CAJ | 학술논문

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-β1（TGF-β1）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（ColⅠ）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、ColⅠ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。
목적：관찰miRNA재대서신소관상피세포（NRK-52E）발생상피-기성섬유세포전분화（EMT）과정중적변화，사선여신소관상피세포전분화상관적miRNA。방법체외배양NRK-52E，용5 ng/ml전화생장인자-β1（TGF-β1）분별작용0、3、6、12、24、48 h，이0 h위공백조（BC조），여위TGF-β1조。도치상차현미경관찰세포형태학변화；real- time PCR법검측세포내α-평활기기동단백（α- SMA）、E개단백（E- cad）、1형효원（ColⅠ）화섬련단백（FN）적 mRNA표체수평；Western blot법검측세포내α- SMA적표체수평；ELISA법검측상청액중FN적함량；경배실시형광정량PCR법검측6조miRNA재세포내적표체수평。결과여BC조비교，TGF-β1조부분세포실거원유적아란석형태，전변성성섬유세포특유적사형；세포내α- SMA、ColⅠ화FN적mRNA표체상조，E- cad적mRNA표체하조，세포내α- SMA표체량증다，세포상청액중FN함량승고（균P＜0.05）；miR-30d、miR-132화miR-21적표체상조，miR-200b、miR-192화miR-10b적표체하조（균P＜0.05）。결론 TGF-β1가유도NRK-52E발생EMT。miR-30d、miR-132、miR-21、miR-200b、miR-192화miR-10b가능삼여대서신소관상피세포EMT，치득진일보연구。
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 per-formed 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 real- time qPCR. Results Some NRK- 52E cells underwent morphological changes after stimulation of TGF- β1, changed from typical cobblestone shape to spindle- like 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.