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目的检测人外周血单个核细胞(PBMC)与肝细胞Na+-牛磺胆酸共转运蛋白(NTCP)基因的表达情况,探讨HBV感染PBMC的可能途径,研究乙型肝炎病毒(HBV)感染PBMC机制。方法用淋巴细胞分离液分离人外周血单个核细胞,用TRIzol提取人外周血单个核细胞与肝细胞总RNA,经逆转录合成cDNA。设计合成一对针对NTCPmRNA的特异性引物,对人外周血单个核细胞及肝细胞的cDNA进行PCR扩增,扩增产物用1.5%琼脂糖凝胶电泳鉴定,以检测NTCP基因在两种细胞的表达情况。结果 RT-PCR电泳显示,用NTCP基因特异性引物对肝细胞cDNA扩增,扩增产物大小在200~300bp之间,与NTCP基因理论值(246bp)相符,即肝细胞NTCP基因表达阳性;人外周血单个核细胞的cDNA扩增后进行电泳鉴定,未出现特异性条带,即NTCP基因表达阴性。结论人外周血单个核细胞不表达NTCP基因,提示其表面的HBV受体可能并非NTCP,或HBV循非受体途径进入PBMC,关于HBV感染PBMC的具体机制尚需进一步研究。
Objective To detect the expression of NTCP gene in peripheral blood mononuclear cells (PBMCs) and hepatocytes of hepatocytes, and to explore possible ways of HBV infection in PBMCs. To study the mechanism of HBV infection in PBMCs . Methods Human peripheral blood mononuclear cells were isolated from lymphocytes and the total RNA of human peripheral blood mononuclear cells and hepatocytes were extracted with TRIzol and cDNA was synthesized by reverse transcription. A pair of primers specific to NTCP mRNA was designed and synthesized to amplify the cDNA of human peripheral blood mononuclear cells and hepatocytes. The amplified product was identified by 1.5% agarose gel electrophoresis to detect the NTCP gene in two kinds of cells Express the situation. The results of RT-PCR electrophoresis showed that using NTCP gene-specific primers to amplify the cDNA of hepatocytes, the size of the amplified product was between 200 and 300bp, which was consistent with the theoretical value of NTCP gene (246bp), ie, the positive expression of NTCP gene in human hepatocytes Peripheral blood mononuclear cells were identified by electrophoresis after amplification of cDNA. There was no specific band, that is, NTCP gene expression was negative. CONCLUSION: NTCP gene is not expressed in human peripheral blood mononuclear cells, suggesting that the surface HBV receptor may not be NTCP, or that HBV non-receptor pathway enters PBMC. The specific mechanism of HBV infection of PBMC needs further study.