干扰素α对乙型肝炎病毒X蛋白诱导小鼠足细胞系凋亡的影响及分子机制研究

Effect and molecular mechanism of interferon-α on podocyte apoptosis induced by hepatitis B virus X protein

摘要:

目的 观察干扰素α（INF-α）对乙型肝炎病毒X （HBx）蛋白诱导的小鼠足细胞系（MPC5）凋亡的影响及其分子机制。方法 以携带HBx基因的pEX质粒转染MPC5细胞，采用逆转录PCR检测不同时间点HBx mRNA表达水平。将MPC5细胞分为对照组：正常条件下培养的MPC5细胞；INF-α组：正常MPC5细胞与INF-α共培养；HBx组：HBx诱导的MPC5细胞；HBx+INF-α组：HBx诱导的MPC5细胞与INF-α共培养。不同实验条件下干预48 h后，采用流式细胞术检测各组MPC5细胞凋亡情况；采用实时荧光定量PCR法和Western blot法检测足细胞裂孔隔膜相关蛋白（nephrin、CD2AP、synaptopodin）和细胞骨架相关蛋白（TRPC6）的mRNA及其蛋白的表达。结果 pEX-HBx转染MPC5细胞后48 h HBx mRNA表达最高（P < 0.05）。HBx组MPC5细胞凋亡率显著高于对照组、INF-α组和HBx+INF-α组（P < 0.05）。与对照组和INF-α组相比，HBx组nephrin、synaptopodin、CD2AP mRNA及其蛋白的表达水平明显降低，TRPC6 mRNA及其蛋白的表达水平明显增高（P < 0.05）。与HBx组相比，HBx+INF-α组nephrin、synaptopodin、CD2AP mRNA及其蛋白的表达水平明显增高，TRPC6 mRNA及其蛋白的表达水平明显降低（P < 0.05）。结论 INF-α可抑制HBx诱导的MPC5细胞凋亡，其机制可能与INF-α可调节HBx诱导的足细胞裂孔隔膜相关蛋白（CD2AP、nephrin、synaptopodin）和细胞骨架相关蛋白（TRPC6）的异常表达恢复正常有关。

Abstract:

Objective To investigate the effect and molecular mechanism of interferon-α (INF-α) on the apoptosis of the mouse podocyte cell line MPC5 induced by hepatitis B virus X (HBx) protein. Methods MPC5 cells were transfected with the pEX plasmid carrying the HBx gene. RT-PCR was used to measure the mRNA expression of HBx at different time points. MPC5 cells were divided into 4 groups:control group (MPC5 cells cultured under normal conditions), INF-α group (MPC5 cells cultured with INF-α), HBx group (MPC5 cells induced by HBx), and HBx+INF-α group (MPC5 cells induced by HBx and cultured with INF-α). After 48 hours of intervention under different experimental conditions, flow cytometry was used to measure the apoptosis of MPC5 cells, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of slit diaphragm-related proteins (nephrin, CD2AP, and synaptopodin) and the cytoskeleton-related protein transient receptor potential cation channel 6 (TRPC6). Results MPC5 cells transfected by pEX-HBx had the highest expression of HBx mRNA at 48 hours after transfection (P < 0.05). Compared with the control, INF-α and HBx+INF-α groups, the HBx group had a significant increase in the apoptosis rate of MPC5 cells (P < 0.05). Compared with the control and INF-α groups, the HBx group had significant reductions in the mRNA and protein expression of nephrin, synaptopodin, and CD2AP and significant increases in the mRNA and protein expression of TRPC6 (P < 0.05). Compared with the HBx group, the HBx+INF-α group had significant increases in the mRNA and protein expression of nephrin, synaptopodin, and CD2AP and significant reductions in the mRNA and protein expression of TRPC6 (P < 0.05). Conclusions INF-α can inhibit the apoptosis of podocytes induced by HBx, possibly through improving the abnormal expression of slit diaphragm-related proteins (CD2AP, nephrin, and synaptopodin) and cytoskeleton-related protein (TRPC6) induced by HBx.