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XPD和P53对HepG2.2.15增殖及乙型肝炎病毒X蛋白表达的影响

丁浩1,张吉翔2   

  1. 1. 南昌大学第二附属医院消化科
    2. 南昌大学第二附医院消化内科
  • 收稿日期:2011-08-08 修回日期:2011-09-19 出版日期:2012-02-15 发布日期:2012-02-15
  • 通讯作者: 丁浩

The effects of XPD and P53 on growth of HepG2.2.15 and the expression of Hepatitis B virus X protein

Hao Ding,   

  • Received:2011-08-08 Revised:2011-09-19 Published:2012-02-15 Online:2012-02-15
  • Contact: Hao Ding

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摘要： 摘 要 目的：探讨人着色性干皮病基因D(xeroderma pigmentosum 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(Hepatitis B virus X protein,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20μM的Pifithrin-α（P53抑制剂）孵育24 h。实验分为5组：（1）空白对照组；（2）pEGFP-N2组；（3）pEGFP-N2/XPD组；（4）pEGFP-N2/XPD + Pifithrin-α组；（5）Pifithrin-α组。用RT-PCR和Western blotting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：RT-PCR和Western blotting检测发现，重组质粒pEGFP-N2/XPD的转染使得XPD表达增高（P均 <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（P 均<0.01）。MTT结果显示，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（P均 <0.001）。流式细胞仪结果显示，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（P均 <0.001）。结论：XPD是通过P53途径抑制肝癌细胞增殖的；XPD可通过P53途径下调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。

关键词: 肝肿瘤, 癌, 细胞系 肿瘤, 转染, 抑制剂 Pifithrin-α, 原癌基因蛋白质 乙型肝炎病毒X蛋白, 细胞增殖

Abstract: Abstract Objective：To investigate the effects of xeroderma pigmentosum D(XPD) and P53 on growth of HepG2.2.15, human hepatoma cell, 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 20μM concentration for 24 h. The experiments were divided into five groups: Blank control group;pEGFP-N2 group;pEGFP-N2/XPD group;pEGFP-N2/XPD + Pifithrin-α group;Pifithrin-α group. Through RT-PCR and Western blotting, the expressions of XPD,HBx , Bcl-2and Bax were detected. The cell growth was detected by MTT. The cell cycles were examined with flow cytometry. Results: RT-PCR and Western blotting results showed that the transfection of pEGFP-N2/XPD increased the expression of XPD（P <0.001,respectively）. The increase of XPD expression down-regulated the expressions of HBx and Bcl-2 and up-regulated the expression of Bax, while Pifithrin-α abolished the above-mentioned effects of XPD（P <0.01,respectively）. MTT results showed that the increase of XPD expression inhibited the cell growth and the inhibition of cell growth by XPD was reduced by Pifithrin-α（P <0.001,respectively）. Flow cytometry results showed that the up-regulation of XPD expression increased the cell number of G1 phase and decreased that of S phase, while the effect of XPD on cell cycle was abolished by Pifithrin-α（P <0.001,respectively）. Conclusions: XPD can suppress growth of hepatoma carcinoma cells, down-regulate the expressions of HBx and Bcl-2 and up-regulate the expression of Bax through P53 pathway. There may be mutual influences among XPD, P53 and HBx that co-regulate hepatocarcinogenesis.

Key words: liver neoplasms, carcinoma, cell line tumor, transfection, inhibitor Pifithrin-α, proto-oncogene protein Hepatitis B virus X protein, cell proliferation