CAJ | 학술논문

目的探讨Erbin在肾脏间质纤维化中表达量的变化及上调Erbin对转化生长因子β1(TGF-β1)诱导大鼠近端肾小管上皮细胞(NRK52E)转分化的影响.方法体内实验采用SD大鼠5/6肾切除法建立肾纤维化动物模型,收集并榆测各组血清中Scr、BUN水平;Masson染色观察肾间质纤维化程度;免疫组化及Western印迹检测Erbin的分布与表达.体外实验采用TGF-β1(10 μg/L)刺激NRK52E细胞72 h建立上皮细胞-间充质转分化(EMT)细胞模型;免疫荧光及Western印迹法检测E钙黏蛋白(E-cadherin)和α平滑肌肌动蛋白(α-SMA)的表达变化;RT-PCR及Western印迹法检测Erbin的表达变化.用脂质体2000将质粒Prk5-myc-Erbin瞬时转染至NRK52E细胞,Western印迹法观察上调Erbin表达后对上述各种指标的影响.结果 (1)假手术组大鼠肾功能正常[Scr(33.96±7.28)μmol/L、BUN(8.11±2.55)mmol/L],Masson染色未见肾间质纤维化,Erbin在肾小管表达较少;模型组大鼠Scr[(140.52±61.11)μmol/L]、BUN[(34.23±7.66)mmol/L]均显著高于假手术组(均P＜0.05),肾间质可见明显纤维化,Erbin在肾小管表达也明显增加,是假手术组的2.9倍(P＜0.01).(2)正常NRK52E细胞表达E-cadherin,少量表达Erbin和α-SMA.TGF-β1刺激后,NRK52E细胞E-cadherin表达显著减少,Erbin和α-SMA则表达增加(均P＜0.05);而转染质粒Prk5-myc-Erbin可逆转TGF-β1诱导的NRK52E细胞E-cadherin表达下调,并可抑制α-SMA表达上调(均P＜0.05).结论 Erbin在肾间质纤维化中表达增加,上调Erbin表达可抑制TGF-β1诱导NRK52E发生EMT,提示Erbin在肾脏纤维化中可发挥保护作用.
목적 탐토Erbin재신장간질섬유화중표체량적변화급상조Erbin대전화생장인자β1(TGF-β1)유도대서근단신소관상피세포(NRK52E)전분화적영향.방법 체내실험채용SD대서5/6신절제법건립신섬유화동물모형,수집병유측각조혈청중Scr、BUN수평;Masson염색관찰신간질섬유화정도;면역조화급Western인적검측Erbin적분포여표체.체외실험채용TGF-β1(10 μg/L)자격NRK52E세포72 h건립상피세포-간충질전분화(EMT)세포모형;면역형광급Western인적법검측E개점단백(E-cadherin)화α평활기기동단백(α-SMA)적표체변화;RT-PCR급Western인적법검측Erbin적표체변화.용지질체2000장질립Prk5-myc-Erbin순시전염지NRK52E세포,Western인적법관찰상조Erbin표체후대상술각충지표적영향.결과 (1)가수술조대서신공능정상[Scr(33.96±7.28)μmol/L、BUN(8.11±2.55)mmol/L],Masson염색미견신간질섬유화,Erbin재신소관표체교소;모형조대서Scr[(140.52±61.11)μmol/L]、BUN[(34.23±7.66)mmol/L]균현저고우가수술조(균P＜0.05),신간질가견명현섬유화,Erbin재신소관표체야명현증가,시가수술조적2.9배(P＜0.01).(2)정상NRK52E세포표체E-cadherin,소량표체Erbin화α-SMA.TGF-β1자격후,NRK52E세포E-cadherin표체현저감소,Erbin화α-SMA칙표체증가(균P＜0.05);이전염질립Prk5-myc-Erbin가역전TGF-β1유도적NRK52E세포E-cadherin표체하조,병가억제α-SMA표체상조(균P＜0.05).결론 Erbin재신간질섬유화중표체증가,상조Erbin표체가억제TGF-β1유도NRK52E발생EMT,제시Erbin재신장섬유화중가발휘보호작용.
Objective To investigate the expression of Erbin in renal interstitial fibrosis (RIF) and the effect of over-expression of Erbin on transforming growth factor β1 (TGF-(β1)-induced epithelial-mesenchymal transition (EMT) in NRK52E cells. Methods In vivo, the model of renal fibrosis was induced by 5/6 subtotal nephrectomy in rat. Scr and BUN was detected and Masson staining was used to evaluate the level of renal tissue fibrosis. The location and expression of Erbin in renal tissue were detected by immunohistochemistry and Western blotting. In vitro, after NRK52E cells were treated by TGF-β1 (10 μg/L) for 72 h, immunofluorescence and Western blotting were used to obverse the expression and distribution of E-cadherin and α-SMA. The expression of Erbin mRNA and protein were detected by RT-PCR and Western blotting respectively. NRK52E cells were transiently transfected with Prk5-myc-Erbin plasmid via lipofectamine 2000, then the expressions of Erbin, E-cadherin and α-SMA were detected by Western blotting. Results (l)Compared to sham group with Scr (33.96±7.28) μmol/L and BUN (8.11±2.55) mmol/L, rats in 5/6 nephrectomy model with Scr (140.52±61.11) μmol/L and BUN (34.23±7.66) mmol/L revealed renal dysfunction. Masson staining indicated kidney interstitial fibrosis, and the expression of Erbin was significantly increased in renal tissue(2.9 folds), especially in tubular epithelia. (2)In vitro, the expressions of Erbin and α-SMA were markedly increased (2.3 folds and 2.1 folds, P<0.05, respectively) and the expression of E-cadherin was dramatically decreased in NRK52E cells stimulated by TGF-β1, which were consistent with immunofluorescence results. TGF-β1-induced E-cadherin suppression and a-SMA induction could be efficiently blocked by over-expression of Erbin (all P <0.05). Conclusions Erbin is up-regulated in renal interstitial fibrosis, and over-expression of Erbin can partly inhibit renal EMT induced by TGF-β1, which indicates Erbin playing an protective role in renal fibrosis.