Notchl信号在乙型肝炎病毒X基因介导的肾小管上皮细胞免疫活性异常中的作用

Effect of Notchl signal on hepatitis B virus X-mediated abnormal immune activity in renal tubularepithelial cells

目的探讨乙型肝炎病毒x基因（HBx）转染人近端肾小管上皮细胞系HK一2细胞后对其表达Notchl信号的影响，并观察Notchl信号在HBx介导的肾小管上皮细胞免疫活性中的作用。方法用分子克隆的方法构建pcDNA3．1／myc．HBx质粒及pcDNA3．1／myc—Notchl质粒，转染HK-2细胞，并应用短发卡RNA（shRNA）沉默Notchl基因表达。将细胞分为7组：①正常培养组；②转染HBx空载质粒组；③转染HBx质粒组；④转染HBx质粒＋Notchl空载质粒组；⑤转染HBx质粒＋Notchl质粒组；⑥转染HBx质粒＋NotchlshRNA空载质粒组；⑦转染HBx质粒＋NotchlshRNA沉默质粒组。实时定量PCR和蛋白印迹法检测细胞中Notchl受体的变化，流式细胞术检测细胞表面人主要组织相容性复合物（MHC）一II、CD40表达的变化，酶联免疫吸附试验（ELISA）检测细胞培养上清中白细胞介素（IL）-4、干扰素（IFN）一1水平。结果HK-2细胞经转染HBx基因后，可高表达HBx，NotchlshRNA在HK-2细胞中的转染效率也高达70％，基因沉默有效；转染HBx基因组细胞Notchl水平高于正常培养组及转染HBx空载质粒组相比，且Notchl过表达组细胞表面MHC。II、CD40表达均明显高于Notchl空载质粒组（9．69％±0．52％比4．90％±0．32％，21．56％±0．71％比15．74％-4-0．20％，均P〈0．05），其上清液中IL_4水平亦高于Notchl空载质粒组（50．2±0．6比28．1±1．2，P〈0．05），而IFN一1水平较低（11．9-4-1．7比18．8±0．8，P〈0．05）。Notchl基因沉默后上述指标结果呈相反变化。结论HBx基因转染HK-2细胞后可引起Notchl表达上调；Notchl又可促进细胞表面免疫分子的表达，并调控细胞因子分泌，最终导致细胞损伤及免疫微环境的紊乱。

Objective To explore the expression of Notchl receptor in renal tubular epithelial cells transfected with hepatitis B virus X （HBx） gene and its role in immunologic activity. Methods The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notchl containing Notchl gene was transiently transfected into HK-2 cells. And the shRNA technique was used to silence Notchl. HK-2 cells were divided into 7 groups, including normal culture, pcDNA3.1/myc, HBx, HBx ＋ pcDNA3.1/myc, HBx ＋ Notchl, HBx ＋ shRNA and HBx ＋ Notchl shRNA. Real-time PCR and Western blotting were used to detect the expression of Notchl. The expressions of MHC- I] and CD40 were examined by flow cytometry. And the supernatant contents of IL-4 and IFN-7 were measured by enzyme-linked immunosorbent assay （ELISA）. Results The results of real-time PCR and Western blotting verified that HBx and Notchl were successfully expressed in HK-2 cells after transfection. The transfection efficiency of shRNA was 70%. Compared with normal culture and pcDNA3.1/myc groups, the expression of Notchl increased. The expressions of MHC-11 and CD40 also significantly increased in the HBx ＋ Notchl group （9.69%±0.52% vs 4.90% ±0.32%,21.56%±0.71% vs 15.74%±0.20%, both P〈O. 05）. The supernatant level of IFN-y was lower in HBx ＋ Notchl group （ 11.9 ± 1.7 vs 18.8 ± O. 8, P 〈 0. 05 ） while the level of IL-4 was higher than control groups （50. 2 ± 0. 6 vs 28. 1 ± 1.2, P 〈 0. 05 ）. And the HBx ±Notchl shRNA group had the opposite results. Conclusions An over-expression of HBx gene may upregulate the expression of Notchl. And Notchl promotes the expression of immune molecules of renal tubular epithelial cell and regulates the secretion of cytokines so as to cause injury of cells and dysfunction of immunomicroenviroment.