RNA干扰对HepG2.2.15细胞中乙型肝炎病毒表达和复制的抑制作用
Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15.
作者信息
Zhao Zhong-Fu, Yang Hui, Han De-Wu, Zhao Long-Feng, Zhang Guo-Ying, Zhang Yun, Liu Ming-She
机构信息
Institute of Hepatology, Changzhi Medical College, Changzhi 046000, Shanxi Province, China.
出版信息
World J Gastroenterol. 2006 Oct 7;12(37):6046-9. doi: 10.3748/wjg.v12.i37.6046.
AIM
To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA (siRNA) pGenesil-HBV X targeting HBV X gene region into HepG2.2.15 cells.
METHODS
pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays (TRFIA). HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry.
RESULTS
The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% (P < 0.01), and by 38.67% (P < 0.05) and 42.86% (P < 0.01) at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR (P < 0.05).
CONCLUSION
In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil-HBV X targeting the HBV X coding region.
目的
通过将靶向乙型肝炎病毒（HBV）X基因区域的载体介导的小干扰RNA（siRNA）pGenesil-HBV X转染至HepG2.2.15细胞中，观察其对HBV复制和表达的抑制作用。
方法
构建pGenesil-HBV X并通过脂质体转染法将其转染至HepG2.2.15细胞。转染后24、48和72小时，采用时间分辨免疫荧光分析法（TRFIA）测定HBV抗原分泌情况。通过荧光定量PCR检测HBV复制情况，采用免疫组织化学法测定细胞质病毒蛋白的表达。
结果
pGenesil-HBV X转染后48小时，上清液中HBsAg和HBeAg的分泌分别被抑制28.5%和32.2%（P<0.01），72小时时分别被抑制38.67%（P<0.05）和42.86%（P<0.01）。转染48小时后，HepG2.2.15细胞中细胞质HBsAg的免疫组织化学染色显示出类似的下降。荧光PCR定量结果显示，转染后48小时和72小时，培养上清液中HBV基因组数量也显著减少（P<0.05）。
结论
在HepG2.2.15细胞中，靶向HBV X编码区的载体介导的siRNA pGenesil-HBV X可抑制HBV的复制和表达。
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