沉默血小板反应蛋白-1基因对亚溶解型C5b-9诱导肾小球系膜细胞分泌细胞外基质的影响

Effects of silencing TSP-1 gene on sublytic C5b-9-induced ECM secretion from glomerular mesangial cell

目的研究沉默血小板反应蛋白-1(thrombospondin-1,TSP-1)基因对于亚溶解型(sublytic)C5b-9复合物诱导大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)分泌细胞外基质(extracellular matrix,ECM)的影响。方法设计合成针对TSP-1基因4个靶点的小发夹RNA(small hair RNA,shRNA),并构建相应的表达质粒,筛选沉默效率最佳的TSP-1shRNA(shTSP-1)后,将shTSP-1转染体外培养的大鼠GMC,再给予sublytic C5b-9刺激。以Westernblot检查TSP-1、纤维连接蛋白(fibronectin,FN)及Ⅳ型胶原蛋白(collagenⅣ)的表达情况。另用ELISA法测定培养上清中总转化生长因子-β1(transforming growth factor-β1,TGF-β1)和活化TGF-β1的含量。结果核酸序列分析证明,4种TSP-1shRNA重组质粒均构建成功。Western blot实验证实,构建的shTSP-1-3(Thbs1-3)具有最佳的沉默效率。将shTSP-1-3转染大鼠GMC后,在沉默TSP-1基因表达的同时,能明显抑制由sublytic C5b-9诱导的TGF-β1活化及细胞外基质(FN和collagenⅣ)的合成。结论 TSP-1基因表达在sublytic C5b-9刺激大鼠GMC诱导TGF-β1活化及ECM分泌的过程中,发挥了重要的促进作用。

Objective To explore the effects of silencing thrombospondin-1(TSP-1) gene on sublytic C5b-9-induced extracellular matrix(ECM) secretion from rat glomerular mesangial cell(GMC).Methods Four pairs of small hair RNA(shRNA) were designed against different regions of TSP-1 gene and the corresponding expression plasmids were constructed;the most optimized TSP-1 shRNA(shTSP-1) was then chosen to silence TSP-1 gene in the following experiment.Cultured rat GMC was transfected with shTSP-1 and stimulated with sublytic C5b-9 subsequently.We assessed the levels of TSP-1,FN and collagen IV protein expressions in GMC as well as the contents of total and active transforming growth factor-β1(TGF-β1) in the cultured supernatant of GMC by Western blot and ELISA,respectively.Results Nucleotide sequencing verified that the four kinds of shRNA expression vectors were all constructed successfully.Western blot assay showed that shTSP-1-3(Thbs1-3) could effectively silence the target gene.Transfection of shTSP-1-3 into rat GMC could not only significantly decrease the expression of TSP-1 gene but also reduce sublytic C5b-9-induced activation of TGF-β1 as well as sublytic C5b-9-induced production of FN and collagen Ⅳ induced by sublytic C5b-9.Conclusion TSP-1 gene expression plays an important role in promoting TGF-β1 activation and ECM secretion in rat GMC.