天津医药

• 论著 • 上一篇    下一篇

人剪切修复基因XPD对P52和P21基因的影响

马果果   

  1. 江西省南昌市民德路1号南昌大学第二附属医院
  • 收稿日期:2010-11-18 修回日期:2011-02-14 出版日期:2011-06-15 发布日期:2011-06-15
  • 通讯作者: 马果果

Biological behavior of HepG2 hepatoma cells transfected by XPD in viro

  • Received:2010-11-18 Revised:2011-02-14 Published:2011-06-15 Online:2011-06-15

PDF (PC)

2181

摘要： 目的 将人剪切修复基因XPD瞬时转染人肝癌细胞HepG2，观察转染后细胞内P52和P21基因表达的变化以及对细胞生长的影响，探讨野生型XPD基因与P52和P21的相互作用及细胞凋亡机制。方法 实验分三组，重组质粒HepG2-pEGFP-N2-XPD、空载质粒HepG2-pEGFP-N2，并用与HepG2-pEGFP-N2-XPD 、HepG2-pEGFP-N2同遗传背景和代数的HepG2细胞作为空白对照。用脂质体转染法瞬时转染细胞。利用荧光显微镜观测绿色荧光蛋白表达，用RT-PCR、Western blot法检测转染XPD基因后细胞内XPD、P52、P21的表达量变化，并用细胞增殖力检测(MTT)法检测细胞增殖情况。结果 ①免疫荧光显微镜下，HepG2-pEGFP-N2-XPD和HepG2-pEGFP-N2细胞中观察到绿色荧光蛋白表达，说明HepG2-pEGFP-N2-XPD重组质粒和HepG2-pEGFP-N2空载质粒成功转染。②RT—PCR检测：HepG2-pEGFP-N2-XPD中P21mRNA、XPD mRNA表达量与HepG2-pEGFP-N2和HepG2相比均明显增高(P<0.01)，P52 mRNA在HepG2-pEGFP-N2-XPD的表达量比两对照组明显降低(P<0．01)，而两对照组各个基因的表达没有明显差异(P>0.05)。③Western blot检测：HepG2-pEGFP-N2-XPD细胞的P21、XPD蛋白表达量较两对照组升高 (P<0．01)，P52的蛋白相对表达量比两对照组降低 (P<0．01)，两对照组差异没有统计学意义(P>0．05)。④MTT检测：HepG2-pEGFP-N2-XPD的细胞增殖力较两对照组减弱(P<0．05)，两对照组差异没有统计学意义(P>0．05)。结论XPD基因可以抑制癌细胞的生长，XPD基因可抑制基因P52的表达,促进P21的表达。

关键词: 癌, 肝细胞, DNA修复, 基因, 转染

Abstract: Objective To investigate the role of wild—type XPD in HepG2 hepatoma cells，its relationship with P52 and P21．And the apoptosis mechanism of HepG2 hepatoma cells． Methods The experiment were classified three groups: pEGFP-N2-XPD group, pEGFP-N2 group, with the same genetic background and the generations of HepG2 cells as normal group. pEGFP-N2 and pEGFP-N2-XPD were transfected into HepG2 cell line by Lipofectamine 2000. The expression of green fluorescent protein(GFP) were observed with fluorescence microscope. The expression of wild-type XPD，P52 ,P21 was detected by RT-PCR，Western blot．Cell growth was detected by MTT. Results ①HepG2-pEGFP-N2-XPD and HepG2-pEGFP-N2 express the green fluorescence protein which could be detected under the immunofluorescence microscope．②RT-PCR The relative expression of P21 mRNA, XPD mRNA in the HepG2, HepG2-pEGFP-N2-XPD and HepG2-pEGFP-N2: the relative expression of P21 mRNA, XPD mRNA in the HepG2-pEGFP-N2-XPD was significantly higher than other two controls(P<0.01). Otherwise, the relative expression of P52 mRNA was significantly lower than other two controls (P<0.01), the difference of two controls was not significant(P>0.05)③Western blot The relative expression of XPD protein,P21 protein were significantly higher than other two controls (P<0．01)．Otherwise，the relative expression of P52 protein was significantly lower than other two controls(P<0.01)，the difference of two controls was not significant(P>0.05)．④MTT The -XPD was much more decreased than other two controls (P<0．05)，the other two controls were not different(P>0．05)． Conclusions The wild-type XPD could inhibit the proliferation of HepG2 cells in vitro. The wild-type XPD could decrease the expression of P52 and increase the expression of P21.

Key words: Carcinoma, hepatocellular, DNA repair, genes, transfection