利用IDO的双向作用重建肝移植后乙肝主动免疫的体外实验

Establishment of Active Immune Against HBV after Liver Transplantation with Ad-IDOEGFP-preS in Vitro

目的观察双目的基因吲哚胺2,3-二氧化酶(IDO)和乙型肝炎病毒前S抗原(HBV preS)在肝癌细胞株HepG2细胞的表达及其对人外周血淋巴细胞(PBLC)的抑制作用。方法将前期制备的重组腺病毒Ad-IDOEGFP-preS转染HepG2细胞,用倒置荧光显微镜观察增强型绿色荧光蛋白(EGFP)的表达,RT-PCR检测目的基因IDO和HBV preS mRNA的表达;与人PBLC共培养,同位素标记法观察IDO对其增殖的抑制作用。结果 HepG2细胞在转染3 d后即可见明显的EGFP表达,7 d后荧光更强,提示目的基因已成功导入HepG2细胞。RT-PCR显示感染Ad-IDOEGFP-preS重组腺病毒的HepG2细胞中含有IDO和HBV preS基因片段,IDO相对表达强度为1.27,HBV preS相对表达强度为1.18,而对照组HepG2细胞未出现IDO和HBV preS基因片段。与其共培养的淋巴细胞每分钟放射线计数(CPM)为(7 471±1 375)次/min,明显低于对照组CPM〔(13 821±1 997)次/min〕,P<0.001。结论重组腺病毒Ad-IDOEGFP-preS能有效地将目的基因IDO和HBV preS导入HepG2细胞并表达,携带该基因的HepG2细胞对PBLC的增殖具有明显的抑制作用,这将为进一步的肝移植乙肝主动免疫动物实验奠定基础。

Objective To detect the expression of indoleamine-2,3-dioxygenase（IDO） and HBV preS mRNA in HepG2 cells and its inhibitory effect on the proliferation of human peripheral blood lymphocyte.Methods The Ad-IDOEGFP-preS was transfected to human hepatocarcinoma cell line HepG2 and the specific fragment of IDO mRNA and HBV preS mRNA in these HepG2 cells were detected by RT-PCR.Then the transfected HepG2 cells were co-cultured with human peripheral blood lymphocyte and the inhibitory effect of IDO on the proliferation of human peripheral blood lymphocyte was observed.Results The IDO and HBV preS mRNA were successfully transferred to HepG2 cells,so the specific fragment of IDO and HBV preS mRNA could be found in the transfected HepG2 cells but not in the control group HepG2 cells by RT-PCR.Furthermore,the relative expression intensity of IDO and HBV preS were 1.27 and 1.18,respectively.When co-cultured with human peripheral blood lymphocyte,the counts per minute in the transfected HepG2 cells（（7 471±1 375） beats/min） was significantly less than that in the control group（（13 821±1 997） beats/min）,P0.001.Conclusion The target gene IDO and HBV preS can be transferred and expressed in HepG2 cells successfully,which can obviously suppress the proliferation of human peripheral blood lymphocyte.