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-Pl(TGF-Pl) 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 (Thbsl-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-Pl 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-Pl activation and ECM secretion in rat GMC.%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) C5 b-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 1 shRNA(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-1 shRNA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.
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