MicroRNA-429对TGF-β1诱导的大鼠胰腺星状细胞活化和上皮-间质转化的调控作用  

Effect of miR-429 on TGF-β1 induced activation and regulation of epithelial mesenchymal transition in rat pancreatic stellate cells

作　　者：徐岷[1] 王国英[1] 赵义[1] 蔡菁[1] 倪鑫[1] 刘鑫[1] 张行行[1] 张尤历[1] 

机构地区：[1]江苏大学附属医院消化内科,江苏镇江212000

出　　处：《中华胰腺病杂志》2015年第3期158-162,共5页Chinese Journal of Pancreatology

基　　金：江苏省自然科学基金（BK20131247）;镇江市“169四期工程”科研项目

摘　　要：目的 探讨miR-429对大鼠胰腺星状细胞（PSCs）活化和上皮-间质转化（EMT）的调控作用.方法 用组织块法提取、培养和分离PSCs,通过免疫荧光染色法检测结蛋白（desmin）、神经胶质原纤维酸性蛋白（GFAP）和α-平滑肌肌动蛋白（α-SMA）的表达鉴定PSCs.取新鲜分离的第2代PSCs,采用脂质体法将miR-429的模拟物（miR-429 mimic）和无意义小分子阴性对照片段（miR-NC）分别转染大鼠PSCs,再用10 μg/L TGF-β1刺激PSCs.采用蛋白质印迹法检测细胞a-SMA、波形蛋白（vimemin）、E-钙黏蛋白（E-cad）及EMT相关蛋白TGF-β1、Smad2/3、p-Smad2/3的表达量;实时荧光定量PCR法检测a-SMA、vimentin、E-cad mRNA和miR-429的表达量.结果 培养后PSCs细胞体积变大,伪足发达,细胞间连接减少,脂滴消失,GFAP、desmin表达阳性,α-SMA表达阴性,符合星状细胞的生物学特征.TGF-β1刺激后,大鼠PSCs α-SMA、vimentin、E-cad蛋白的表达量分别为对照组的（1.369 ±0.878）、（1.518±0.041）、（0.549±0.076）倍,差异均有统计学意义（P值均＜0.05）.转染miR-429 mimic且经TGF-β1刺激后,PSCs的α-SMA、vimentin、E-cad mRNA表达量是转染miR-NC组的（0.423±0.038）、（0.652±0.043）、（2.235±0.045）倍,蛋白表达量是（0.683±0.053）、（0.548±0.049）、（1.548±0.038）倍,差异均有统计学意义（P＜0.05或＜0.01）;TGF-β/Smad通路的TGF-β1、p-Smad2/3的相对表达量是miR-NC组的（0.459±0.038）、（0.525±0.029）倍,差异均有统计学意义（P ＜0.05或＜0.01）.结论 上调miR-429的表达可减弱TGF-β1对大鼠PSCs活化的影响及逆转EMT,其可能的机制是抑制TGF-β1介导的TGF-β/Smad信号转导通路。Objective To investigate the effect of miR-429 on activation of pancreatic stellate cells （PSCs） in rat and regulation of epithelial mesenchymal transition （EMT）.Methods PSCs were isolated and cultured with tissue culture method and desmin,gila1 fibrillary acidic protein （GFAP） and α smooth muscular actin （α SMA） were determined by immunofluorescence staining to identify PSCs.Freshly isolated PSCs of 2nd generation were used.MicroRNA-429 mimic or nonsense small molecular fragments （negative control,NC） were transfected into PSCs by liposomes.10 μg/L TGF-β1 was applied to stimulate PSCs.The expression of α-SMA,vimentin,E-cad and EMT-related proteins,TGF-β1,Smad2/3,P-Smad2/3 protein was measured by Western blot.The expression of α-SMA,vimentin,E-cad mRNA and miR-429 was detected by quantitative real time PCR.Results After culture,the cell volume of PSCs increased,pseudopodia were developed,intercell connections were reduced,and lipid droplets disappeared.GFAP,desmin were positively expressed,and α-SMA was negatively expressed,which were consistent with PSCs biological properties.After TGF-β1 stimulation,the expression of α-SMA,vimentin,E-cad in rat PSCs was （1.369 ± 0.878）,（1.518 ± 0.041）,（0.549 ± 0.076） folds higher than those in control group,and the difference between the two groups was statistically significant （P 〈0.05）.After miR-429 mimic transfeetion and TGF-β1 stimulation,the expression of α-SMA,vimentin,E-cad mRNA was （0.423 ±0.038）,（0.652 ±0.043）,（2.235 ±0.045） folds higher than those in miR-NC group,and the expression of proteins was （0.683 ± 0.053）,（0.548 ± 0.049）,（1.548 ± 0.038）folds higher than those in miR-NC group,and the difference between the two groups was statistically significant （P 〈 0.05 or 〈 0.01）.The relative expression of TGF-β1,p-Smad2/3 were （0.459 ± 0.038）,（0.525 ± 0.029） folds of those in miR-NC group,and the difference between the two groups was statistically significant （P 〈 0.05

关 键 词：微RNAS 转化生长因子β1 胰腺星形细胞 上皮-间质转化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]