中文摘要：

目的研究HCV F蛋白的生物学功能。方法扩增目的基因片段，通过酶切连接方法将编码HCVF蛋白的基因片段克隆至载体pSEB-3Flag中，用构建好的重组质粒pSEB-3Flag-F转染Huh7、SMMC7721细胞，经Blasticidine抗性筛选，拟建立HCVF蛋白稳定表达的细胞株，用RT-PCR方法检测HCV-F基因的表达。通过MTS、细胞计数、结晶紫以及流式细胞检测实验观察F蛋白对转染细胞增殖和细胞周期的影响。计量资料分析采用t检验。结果成功建立了HCVF基因稳定表达的细胞株Huh7-F和SMMC7721-F，MTS、细胞计数实验均证实Huh7-F和SMMC7721-F细胞较对照细胞增殖速度减慢，Huh7-F稳定细胞株结晶紫定量爿值为1．072±0．070，对照组为1．387±0．005（f=-6．347，P〈0．05）；SMMC7721-F稳定细胞株4值为1．594±0．007，对照组为1．921±0．090（t=-4．81，P〈0．05）。流式细胞仪检测Huh7-F稳定细胞株48hS期细胞百分比分别为47．1妣±0．040／0，对照组S期细胞数为55．32％±1．45％（f=-7．99，P〈0．05）；SMMC7721-F稳定细胞株S期细胞百分比分别为30．75％±0．09％，对照组S期细胞数为33．23％±0．28％（f=-5．91，P〈0．05）。结论稳定转染HCVF基因可以明显抑制Huh7细胞、SMMC7721细胞的增殖。

英文摘要：

Objective To investigate the biological function of the hepatitis C virus （HCV）-encoded F protein in hepatocytes. Methods The full-length F gene was amplified by PCR from HCV genotype la and cloned into plasmid pSEB-3Flag by restriction enzyme digestion and ligation. Hepatoma cell lines, Huh7 and SMMC7721, were transfected with the resultant recombinant pSEB-3Flag-F or the original pSEB-3Flag （negative control） and screened with the selective antibiotic, blasticidine. Stable F gene and protein expression was verified by RT-PCR analysis. Analysis of cell growth and cell cycle was carried out by MTS assay, crystal violet staining and flow cytometry. Results Huh7 and SMMC7721 cells transfected with pSEB-3Flag-F plasmid （Huh7-F and SMMC7721-F, respectively） uniquely expressed the F gene and protein. The Huh7-F and SMMC7721-F cells showed significantly decreased proliferation rates, compared to the respective control groups. A similar HCV F-mediated growth-inhibiting activity was observed by the cell viability assay. Furthermore, cell cycle analysis revealed that the S-phase distribution was much lower in Huh7-F （47.12%） and SMMC7721-F （30.75%） cells than in the respective controls （55.35% and 33.23%, respectively） （P〈 0.05）. Conclusion Stable expression of the HCV F gene reduced the in vitro proliferation rate of hepatoma cell lines, indicating that the F protein may function as a growth inhibitor of infected cells.