摘要目的 探讨转化生长因子β(TGF-β)Ⅰ型受体(R Ⅰ)、Ⅱ型(RⅡ)受体以及下游Smad蛋白在单侧输尿管梗阻(UUO)大鼠模型肾脏中表达及意义.方法 90只雌性Wistar 大鼠随机分为正常对照组(CON组)、假手术组(SOR组)和单侧输尿管梗阻组(UUO组),分别于术后1、3、7、14、21 d处死,检测各组大鼠肾功能；PAS与Masson染色观察大鼠肾间质病理形态改变；实时定量PCR基因芯片分析正常大鼠和肾间质纤维化大鼠肾组织TGF-β Ⅰ、Ⅱ、Ⅲ型受体及Smad蛋白家族表达.筛选出差异表达的受体亚型,进一步应用实时荧光定量PCR、蛋白免疫印迹法、免疫荧光法检测和验证筛选出的差异受体亚型在不同分期肾间质纤维化大鼠肾组织的分布和表达.结果 与CON组相比,UUO组大鼠的Scr及BUN于术后3d开始升高(P＜0.05),第21天达峰值(P＜0.01)；UUO组术后3d肾间质可见明显炎性细胞浸润；14 d后出现明显肾小管萎缩；21 d可见明显肾间质纤维化.UUO组肾组织TGF-β Ⅰ型受体ALK-5、ALK-7和TGF-βRⅡ的mRNA表达于术后3d上升并随梗阻时间延长逐渐增加(P＜0.05),于14 d达到峰值(均P＜0.01)；ALK-6的mRNA表达于术后3d下降(P＜0.05)并随梗阻时间延长逐渐减少,于14 d达谷值(P＜0.01).ALK-5、ALK-6、ALK-7和TGF-βRⅡ蛋白表达与基因表达一致.Smad2/3及磷酸化(p)-Smad2/3的蛋白表达于术后3d上升(均P＜0.05)并随梗阻时间延长逐渐增加,于14 d达到峰值(均P＜0.01).结论 在肾间质纤维化进展中不同TGF-β受体亚型存在不同的变化规律并与肾间质纤维化进展密切关联.

abstractsObjective To investigate the influence of TGF-β receptor subtypes expression and their downstream signaling Smad proteins on rat renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Methods A total of 90 rats were randomly divided into three groups:normal control (CON),sham operation (SOR) and UUO group,and sacrificed 1,3,7,14 and 21 days after operation.Serum creatinine and urea nitrogen were detected to assess renal function.PAS and Masson staining were performed to observe histological damage in the kidneys.Quantitative RT-PCR was used to define expression of mRNA encoding TGF-β receptor subtypes and their downstream signaling Smad proteins in kidney tubular cells.Real-time PCR,Western blotting and immunofluorescence were used to monitor the time-related expression of the TGF-β receptor subtypes and their downstream signaling Smad proteins in kidney. Results Compared with the CON group,serum creatinine and urea nitrogen in UUO groups increased at day 3 after operation (P＜0.05) and reached their peak 21 days after operation (P＜0.01).Obvious inflammatory cell infiltration was observed in UUO group 3 days after operation,while renal tubular atrophy and renal interstitial fibrosis were observed in UUO group 14 days after operation.The mRNA expressions of ALK-5,ALK-7 and TGF-βR Ⅱ increased significantly in UUO group 3 days after operation (all P＜0.05) and reached their peaks 14 days after operation (all P＜0.01).The mRNA expression of ALK-6 decreased significantly in UUO group 3 days after operation (P＜0.05) and reached its lowest level 14 days after operation (P＜0.01).The changes in the protein level of those receptors were consistent with their mRNA expressions.The protein expressions of Smad2/3 and p-Smad2/3 increased significantly in UUO group at day 3 (all P＜0.05) and reached their peak at day 14 after operation (all P＜0.01). Conclusion Expressions of TGF-β receptor subtypes ALK-5,ALK-6,ALK-7,TGF-βR Ⅱ and their downstream signaling Smad2 and Smad3 proteins may influence the progress of renal interstitial fibrosis,tubular atrophy and inflammatory cell infiltration in UUO model rats.