乙型肝炎病毒全基因转染L02细胞模型的建立及其HLA-A,B,C和MHCⅠ类链相关基因A／B分子的表达  

Establishing a L02 cell model with whole HBV gene transfection and analyzing its expressions of HLA- A, B, C and MICAIB

作　　者：陈敏[1] 彭明利[1] 石庆凤[1] 任红[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所、教育部省部共建感染性疾病分子生物学重点实验室,400010

出　　处：《中华肝脏病杂志》2007年第6期417-421,共5页Chinese Journal of Hepatology

摘　　要：目的 以双拷贝HBV全基因质粒pEcob6转染非癌性肝源细胞株L02细胞建立有HBV分泌的细胞转染模型,通过检测细胞表面HLA-A,B,C及MHC Ⅰ类链相关基因（MIC）A／B分子,分析HBV对肝细胞HLA-A,B,C和MICA／B表达的影响。方法 用EcoRⅠ酶切掉pEcob6的HBV基因并连接,构建无HBV基因的对照空质粒;用脂质体法以pEcob6或空质粒与pcDNA3．1-neo-共转染L02细胞株,筛选抗性细胞株并稳定传代;用Abbott EIA试剂和免疫荧光法检测细胞上清液和（或）细胞的HBsAg、HBcAg和HBeAg表达,实时荧光定量PCR检测HBV DNA含量;用流式细胞仪检测细胞表面HLA-A,B,C和MICA／B分子的表达,分析两种分子表达的变化。结果 经G418抗性克隆筛选,pEcob6稳定转染细胞株（L02-HBV）的上清液HBsAg和HBeAg可达24．78 （S／N）和4．117（S／N）,HBV DNA为9．67×10^4拷贝／ml;而对照空质粒转染细胞株（L02 mock）和L02的表达均为阴性。共聚焦显微镜观察,L02-HBV细胞内的HBsAg强表达于细胞质内,而HBcAg弱表达于胞核或胞质内。流式细胞检测结果显示,L02和L02-mock细胞有HLA-A,B, C的表达,基本无MICA／B的表达,L02-HBV细胞HLA-A,B,C和MICA／B的表达均增强,差歼有统计学意义（P〈0．05）。结论 以HBV全基因质粒转染L02细胞经筛选、克隆、传代,可获得有HBV相关抗原表达及HBV DNA分泌的细胞模型。HBV基因的转录或复制可增强肝细胞株L02的HLA-A-A,B,C及MICA／B的表达,可能与肝细胞的免疫损伤有关。Objective To establish a cell model with HBV secretion by plasmid transfection with whole HBV genes into hepatic L02 cells, and to analyze the effect of HBV on the expression of HLA-A, B, C and MICA/B in L02 cells. Methods The mock control plasmid was built by digesting pEcob6 plasmid with EcoR Ⅰ at the HBV DNA site and ligating the fragment without HBV DNA. The L02 cells were transfected with pEcob6 or mock plasmid and pcDNA3 1-neo by liposome. The expressions of HBsAg, HBcAg/HBeAg and HBV DNA were detected by immunofluorescence assay, Abbott enzymoimmunoassay, or FQ-PCR. The expressions of HLA-A, B, C and MICA/B were determined by FACS and the differences in the two molecules were analyzed. Results After the transfected cells were selected by G418, the HBsAg and HBeAg in the supematant of L02-HBV cells were 24.78（S/N） and 4.117（S/N）. The quantity of HBV DNA was 9.67 × 10^4 copies/ml, but in L02-mock and in L02 cells they were all negative. Under a confocal microscope HBsAg was brightly shown in the cytoplasm while HBcAg showed dimly in the cytoplasm or in the nuclei. By using FACS, the L02 and L02-mock cells showed some low expressions of HLA-A, B, C and a little expression of MICA/B, while the expression of the two molecules was higher in L02-HBV and the differences were significant （P 〈 0.05）. Conclusion A cell model with the expression of HBV antigens and the secretion of HBV DNA was established by pEcob6 transfection into L02 cells, the transcription or replication of HBV gene might induce stronger expressions of HLA-A, B, C and MICA/B on hepatic cells. They might be related to the immune injuries of hepatic cells.

关 键 词：肝炎病毒 乙型 转染 HLA抗原 MHC Ⅰ类链相关基因 

分 类 号：R686[医药卫生—骨科学]