摘要
目的观察miRNA在大鼠肾小管上皮细胞(NRK-52E)发生上皮-肌成纤维细胞转分化(EMT)过程中的变化,筛选与肾小管上皮细胞转分化相关的miRNA。方法体外培养NRK-52E,用5 ng/ml转化生长因子-13 1(TGF-13 1)分别作用0、3、6、12、24、48 h,以0 h为空白组(BC组),余为TGF-β1组。倒置相差显微镜观察细胞形态学变化;real-time PCR法检测细胞内α-平滑肌肌动蛋白(α-SMA)、E钙蛋白(E-cad)、1型胶原(Col 1)和纤连蛋白(FN)的mRNA表达水平;Western blot法检测细胞内α-SMA的表达水平;ELISA法检测上清液中FN的含量;茎环实时荧光定量PCR法检测6条miRNA在细胞内的表达水平。结果与BC组比较,TGF-β1组部分细胞失去原有的鹅卵石形态,转变成成纤维细胞特有的梭形;细胞内α-SMA、Col I和FN的mRNA表达上调,E-cad的mRNA表达下调,细胞内α-SMA表达量增多,细胞上清液中FN含量升高(均P<0.05);miR-30d、miR-132和miR-21的表达上调,miR-200b、miR-192和miR-10b的表达下调(均P<0.05)。结论 TGF-β1可诱导NRK-52E发生EMT。miR-30d、miR-132、miR-21、miR-200b、miR-192和miR-10b可能参与大鼠肾小管上皮细胞EMT,值得进一步研究。
Objective To screen microRNA （miRNA） related to epithelial- myofibroblast transdifferentiation （EMT） of renal tubular epithelial cells. Methods Normal rat kidney tubular epithelial NRK- 52E cells were cultured and stimulated with 5 ng/ml TGF- β1 for 0, 3, 6, 12, 24 and 48h. Morphological changes of NRK- 52E cells were observed with inverted phase contrast mi-croscope at different time points after stimulation. The expression of α- smooth muscle actin （α- SMA） mRNA, E- cadherin （E- cad） mRNA, col agen I （Col I） mRNA and fibronectin （FN） mRNA was detected with real time qPCR. Western blot was performed to analyze the expression ofα- SMA and ELISA was used to quantitatively detect the FN in the supernatant.Six selected miRNAs were examined by stem- loop realtime qPCR. Results Some NRK- 52E cells underwent morphological changes after stimulation of TGF- β1, changed from typical cobblestone shape to spindlelike in appearance. Compared with BC group, the expression ofα- SMA mRNA, Col I mRNA and FN mRNA was enhanced after stimulation of TGF- β1（P〈0.05）, while the expres-sion of E- cadherin mRNA was decreased （P〈0.05）. Theα- SMA content in cells and FN content in supernatant was significantly increased （P〈0.05） after the stimulation of TGF- β1. Compared with BC group, miR- 30d, miR- 132 and miR- 21 were up- regu-lated （P〈0.05）, while miR- 200b, miR- 192 and miR- 10b were down- regulated （P〈0.05）. Conclusion EMT of NRK- 52E cells can be induced by TGF- β1 stimulation. miR- 30d, miR- 132, miR- 21, miR- 200b, miR- 192 and miR- 10b may be related to EMT in renal tubular cells.
出处
《浙江医学》
CAS
2014年第17期1434-1437,1442,共5页
Zhejiang Medical Journal
基金
温州市科技计划项目(Y20080116)
浙江省自然科学基金资助项目(Y12H050017)
浙江省医药卫生科技计划项目(No.2010KYA135)
