中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2013年
1期
39-43
,共5页
郑晓玉%焦素敏%王力宁%陈莹%杨雪%张瑾%杨爽%高鑫然%范秋灵
鄭曉玉%焦素敏%王力寧%陳瑩%楊雪%張瑾%楊爽%高鑫然%範鞦靈
정효옥%초소민%왕력저%진형%양설%장근%양상%고흠연%범추령
转化生长因子β1%转染%RNA,小分子干扰%肾小球系膜细胞%高糖%信号转导与转录激活子
轉化生長因子β1%轉染%RNA,小分子榦擾%腎小毬繫膜細胞%高糖%信號轉導與轉錄激活子
전화생장인자β1%전염%RNA,소분자간우%신소구계막세포%고당%신호전도여전록격활자
Transforming growth factor beta 1%Transfection%RNA,small interfering%Glomerular mesangial cells%High glucose%Signal transducer and activator of transcription
目的 观察信号转导与转录激活子1(STAT1) siRNA转染后高糖培养人肾小球系膜细胞(HMC)的增殖情况及细胞内STAT1、磷酸化(p)STAT1、STAT3、p-STAT3蛋白及下游TGF-β31的表达变化,探讨高糖状态下STAT1和STAT3活性变化及STAT亚型变化对TGF-β1的影响.方法 设计并合成针对STAT1基因的3个特异性siRNA序列(STAT1-siRNA),应用Lipofectamine 2000转染试剂将STAT1-siRNA转染入HMC.激光共聚焦显微镜鉴定转染效率,应用Western印迹、实时定量PCR法筛选最有效抑制STAT1表达的干扰序列用于后续实验.转染有效干扰序列24 h后,用25 mmol/L葡萄糖刺激24、48、72 h,MTT法检测各组系膜细胞的增殖情况；Western印迹法检测各组细胞STAT1、p-STAT1、STAT3、p-STAT3蛋白的表达；ELISA法检测TGF-β1的表达.结果 与对照组相比,高糖可刺激HMC增殖,HMC的p-STAT1、p-STAT3及TGF-β1表达上调(P＜0.05).转染STAT1-siRNA后高糖刺激HMC,p-STAT3及TGF-β1的表达进一步增高(P＜0.05).结论 高糖可以通过磷酸化方式激活HMC的JAK-STAT信号转导通路.转染STAT1-siRNA后,系膜细胞增殖增多,STAT3活性增强.高糖促进HMC分泌TGF-β1,转染STAT1-siRNA后,TGF-β1分泌进一步增加,与肾脏纤维化有关.
目的 觀察信號轉導與轉錄激活子1(STAT1) siRNA轉染後高糖培養人腎小毬繫膜細胞(HMC)的增殖情況及細胞內STAT1、燐痠化(p)STAT1、STAT3、p-STAT3蛋白及下遊TGF-β31的錶達變化,探討高糖狀態下STAT1和STAT3活性變化及STAT亞型變化對TGF-β1的影響.方法 設計併閤成針對STAT1基因的3箇特異性siRNA序列(STAT1-siRNA),應用Lipofectamine 2000轉染試劑將STAT1-siRNA轉染入HMC.激光共聚焦顯微鏡鑒定轉染效率,應用Western印跡、實時定量PCR法篩選最有效抑製STAT1錶達的榦擾序列用于後續實驗.轉染有效榦擾序列24 h後,用25 mmol/L葡萄糖刺激24、48、72 h,MTT法檢測各組繫膜細胞的增殖情況；Western印跡法檢測各組細胞STAT1、p-STAT1、STAT3、p-STAT3蛋白的錶達；ELISA法檢測TGF-β1的錶達.結果 與對照組相比,高糖可刺激HMC增殖,HMC的p-STAT1、p-STAT3及TGF-β1錶達上調(P＜0.05).轉染STAT1-siRNA後高糖刺激HMC,p-STAT3及TGF-β1的錶達進一步增高(P＜0.05).結論 高糖可以通過燐痠化方式激活HMC的JAK-STAT信號轉導通路.轉染STAT1-siRNA後,繫膜細胞增殖增多,STAT3活性增彊.高糖促進HMC分泌TGF-β1,轉染STAT1-siRNA後,TGF-β1分泌進一步增加,與腎髒纖維化有關.
목적 관찰신호전도여전록격활자1(STAT1) siRNA전염후고당배양인신소구계막세포(HMC)적증식정황급세포내STAT1、린산화(p)STAT1、STAT3、p-STAT3단백급하유TGF-β31적표체변화,탐토고당상태하STAT1화STAT3활성변화급STAT아형변화대TGF-β1적영향.방법 설계병합성침대STAT1기인적3개특이성siRNA서렬(STAT1-siRNA),응용Lipofectamine 2000전염시제장STAT1-siRNA전염입HMC.격광공취초현미경감정전염효솔,응용Western인적、실시정량PCR법사선최유효억제STAT1표체적간우서렬용우후속실험.전염유효간우서렬24 h후,용25 mmol/L포도당자격24、48、72 h,MTT법검측각조계막세포적증식정황；Western인적법검측각조세포STAT1、p-STAT1、STAT3、p-STAT3단백적표체；ELISA법검측TGF-β1적표체.결과 여대조조상비,고당가자격HMC증식,HMC적p-STAT1、p-STAT3급TGF-β1표체상조(P＜0.05).전염STAT1-siRNA후고당자격HMC,p-STAT3급TGF-β1적표체진일보증고(P＜0.05).결론 고당가이통과린산화방식격활HMC적JAK-STAT신호전도통로.전염STAT1-siRNA후,계막세포증식증다,STAT3활성증강.고당촉진HMC분비TGF-β1,전염STAT1-siRNA후,TGF-β1분비진일보증가,여신장섬유화유관.
Objective To observe the cell proliferation and the protein expression of STAT1,phosphorylation of STAT1 (p-STAT1),STAT3,p-STAT3 and transforming growth factor β1 (TGF-β1)in human glomerulur mesangial cells (HMCs) induced by high glucose after STAT1-siRNA transfection.Methods Three STAT1-siRNA sequences were designed and synthetized.HMCs in 6-well plate were transiently transfected with STAT1-siRNA using Lipofectamine 2000.After transfection for 48 h or 72 h,STAT1 mRNA and protein expression were detected by real-time PCR and Western blotting,respectively,to choose the effective sequence in later experiments.After transfection for 24 h and stimulated with 25 mmol/L glucose for 24 h,48 h,72 h,cell proliferation was measured by MTT assays,the protein expressions of STAT1,p-STAT1,STAT3 and p-STAT3 were detected by Western blotting,the expression of TGF-β1 was detected by ELISA in each group.Results High glucose could stimulate HMCs proliferation.The protein expressions of p-STAT1,p-STAT3 and TGF-β1 were increased in the group stimulated by high glucose (P ＜ 0.05).The protein expressions of p-STAT3 and TGF-β1 were further increased in HMCs induced by high glucose after STAT1-siRNA transfection (P ＜ 0.05).Conclusions Under high glucose conditions,JAK-STAT signal transduction pathway of HMCs can be activated,then it is far greater when HMCs are induced by high glucose after STAT1-siRNA transfection.The secretion of TGF-β1 is increased in HMCs under the state of high glucose,and it is further increased after STAT1-siRNA transfection,which is related to the kidney fibrosis.
