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为了研究HBV感染对肝癌细胞凋亡的影响,我们首先观察肝癌细胞HepG2和HepG2.2.15细胞凋亡相关蛋白的表达差异,继而构建了HBV全长基因组以及HBV各元件HBx、HBc、HBs、HBp的表达载体,并将其分别转染到HepG2细胞。采用流式细胞术和普通PCR的方法检测不同转染组中Fas蛋白水平及基因水平表达;Annexin V/PI双染法检测细胞的凋亡,定量PCR检测凋亡相关基因mRNA的表达变化;并用CFSE/7AAD方法检测HepG2细胞对NK杀伤敏感性的变化。数据显示:含有HBV全基因组的HepG2.2.15Fas表达明显低于无HBV感染的HepG2细胞;转染HBp元件后,HepG2细胞中Fas表达下调最明显;并且细胞凋亡比例显著下降,抗凋亡基因Bcl-2和Bcl-xl的表达上升,促凋亡基因Bax、Bak和Bad的表达下降。而且,转染HBp元件显著降低HepG2细胞对NK细胞杀伤的敏感性。这说明在HBV病毒感染过程中,HBp基因除发挥DNA聚合酶的作用外,还可通过下调Fas表达抑制肝细胞的凋亡、降低其对NK细胞的杀伤敏感性,此可能为HBV+肝癌发生发展更易发生免疫逃逸的原因之一。
To investigate the effect of HBV infection on the apoptosis of hepatocellular carcinoma cells, we first observed the differences in the expression of apoptosis-related proteins in HepG2 and HepG2.2.15 cells, then constructed the full-length HBV genome and the HBx, HBc, HBs, HBs The vector was transfected into HepG2 cells. The expression of Fas protein and gene in different transfection groups were detected by flow cytometry and normal PCR. Apoptosis was detected by Annexin V / PI double staining. The mRNA expression of apoptosis related gene was detected by quantitative PCR. Changes of Susceptibility of HepG2 Cells to NK Toxicity by CFSE / 7AAD Method. The data showed that the expression of HepG2.2.15Fas in HBV genome was significantly lower than that in HepG2 cells without HBV infection. The expression of Fas in HepG2 cells was most obviously down-regulated after HBp transfection, and the percentage of apoptosis was significantly decreased. The anti-apoptotic gene The expression of Bcl-2 and Bcl-xl increased, and the expression of pro-apoptotic genes Bax, Bak and Bad decreased. Moreover, transfection of HBp elements significantly reduced the sensitivity of HepG2 cells to NK cell killing. This shows that in the process of HBV virus infection, HBp gene in addition to play the role of DNA polymerase, but also by down-regulating Fas expression inhibition of hepatocyte apoptosis, reduce its sensitivity to NK cell killing, which may be HBV + liver cancer development One of the reasons why immune escape is more likely.