上调miR-200 a的表达减弱TGF-β1刺激的大鼠胰腺星状细胞活化和胶原合成

摘要

目的：探讨miR-200a对转化生长因子β1（TGF-β1）刺激的大鼠胰腺星状细胞（PSCs）活化和胶原蛋白合成的影响。方法用组织块法培养分离PSCs，免疫荧光染色检测结蛋白（ 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；取新鲜培养的第2代PSCs，设空白对照组、TGF-β1组、TGF-β1＋miR-NC组、TGF-β1＋miR-200 a mimic组，Western blot法和细胞免疫荧光染色法检测α-SMA和Ⅰ型胶原蛋白（ col-lagen Ⅰ）的表达，荧光定量PCR检测α-SMA、collagen Ⅰ mRNA及miR-200 a的表达。结果 TGF-β1可刺激大鼠PSCs活化及促进胶原蛋白的合成（ P＜0.05），且呈时间依赖性；转染miR-200 a mimic后，在相同浓度的TGF-β1刺激下，α-SMA和collagen Ⅰ的蛋白和mRNA表达明显降低（P＜0.01）。结论上调miR-200a的表达，可减弱TGF-β1对大鼠PSCs活化和胶原蛋白合成的刺激作用，其可能的机制是抑制TGF-β1的生物学作用。%Objective To investigate the effect of miR-200 a mimic on transforming growth factor β1-mediated acti-vation and collagen secretion of rat pancreatic stellate cells .Methods PSCs were isolated and cultured from pan-creatic tissue and identified by desmin , GFAP and α-SMA immunofluorescence staining .PSCs of 2nd generation were divided into control group , TGF-β1 group, TGF-β1+miR-NC group and TGF-β1+miR-200a mimic group.α-SMA and collagen Ⅰ protein were measured by Western blot and immunofluorescence staining .The mRNA ofα-SMA and collagen Ⅰ and the expression of miR-200a were detected by quantitative real-time PCR.Results TGF-β1 stimulates the activation of PSCs and promote collagen synthesis in time-dependment manner ( P<0.05 ) . After transfection of the mimic , treating with the same concentration of TGF-β1, the expressions of protein and mR-NA of both α-SMA and collagen Ⅰ decreases significantly ( P<0.01 ) .Conclusions Over-expression of miR-200 a significantly attenuates α-SMA activity and further affects the collagen synthesis of TGF-β1-dependent activa-tion of PSCs.The mechanisms are potentially related to the biological effects of TGF-β1.