CAJ | 학술논문

目的研究尿酸对肾小管上皮细胞间充质转分化的影响及microRNA-101a在其中的作用。方法体外培养大鼠肾小管上皮细胞（NRK-52E），分别以0、10、100、500μmol/L的尿酸诱导细胞，采用实时定量PCR检测各组α-平滑肌肌动蛋白（α- SMA）、I型胶原（ColⅠ）及E钙蛋白mRNA的表达水平；Wersten blot检测各组中α- SMA的表达，免疫荧光检测各组中胶原蛋白I（COL）表达水平，观察细胞是否发生转分化。再用实时定量PCR检测各组中miR-101a及COX-2mRNA的表达水平，Wersten blot检测各组中COX-2的表达。进一步实验选择100μmol/L尿酸诱导细胞，并转染miR-101a mimics，分为3组，miR-101a组：先转染miR-101a mimics，再以100μmol/L尿酸培养基培养；空白对照组：以100μmol/L尿酸培养基培养；阴性对照组：细胞转染随机合成的miRNA NC片段，再以100μmol/L尿酸培养基培养。检测各组中α- SMA、ColⅠ、E钙蛋白及COX-2mRNA的表达量。结果培养基中尿酸浓度越高，α- SMA、ColⅠ表达水平也越高，E钙蛋白越低，提示细胞发生转分化，同时miR-101a表达减少，COX-2mRNA表达增多。miR-101a组与空白对照组和阴性对照组比较，α- SMA、ColⅠ及COX-2的表达明显降低（P＜0.05），E钙蛋白明显升高（P＜0.05），空白对照组与阴性对照组间差异无统计学意义（P＞0.05）。结论尿酸诱导肾小管上皮细胞转分化可能呈浓度依赖性， miR-101a及COX-2可能参与尿酸诱导肾小管上皮细胞转分化的调节过程。
목적연구뇨산대신소관상피세포간충질전분화적영향급microRNA-101a재기중적작용。방법체외배양대서신소관상피세포（NRK-52E），분별이0、10、100、500μmol/L적뇨산유도세포，채용실시정량PCR검측각조α-평활기기동단백（α- SMA）、I형효원（ColⅠ）급E개단백mRNA적표체수평；Wersten blot검측각조중α- SMA적표체，면역형광검측각조중효원단백I（COL）표체수평，관찰세포시부발생전분화。재용실시정량PCR검측각조중miR-101a급COX-2mRNA적표체수평，Wersten blot검측각조중COX-2적표체。진일보실험선택100μmol/L뇨산유도세포，병전염miR-101a mimics，분위3조，miR-101a조：선전염miR-101a mimics，재이100μmol/L뇨산배양기배양；공백대조조：이100μmol/L뇨산배양기배양；음성대조조：세포전염수궤합성적miRNA NC편단，재이100μmol/L뇨산배양기배양。검측각조중α- SMA、ColⅠ、E개단백급COX-2mRNA적표체량。결과배양기중뇨산농도월고，α- SMA、ColⅠ표체수평야월고，E개단백월저，제시세포발생전분화，동시miR-101a표체감소，COX-2mRNA표체증다。miR-101a조여공백대조조화음성대조조비교，α- SMA、ColⅠ급COX-2적표체명현강저（P＜0.05），E개단백명현승고（P＜0.05），공백대조조여음성대조조간차이무통계학의의（P＞0.05）。결론뇨산유도신소관상피세포전분화가능정농도의뢰성， miR-101a급COX-2가능삼여뇨산유도신소관상피세포전분화적조절과정。
Objective To investigate the role of microRNA- 101a (miR- 101a) in epithelial- mesenchymal transition (EMT) of rat renal tubular epithelial cel s induced by uric acid. Methods Cultured renal tubular epithelial NRK- 52E cel s were treated with medium containing various concentration of uric acid (0μmol/L, 10μmol/L, 100μmol/L, 500μmol/L). The expres-sion ofα- smooth muscle actin(α- SMA) mRNA and protein was detected by real time PCR and Western Blot, expression of col-lagen I (ColⅠ) mRNA and protein by real time PCR and immunofluorescence, and expression of E- cadherin mRNA by real time PCR. The expression of miR- 101a, cycloxygenase- 2(COX- 2) was detected by real time PCR or Western Blot. Before treated with 100μmol/L uric acid, the cultured NRK- 52E cel s were transfected with miR- 101a (miR- 101a group), transfected with randomly synthesized miRNA (negative control group) or not transfected (blank control group). Real time PCR was preformed to examine efficiency of transfection. Then the expression ofα- SMA, ColⅠ, E- cadherin and COX- 2 was determined. Results With the in-creasing of uric acid concentration, the expression of α- SMA and ColⅠ was increased,and E- cadherin decreased, while the expression of miR- 101a decreased and COX- 2 increased. The expression of α- SMA, ColⅠ and COX- 2 in miR- 101a group was significantly decreased, E- cadherin increases, compared with blank control group and negative control group (P<0.05), while there was no significant difference between the latter two groups. Conclusion Uric acid induces EMT of rat real tubular ep-ithelial NRK- 52E cel s in a concentration- dependent manner, miR- 101a and inflammation may be involved in EMT of NRK- 52E cel s induced by uric acid.