培元固肾方含药血清对已转染HBV质粒的HK-2细胞中TGF-β1、α-SMA、E-cadherin蛋白的影响

Effects of Peiyuan Gushen Fang serum on TGF-beta 1,alpha-SMA and E-cadherin proteins in HBV-transfected HK-2 cells

关键词:  乙型肝炎病毒相关性肾炎  HK-2细胞  肾间质纤维化  培元固肾方

Key words:  hepatitis B virus associated glomerulonephritis  HK-2 cell  renal interstitial fibrosis  Peiyuan Gushen Fang

摘要:

[目的] 通过培元固肾方含药血清对已转染乙型肝炎病毒（HBV）质粒的人肾皮质近曲小管上皮细胞（HK-2细胞）的干预，观察其对转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）、E-cadherin蛋白表达的影响，探讨培元固肾方抗肾间质纤维化的作用机制。[方法] 体外培养HK-2细胞，应用脂质体转染法，将扩增、鉴定后的质粒转染至HK-2细胞。将细胞分为7组，分别为：Mock组（常规培养的HK-2细胞组）；Control组（转染空载质粒pHY106的HK-2细胞组）；HBV组（转染空质粒与HBV复合物（PHY106-HBV）的HK-2细胞组）；HBV+ACEI组（贝那普利组）；HBV+PYGS Low组（培元固肾方低剂量组）；HBV+PYGS Middle组（培元固肾方中剂量组）；HBV+PYGS High组（培元固肾方高剂量组），实验分3个独立样本（n=3）。经HBV转染6 h后，予各组相应的药物进行干预。药物干预48 h后，应用酶联免疫吸附实验（ELISA）法检测各组细胞裂解液中乙型肝炎表面抗原（HBsAg）、乙型肝炎病毒e抗原（HBeAg）及TGF-β1的表达量；应用免疫细胞化学法检测α-SMA、E-cadherin的表达。[结果] 培元固肾方各干预组对TGF-β1表达均有下调（P<0.001），对α-SMA蛋白表达明显下调，差异有统计学意义（P<0.001），对E-Cadherin蛋白表达上调，差异有统计学意义（P<0.05）。[结论] 培元固肾方可能通过抑制促纤维化因子TGF-β1的表达抑制乙型肝炎病毒相关性肾炎（HBV-GN）肾小管上皮细胞的表型转化，对上皮细胞标志性蛋白E-cadherin的表达具有上调作用，对肌成纤维细胞标记蛋白α-SMA的表达具有下调作用，从而防治HBV-GN肾小管间质纤维化。

Abstract:

[Objective] To explore the mechanism of Peiyuan Gushen Fang in the treatment of renal interstitial fibrosis,the effects of serum containing Peiyuan Gushen Fang on the expression of TGF-beta 1,alpha-SMA and E-cadherin in HBV-transfected HK-2 cells were observed.[Methods] HK-2 cells were cultured in vitro. The amplified and identified plasmids were transfected into HK-2 cells by liposome transfection. According to the experimental purpose,the cells were divided into 7 groups,including Mock group (HK-2 cell group in conventional culture),control group (HK-2 cell group transfected with empty plasmid pHY106),HBV group (HK-2 cell group transfected with empty plasmid and HBV complex (PHY106-HBV);HBV+ACEI group (Benazepril group),HBV+PYGS low group (low concentration group of Peiyuan Gushen Fang),HBV+PYGS middle group (medium concentration group of Peiyuan Gushen Fang);HBV+PYGS high group (high concentration group of Peiyuan Gushen Fang). The experiment was divided into three independent samples (n=3). After 6 hours of HBV transfection,the corresponding drugs in each group were intervened. After 48 hours of drug intervention,the expression levels of HBsAg,HBeAg and TGF-beta 1 in cell lysates were detected by ELISA. Immunocytochemistry was used to detect the expression of alpha-SMA and E-cadherin.[Results] The expression of TGF-beta 1 was down-regulated in all intervention groups of Peiyuan Gushen Fang (P<0.001),and the expression of alpha-SMA was down-regulated significantly (P<0.001),while the expression of E-Cadherin was up-regulated (P<0.05).[Conclusion] Peiyuan Gushen Fang may inhibit the phenotypic transformation of HBV-GN renal tubular epithelial cells by inhibiting the expression of TGF-beta 1. It can up-regulate the expression of E-cadherin in epithelial cells and down-regulate the expression of alpha-SMA in myofibroblasts,to prevent and treat tubulointerstitial fibrosis of HBV-GN.