XPD和P53对HepG2.2.15增殖及乙型肝炎病毒X蛋白表达的影响

Effects of XPD and P53 on Proliferation of HepG2.2.15 and Expression of Hepatitis B Virus X Protein

目的:探讨人着色性干皮病基因D(XPD)和P53对人肝癌细胞株HepG2.2.15增殖及乙型肝炎病毒X蛋白(HBx)、Bcl-2和Bax基因表达的影响。方法:用脂质体转染法将重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2转染HepG2.2.15细胞,转染后第2天给予20μmol/L的Pifithrin-α(P53抑制剂)孵育24h。分为空白对照组、pEGFP-N2组、pEGFP-N2/XPD组、pEGFP-N2/XPD+Pifithrin-α组及Pifithrin-α组。用RT-PCR和Westernblotting方法检测XPD、HBx、Bcl-2和Bax表达的变化;用MTT法观察细胞增殖活力;用流式细胞仪检测细胞周期。结果:(1)重组质粒pEGFP-N2/XPD的转染使得XPD表达增高(均P<0.001);XPD表达增高使得HBx和Bcl-2表达降低,同时使得Bax表达增高,而Pifithrin-α能抑制XPD这一作用(均P<0.01)。(2)XPD表达增高抑制了细胞增殖活力,而Pifithrin-α能抑制XPD降低细胞活力的作用(均P<0.001)。(3)XPD表达增高引起了细胞G1期增加、S期减少,而Pifithrin-α能抑制XPD这一作用(均P<0.001)。结论:XPD是通过P53途径抑制肝癌细胞增殖,下调HBx和Bcl-2的表达并上调Bax的表达;XPD、P53和HBx三者之间能相互作用、相互影响,共同调节肝癌的发生与发展。

Objective： To investigate effects of xeroderma pigmentosum D （XPD） and P53 on the proliferation of HepG2.2.15, human hepatoma cells and the expressions of hepatitis B virus X protein （HBx）, Bcl-2 and Bax. Methods： Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 by liposome. On the next day, these cells were incubated with Pifithrin-α （P53 inhibitor） at a 20 μ mol/L concentration for 24 h. Cells were divided into five groups, blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD ＋ Pifithrin-α group and Pifithrin-ot group. The expressions of XPD, HBx, Bcl-2 and Bax were detected by RT-PCR and Western blotting. The cell proliferation was detected by MTT. The cell cycles were examined with flow cytometry. Results： （1） The expression of XPD increased by transfection of pEGFP-N2/XPD （P 〈 0.001）. The increased XPD expression down-regulated the expres- sions of HBx and Bcl-2, and up-regulated the expression of Bax, while Pifithrin-α abolished the above effects of XPD（P 〈 0.01, respectively）. （2） MTT results showed that the increased XPD expression inhibited the cell proliferation. This inhibition induced by XPD can be inhibited by Pifithrin-α （P 〈 0.001）. （3） Flow cytometry results showed that the up-regulated XPD ex- pression increased the cell number of G, phase and decreased that of S phase, while the effect of XPD on cell cycle was abol- ished by Pifithrin-α （P 〈 0.001, respectively）. Conclusion： XPD can suppress the proliferation of hepatoma carcinoma cells, down-regulate the expression of HBx and Bcl-2, and up-regulate the expression of Bax through P53 pathway. There may bemutual influences among XPD, P53 and HBx that co-regulate hepatocarcinogenesis.