CAJ | 학술논문

目的探讨α-1,6核心岩藻糖基转移酶(FUT8)小分子干扰RNA( siRNA)对肾小管上皮细胞转化生长因子( TGF) β-Smad2/3信号通路的影响.方法 HK-2细胞共分为6组:正常组、阴性对照组、TGF-β1组、TGF-β1加FUT8干扰组、TGF-β1加阴性对照组、FUT8干扰组.利用外源性TGF-β1激活HK-2细胞TGF-β-Smad2/3信号通路.应用siRNA技术沉默FUT8.用免疫荧光方法测定细胞表面核心岩藻糖链表达；用免疫沉淀、凝集素免疫印迹以及免疫双染的方法测定FUT8基因沉默后TGF-βⅡ型受体(TGF-βRⅡ)、TGF-β Ⅰ型受体(ALK-5)的核心岩藻糖基化变化,并检测Smad2/3蛋白表达和磷酸化(p)-Smad2/3蛋白表达及其核转位的变化.结果与正常组及阴性对照组相比,加入5 μg/L的TGF-β1孵育HK-2细胞48 h,能显著上调TGF-βRⅡ和ALK-5蛋白表达水平(P＜0.05),导致p-Smad2/3表达水平明显升高(P＜0.05),并促使其发生核转位.HK-2细胞表面存在核心岩藻糖链表达.与正常组及阴性对照组相比,TGF-β1孵育后,TGF-βRⅡ与ALK-5两种受体的核心岩藻糖链均显著升高(P＜0.05),FUT8 siRNA能显著抑制TGF-βRⅡ与ALK-5核心岩藻糖链表达(P＜0.05),从而抑制p-Smad2/3表达升高(P＜0.05)及其核转位,但不影响TGF-βRⅡ和ALK-5的蛋白表达(P＞0.05).结论在肾小管上皮细胞中,TGF-βRⅡ和ALK-5蛋白翻译后的核心岩藻糖基化修饰是它们发挥生物学功能所需要的,阻止TGF-βRⅡ和ALK-5的核心岩藻糖基化,能抑制TGF-β-Smad2/3信号转导通路的活化.
목적 탐토α-1,6핵심암조당기전이매(FUT8)소분자간우RNA( siRNA)대신소관상피세포전화생장인자( TGF) β-Smad2/3신호통로적영향.방법 HK-2세포공분위6조:정상조、음성대조조、TGF-β1조、TGF-β1가FUT8간우조、TGF-β1가음성대조조、FUT8간우조.이용외원성TGF-β1격활HK-2세포TGF-β-Smad2/3신호통로.응용siRNA기술침묵FUT8.용면역형광방법측정세포표면핵심암조당련표체；용면역침정、응집소면역인적이급면역쌍염적방법측정FUT8기인침묵후TGF-βⅡ형수체(TGF-βRⅡ)、TGF-β Ⅰ형수체(ALK-5)적핵심암조당기화변화,병검측Smad2/3단백표체화린산화(p)-Smad2/3단백표체급기핵전위적변화.결과 여정상조급음성대조조상비,가입5 μg/L적TGF-β1부육HK-2세포48 h,능현저상조TGF-βRⅡ화ALK-5단백표체수평(P＜0.05),도치p-Smad2/3표체수평명현승고(P＜0.05),병촉사기발생핵전위.HK-2세포표면존재핵심암조당련표체.여정상조급음성대조조상비,TGF-β1부육후,TGF-βRⅡ여ALK-5량충수체적핵심암조당련균현저승고(P＜0.05),FUT8 siRNA능현저억제TGF-βRⅡ여ALK-5핵심암조당련표체(P＜0.05),종이억제p-Smad2/3표체승고(P＜0.05)급기핵전위,단불영향TGF-βRⅡ화ALK-5적단백표체(P＞0.05).결론 재신소관상피세포중,TGF-βRⅡ화ALK-5단백번역후적핵심암조당기화수식시타문발휘생물학공능소수요적,조지TGF-βRⅡ화ALK-5적핵심암조당기화,능억제TGF-β-Smad2/3신호전도통로적활화.
Objective To investigate the effect of FUT8-siRNA on transforming growth factor β (TGF-β)-Smad2/3 signalling pathway in renal tubular epithelial cells. Methods HK-2 cells were divided into six groups:normal group,negative control group,TGF-β1 group,TGF-β1 with FUT8 interference group,TGF-β1 with negative control group,FUT8 interference group.RNAi was performed to silence the expression of FUT8 gene,then immunofluorescent analysis was used to detect the expression of core fucose in the HK-2,immunoprecipitation and lectin blotting were performed to detect the core fucosylation of TGF-βR Ⅱ and ALK-5,and detect the change of Smad2/3 and p-Smad2/3 in HK-2 cells after FUT8 gene was silenced. Results Compared with the normal and negative control group,incubation with 5 μg/L TGF-β1 for 48 h could significantly up-regulate the core fucosylation of HK-2 cells,enhance the protein expression of TGF-βR Ⅱ and ALK-5 (P＜0.05),markedly increase the expression level of p-Smad 2/3 (P＜0.05) and cause it to nuclear translocation in HK-2 cells.While FUT8siRNA could inhibit the above up-regulation of TGF-βR Ⅱ and ALK-5 (P＜0.05),suppress the increase of p-Smad 2/3 (P＜0.05) and its nuclear translocation without disturbing the protein expression of TGF-βR Ⅱ and ALK-5. Conclusion FUT8-catalized core fucosylation of TGF-βR Ⅱ and ALK-5 is needed to fulfill their functions,and blocking core fucosylation of TGF-βR Ⅱ and ALK-5 leads to the inhibition of TGF-β-Smad2/3signalling pathway in HK-2 cells.