沉默PAX2基因对肾间质纤维化大鼠的影响

Influence of PAX2 gene silencing on renal interstitial fibrosis in rats

摘要:

目的 探讨体内沉默PAX2基因对肾间质纤维化大鼠肾小管上皮细胞转分化(EMT)的影响。方法 64只Wistar大鼠麻醉后,单侧输尿管结扎法制作肾间质纤维化大鼠模型,随机分为阴性对照组和PAX2基因沉默组,每组32只。将200 μL NC-siRNA-in vivo jetPEI^TM混合液转染阴性对照组大鼠,将200 μL PAX2-siRNA-in vivo jetPEI^TM混合液转染PAX2基因沉默组大鼠,各组分别于转染后3、5、7、14 d分为4个亚组,每组8只。留取各组肾组织标本,应用Real-Time PCR及Western blot法检测肾皮质PAX2 mRNA及其蛋白的沉默情况,以及E-钙粘连素(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)mRNA和蛋白表达情况。结果 PAX2基因沉默组PAX2 mRNA和蛋白表达量较阴性对照组均降低 (P < 0.05)。随着梗阻时间的延长,两组E-cadhelin mRNA和蛋白表达量逐渐下降,α-SMA mRNA和蛋白表达量逐渐升高;在转染14 d时,PAX2基因沉默组E-cadhelin mRNA和蛋白相对表达量明显高于阴性对照组 (P < 0.05),而α-SMA mRNA和蛋白相对表达量明显低于阴性对照组 (P < 0.05)。结论 沉默PAX2基因在肾间质纤维化晚期大鼠中可明显抑制肾小管EMT进程,可能对肾间质纤维化有治疗作用。

Abstract:

Objective To investigate the influence of silencing PAX2 gene in vivo on epithelial-mesenchymal transition (EMT) of renal tubular cells in rats with renal interstitial fibrosis. Methods A total of 64 Wistar rats were anaesthetized, and unilateral ureteral obstruction (UUO) was performed to establish a rat model of renal interstitial fibrosis. The 64 rats were randomly divided into negative control and PAX2 gene silencing groups (n = 32 each). The rats in the control group were transfected with 200 μL NC-siRNA-in vivo jetPEI^TM solution. Those in the PAX2 gene silencing group were transfected with 200 μL PAX2-siRNA-in vivo jetPEI^TM solution. Each group was further divided into 4 subgroups based on the post-transfection time (3, 5, 7 and 14 days after transfection), with 8 rats in each subgroup. Renal tissue samples were harvested in each group. Real-time PCR and Western blot were used to measure the mRNA and protein expression of PAX2 in the renal cortex, as well as the mRNA and protein expression of E-cadherin and α-SMA. Results Compared with the control group, the PAX2 gene silencing group showed significantly lower mRNA and protein expression of PAX2 (P<0.05). In the two groups, the mRNA and protein expression levels of E-cadherin were gradually reduced over the time of obstruction, while those of α-SMA gradually increased. At 14 days after transfection, the PAX2 gene silencing group had significantly higher mRNA and protein expression of E-cadherin but lower mRNA and protein expression of α-SMA compared with the control group (P<0.05). Conclusions PAX2 gene silencing can significantly inhibit the process of EMT of renal tubular cells in rats with advanced fibrosis, suggesting that PAX2 gene silencing may have a therapeutic effect on renal interstitial fibrosis.