8．00mm01]L染毒组SET缺陷细胞与L-02肝细胞比较，增殖水平呈下降趋势，凋亡率呈上升趋势，且差异均有统计学意义（t值分别为-4．362和23．950，P〈0．05）。TCE处理L-02肝细胞和SET缺陷细胞后均引起细胞增殖水平的下降和凋亡率的升高，当TCE浓度达到8．00mmol／L时，两组细胞间的增殖水平和凋亡率的差异有统计学意义（t值分别为-4．362，和23．950，P值均小于0．05）。使用0，0．25，0．50，1．00，2．00，4．00和8．00mmol／L TCE处理L-02细胞和SET缺陷细胞24h后，两种细胞的组蛋白去乙酰化酶活力水平均呈现上升趋势。其中，当TCE染毒浓度到达0．50mmol／L时，与L-02细胞对照组比较，L-02细胞组组蛋白去乙酰化酶活力明显升高，差异有统计学意义（F值为403．26，P〈0．01）；TCE为1．00mmol/L时酶活力最高。与SET缺陷细胞对照组比较，当TCE达到1．00mmol/L时，SET缺陷细胞组蛋白去乙酰化酶活力明显升高，差异有统计学意义（F值为44．01，P〈0．01）。与同染毒剂量的L-02细胞比较，当TCE染毒剂量到达0．50mmol／L时，SET缺陷细胞的组蛋白去乙酰化酶活力均低于L-02细胞，差异有统计学意义（P〈0．05）。与L-02细胞对照组比较，TCE染毒的L-02细胞HDAC2的蛋白表达水平明显上调，差异有统计学意义（F值为79．99，P〈0．01）；TCE染毒的SET缺陷细胞HDAC2蛋白表达水平变化无明显规律。结论TCE染毒可以诱发L-02细胞细胞增殖水平、凋亡率、组蛋白去乙酰化酶活力及相关蛋白表达水平的改变，SET缺陷可以明显抑制TCE染毒引起的细胞增殖、凋亡以及组蛋白修饰的异常。

Objective To compare the trichloroethylene（TCE）-induced alteration in cell proliferation, cell apoptosis, histone deaeetylase activity and expression levels in human hepatic L-02 ceils （L-02 cells） and SET deficient ceils, and reveal the TCE-induced effect in histone modification and the role of SET on epigenetic pathway. Methods The L-02 cells and preestablished SET deficient cells were treated with different TCE concentrations. For the changes of cell proliferation level and apoptosis rate, The L-02 cells and SET deficiency ceils without TCE treatment were served as the control group, the TCE treatment was in the concentration of 2.0 and 8.0 mmol/L for 24 h. For histone deacetylase activity and expression levels, the TCE treatment was in the concentration of 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 mmol/L for 24 h. Results After treatment with TCE for 24 h, the cell proliferation level was significantly decreased and the apoptotie rate was significantly increased in both cell lines. When concentration of TCE were reached to 8.0 mmol/L, the difference of cell proliferation level and apoptotic rate between two groups was statistically significant （t=-4.362 for proliferation level and t =23.950 for apoptotic rate, both P〈0.05 ）. After treatment with TCE for 24 h in various concentration（0, 0.25, 0.50, 1.00, 2.00, 4.00 and 8.00 retool/L）, the activity of histone deacetylases was significantly increased in both cell lines. When the TCE concentration were high than 0.50 mmol/L, compared with control group of L-02 cells, the enzymes activity were significantly increased （F=403.26, P〈 0.001 ）. When TCE concentration was reached 1.00 mmol/L, the enzyme activity is highest. Compared with control group of SET deficiency cells, the enzyme activity was significantly increased when TCE concentration was reached 1.00 mmol/L （F=44.01, P〈0.001）. When concentration of TCE reached 0.50 mmol/L, the difference of enzyme activity between two groups was statistically significant. For the protein expression, compared with control group of L-02 cells, TCE exposure can induced a significant increased expression level of HDAC2 in TCE-treated L-02 cells（Fvalues were 79.99, P〈0.001 ）. But the alteration in SET deficiency cells was not significant. Conclusion TCE exposure can induce a significant alteration on cell proliferation, apoptotic rate and, the activity and expression on histone deacetylases. SET deficiency can attenuate the TCE- induced alteration in histone modification in L-02 cells. Our results indicated that SET is involved in the mechanism of TCE-induced cytotoxicity and epigenetic regulation in L-02 cells.