CAJ | 학술논문

目的观察高葡萄糖作用下蛋白激酶C(PKC)δ对人肾小管上皮细胞株(HK-2)p66Shc转录后蛋白磷酸化及线粒体转位的影响.方法以HK-2细胞作为体外观察的细胞,采用不同浓度的D-葡萄糖进行刺激,分别用实时定量PCR(RT-PCR)和Western印迹法检测PKCδ和p66Shc基因表达及蛋白磷酸化水平;然后将HK-2细胞分为3组:5 mmol/L低葡萄糖组、30 mmol/L高葡萄糖组、30 mmol/L高葡萄糖+粗糠紫苦素(Rottlerin) 1.0 μmol/L组.采用Western印迹法和细胞免疫荧光方法检测p66Shc蛋白磷酸化水平及其线粒体转位情况.结果高葡萄糖处理HK-2细胞后,PKCδ和p66Shc mRNA及蛋白磷酸化水平明显高于对照组[其中PKCδ mRNA及其磷酸化蛋白的表达量随着D-葡萄糖浓度(15、30、45 mmol/L)的增加分别上调0.9、1.3和1.6倍及0.4、1.5和2.0倍,均P＜0.05],且呈剂量和时间依赖;高糖+Rottlerin组p66Shc蛋白磷酸化水平及其线粒体转位明显低于对照组,(高糖+ Rottlerin组在180 min时线粒体p66Shc磷酸化蛋白表达量下调3.1倍,P＜0.01).结论高葡萄糖可诱导HK-2细胞PKCδ和p66Shc的转录及磷酸化,PKCδ可促进高葡萄糖诱导的p66Shc磷酸化及其线粒体转位.
목적 관찰고포도당작용하단백격매C(PKC)δ대인신소관상피세포주(HK-2)p66Shc전록후단백린산화급선립체전위적영향.방법 이HK-2세포작위체외관찰적세포,채용불동농도적D-포도당진행자격,분별용실시정량PCR(RT-PCR)화Western인적법검측PKCδ화p66Shc기인표체급단백린산화수평;연후장HK-2세포분위3조:5 mmol/L저포도당조、30 mmol/L고포도당조、30 mmol/L고포도당+조강자고소(Rottlerin) 1.0 μmol/L조.채용Western인적법화세포면역형광방법검측p66Shc단백린산화수평급기선립체전위정황.결과 고포도당처리HK-2세포후,PKCδ화p66Shc mRNA급단백린산화수평명현고우대조조[기중PKCδ mRNA급기린산화단백적표체량수착D-포도당농도(15、30、45 mmol/L)적증가분별상조0.9、1.3화1.6배급0.4、1.5화2.0배,균P＜0.05],차정제량화시간의뢰;고당+Rottlerin조p66Shc단백린산화수평급기선립체전위명현저우대조조,(고당+ Rottlerin조재180 min시선립체p66Shc린산화단백표체량하조3.1배,P＜0.01).결론 고포도당가유도HK-2세포PKCδ화p66Shc적전록급린산화,PKCδ가촉진고포도당유도적p66Shc린산화급기선립체전위.
Objective To observe the effect of PKCδ on the phosphorylation of p66Shc and its mitochondrial translocation in human proximal tubular cells (HK-2) under high glucose (HG).Methods HK-2 cells were incubated with different concentrations of D-glucose (5-45 mmol/L) for indicated time (0-48 h).Then the mRNA expressions of PKCδ and p66Shc and the phosphorylation levels of PKCδ (p-PKCδ) and p66Shc (p-p66Shc) were determined by real-time polymerase chain reaction (PCR) and Western blot analysis respectively.In addition,the effect of PKCδ inhibitor on the phosphorylation and mitochondrial translocation of p66Shc in HK-2 cells exposed to HG was also observed.HK-2 cells were divided into 3 groups of 5 mmol/L glucose,30 mmol/L glucose and 30 mmol/L glucose + 1.0 μmol/LRottlerin.Cell immunofluorescence and Western blotting were used to observe the phosphorylation and mitochondrial translocation of p66Shc.Results Both mRNA expression and phosphorylation level of p66shc and PKCδ significantly increased in HK-2 cells after exposure to HG (15,30,45 mmol/L).And it was in a concentration-and time-dependent manner as compared with control group (up-regulated 0.9,1.3 and 1.6-fold in mRNA of PKCδ,0.4,1.5 and 2.0-fold in protein of p-PKCδ respectively (all P ＜ 0.05).PKCδ inhibitor Rottlerin dramatically inhibited the phosphorylation and mitochondrial translocation of p66Shc induced by HG in HK-2 cells (down-regulated 3.1 folds in protein of p-p66Shc in mitochondria,P ＜0.01).Conclusions HG increases the transcription and phosphorylation of PKCδ and p66Shc in HK-2 cells.And PKCδ may modulate the phosphorylation and mitochondrial translocation of p66Shc under HG.