沉默TSP-1基因对亚溶解型C5b-9复合物诱导大鼠肾小球系膜细胞增殖的影响  

Roles of thrombospondin-1 gene in glomerular mesangial cell proliferation induced by sublytic C5b-9

作　　者：吴宜琴[1] 车楠[1] 冯雪凤[1] 邱文[1] 王迎伟[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《江苏大学学报（医学版）》2011年第5期369-374,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(31000396;81072402);江苏省高校自然科学基金资助项目(10KJB310006);南京医科大学科技发展基金面上资助项目(09NJMUM003);南京医科大学基础医学院青年教师培养基金资助项目(09JC007)

摘　　要：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)增殖的影响。方法:设计合成针对TSP-1基因的发夹式siR-NA(small hair RNA,shRNA),并构建相应的表达质粒,将TSP-1 shRNA(shTSP-1)转染体外培养的大鼠GMC,再给予亚溶解型C5b-9刺激。以实时PCR和蛋白质印迹法检查各组GMC中TSP-1、细胞周期蛋白D2(cyclin D2)及增殖细胞核抗原(proliferative cell nuclear antigen,PCNA)的mRNA及蛋白表达情况。另用3H-胸腺嘧啶核苷酸掺入法(3H-thymi-dine incorporation,3H-TdR)和CCK-8(cell counting kit-8)方法分别检查GMC的DNA合成及细胞数量的改变。最后,观察各组GMC中α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达情况。结果:使用shTSP-1沉默TSP-1基因,对于亚溶解型C5b-9刺激GMC后18 h诱导的cyclin D2和PCNA表达及DNA合成均无显著影响,但是shTSP-1能够抑制亚溶解型C5b-9刺激GMC 54 h时细胞数量的增加,同时能下调由亚溶解型C5b-9刺激GMC引起的α-SMA表达。结论:用shTSP-1沉默GMC中的TSP-1基因,可抑制亚溶解型C5b-9刺激GMC的增殖和α-SMA的合成,shTSP-1对GMC增殖的影响可能与其下调α-SMA的表达有一定关系。Objective： To explore the effects of silencing thrombospondin-1（TSP-1） gene on sublytic C5b-9 complex-induced glomerular mesangial cell（GMC） proliferation.Methods： Small hair RNA（shRNA） against TSP-1 gene was designed and the corresponding expression plasmids were constructed.Cultured rat GMC was transfected with TSP-1 shRNA（shTSP-1）,and then stimulated with sublytic C5b-9 complexes.The expression of TSP-1,cyclin D2 and proliferative cell nuclear antigen（PCNA） at mRNA and protein levels in different groups of GMC was assessed by real-time PCR and Western blot respectively.In addition,DNA synthesis and GMC number were detected by using 3H-thymidine incorporation（3H-TdR） and cell counting kit-8（CCK-8）.Finally,the expression of α-smooth muscle actin（α-SMA） in GMC was examined in different groups.Results： TSP-1 gene knockdown by using shTSP-1 in rat GMC had no significant effect on the expression of cyclin D2 and PCNA,and the synthesis of DNA in the GMC induced by sublytic C5b-9 for 18 h.But,shTSP-1 could suppress the increase of GMC number due to sublytic C5b-9 stimulation for 54 h and production of α-SMA at 18 h after sublytic C5b-9 stimulation.Conclusion： Silencing TSP-1 gene in GMC with shTSP-1 can inhibit sublytic C5b-9-induced GMC proliferation and α-SMA production.The proliferation of GMC treated with sublytic C5b-9 was possibly mediated in part by α-SMA production induced by TSP-1 gene activation.

关 键 词：肾小球系膜细胞 亚溶解型C5b-9复合物 血小板反应蛋白-1 增殖 Α-平滑肌肌动蛋白 

分 类 号：R392.31[医药卫生—免疫学]