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目的通过在小鼠肝癌细胞表面串联表达HCV受体分子,在二维结构的基础上研究HCV受体分子的排列顺序对病毒入侵的影响,为研究HCV的早期感染机制奠定基础。方法将已构建的慢病毒表达载体p CDHh LDLR-h SR-BⅠ-h CD81-GFP、p CDH-h LDLR-h CD81-h SR-BⅠ和p CDH-h CLDN-1-h OCLN-DsRed与包装质粒共转染包装细胞293FT,包装慢病毒,用收集浓缩的慢病毒攻击小鼠肝癌细胞系Hepa1-6,抗生素G418加压筛选得到串联表达人紧密链接区域分子CLDN-1和OCLN的转基因细胞株h CLDN-1-OCLN/Hepa1-6(CO/Hepa1-6);重组慢病毒p CDHh LDLR-h SR-BⅠ-h CD81-GFP和p CDH-h LDLR-h CD81-h SR-BⅠ攻击转基因细胞株CO/Hepa1-6,通过嘌罗霉素puro和G418双抗加压及流式细胞术,最终筛选获得5种分子h LDLR、h SR-BⅠ、h CD81、h CLDN-1和h OCLN共表达且具有不同受体排列顺序的转基因人源化小鼠肝癌细胞株LSCCO/Hepa1-6和LCSCO/Hepa1-6;利用HCV阳性血清直接感染方法分析LSCCO/Hepa1-6和LCSCO/Hepa1-6对HCV的易感性,研究HCV受体分子的排列顺序对病毒入侵的影响。结果两种细胞株LSCCO/Hepa1-6和LCSCO/Hepa1-6对HCV结合均有增加,但LSCCO/Hepa1-6细胞株细胞表面的HCV受体排列顺序更有利于HCV入侵。结论在二维结构的基础上,HCV在感染早期要优先与B族Ⅰ型清道夫受体SR-BⅠ作用。
OBJECTIVE: To study the effect of the order of HCV receptor molecules on the virus invasion on the basis of two-dimensional structure by expressing HCV receptor molecules in tandem on the surface of mouse hepatoma cells, and to lay a foundation for studying the mechanism of early infection of HCV. Methods The constructed lentiviral expression vectors p CDHh LDLR-h SR-BI-h CD81-GFP, p CDH-h LDLR-h CD81-h SR-BI and p CDH-h CLDN-1-h OCLN-DsRed and The packaging plasmids were co-transfected into 293FT packaging cells and the lentivirus was packaged. The mouse hepatoma cell line Hepa1-6 was challenged with concentrated lentivirus. The antibiotic G418 was screened by pressure to obtain transgenic cells expressing tandemly linked human CLDN-1 and OCLN Strain h CLDN-1-OCLN / Hepa 1-6 (CO / Hepa 1-6); recombinant lentivirus p CDHh LDLR-h SR-BHI CD81-GFP and p CDH-h LDLR-h CD81-h SR- Transgenic cell line CO / Hepa1-6 was screened by puromycin puro and G418 double anti-pressure and flow cytometry. Five molecules of h LDLR, h SR-BⅠ, h CD81, h CLDN-1 and h OCCN and LSCCO / Hepa1-6 and LCSCO / Hepa1-6, which were co-expressed in OCLN and had different receptor order, were used to analyze the expression of LSCCO / Hepa1-6 and LCSCO / Hepa1-6 by LC- 6 susceptibility to HCV to study the influence of the sequence of HCV receptor molecules on the virus invasion. Results The two cell lines LSCCO / Hepa1-6 and LCSCO / Hepa1-6 increased the binding of HCV, but the order of HCV receptor on the cell surface of LSCCO / Hepa1-6 cells was more favorable for HCV invasion. Conclusion Based on the two-dimensional structure, HCV has priority to SR-BⅠ, a type B scavenger receptor, in the early stage of infection.