目的：探讨人剪切修复基因XPD对肝癌细胞P53和Ets-1基因的影响。方法使用脂质体瞬时转染技术，将pEGFP-N2-XPD重组质粒转染至肝癌细胞HepG2，24 h后加入20μmol/L的P53抑制剂Pifithrin-α，孵育24 h，并以未经转染的HepG2细胞作为空白对照。 RT-PCR、Western blot 检测细胞中XPD、P53、phospho -P53（ser-15）、Ets-1的mRNA和蛋白的表达变化，MTT法观察细胞增殖的活力，流式细胞仪检测细胞周期变化。结果与空白对照比较，转染pEGFP-N2-XPD重组质粒后，HepG2细胞XPD、P53 mRNA和蛋白表达上调（ P＜0.01），而Ets-1 mRNA和蛋白表达下调（P＜0.01），加入Pifithrin-α后，XPD、P53 mRNA和蛋白表达下调，Ets-1 mRNA和蛋白表达上调（ P＜0.01）。转染pEGFP-N2-XPD重组质粒后，HepG2细胞进入S期发生阻滞，停滞在G1期（P＜0.01），加入Pifithrin-α后，HepG2细胞G1期细胞减少，S期细胞增多（P＜0.01）。转染pEGFP-N2-XPD重组质粒后，HepG2细胞增殖活力下降（ P＜0.01），加入Pifithrin-α后， HepG2细胞增殖活力上升（ P＜0.01）。结论野生型XPD基因在转染至肝癌HepG2细胞后，可以上调P53的表达，通过P53途径抑制Ets-1的表达，促使肝癌细胞凋亡。%Objective To investigate the effects of xeroderma pigmentosum D ( XPD) on the growth of hepatoma cells and the expressions of P53 and Ets-1.Methods Human hepatoma cells (HepG2) were transfected with the plas-mids of pEGFP-N2-XPD using Lipofectamine 2000, and subsequently incubated with 20μmol/L Pifithrin-α( P53 in-hibitor) for 24 h.Blank control group (no transfecting and adding medicine ) was set.The expressions of XPD, P53, phospho-P53 (ser-15) and Ets-1 were assessed by RT-PCR and Western blot.Flow cytometry was used to examine the cell cycle and apoptosis .MTT assay was used to detect the cell proliferation .Results After transfected with the pEG-FP-N2-XPD plasmid, the mRNA and protein expression of XPD and P53 were significantly up -regulated, while the expression of Ets-1 was significantly down -regulated (P<0.01).Moreover, the cell proliferation was inhibited and the apoptosis was accelerated with pEGFP -N2-XPD plasmid transfected , which was reversed by Pifithrin -α.Conclusion Wild-XPD gene can restrain the proliferation, down -regulate Ets -1 via P53 pathway, and impel the apoptosis ofhepatoma cells.
展开▼
