纤维母细胞生长因子对肾脏足细胞增殖、骨形态蛋白-7以及细胞骨架蛋白表达的调节作用研究  

The regulation effects of fibroblast growth factor on podocyte proliferation and expressions of bone morphogenetic protein-7 and cytoskeletal proteins in podocytes

作　　者：王松[1] 税朝祥 王尧尧 阿拉塔[1] 张爱华[1] 王悦[1] 范敏华[1] 

机构地区：[1]北京大学第三医院肾内科,北京100083 [2]清华大学第一附属医院中心实验室,北京100016

出　　处：《中国血液净化》2008年第5期270-274,共5页Chinese Journal of Blood Purification

基　　金：国家自然科学基金30570853

摘　　要：目的研究纤维母细胞生长因子(fibroblast growth factor,bFGF)对肾脏足细胞增殖、骨形态蛋白-7(bone morphogenetic protein-7,BMP-7)以及细胞骨架蛋白表达的调节作用。方法利用SV40肿瘤抗原转染所获得的永生性小鼠足细胞系培养,以集落形成法和噻唑蓝MTT还原实验测定不同浓度bFGF刺激下的足细胞增殖,采用SDS-聚丙烯酰胺凝胶电泳和Western blotting检测BMP-7、alpha-平滑肌肌动蛋白(α-SMA)、以及波形蛋白(vimentin)的蛋白质水平表达,同时以甲基兰染色观察细胞形态。结果bFGF(1.25～20 ng/ml)处理10天后明显增加足细胞集落生长,并呈现浓度依赖关系(r=0.844),当bFGF达到20ng/ml时,集落数为对照培养的4.2倍,MTT实验亦获得一致的结果。甲基兰染色发现,bFGF刺激后的集落生长更加致密,直径增大,但同时出现集落形成细胞的空泡样改变。Western blotting免疫印迹法检测到,bFGF(1.25～20ng/ml)处理96h后能显著抑制BMP-7的表达并呈浓度依赖关系,20ng/ml bFGF抑制>90%。96h bFGF处理也抑制α-SMA表达,但对vimentin表达无明显作用。结论以上结果表明bFGF刺激足细胞增殖的同时,对BMP-7的表达呈现下调作用,并引起空泡形成及α-SMA减少等细胞内部结构改变。基于BMP-7的肾保护作用,其降低可能是介导bFGF引起肾损伤的重要机制。Objective To investigate the effect of fibroblast growth factor （bFGF） on podocyte proliferation and on expressions of bone morphogenetic protein-7 （BMP-7） and cytoskeletal proteins in podocytes. Method We employed a cell line of mouse podocytes immortalized with the simian virus 40 large tumor antigen gene, and examined the cell proliferation using colony-forming and MTT assays following treatment with various concentrations of bFGF. BMP-7, alpha-smooth muscle actin （α-SMA）, and vimentin in the podocytes were detected by western blot. Methyl blue staining was used for the observation of cell morphology. Results After being treated with 1.25 to 20 ng/ml bFGF for 10 days, the podocytes showed significant increase of colony formation in a dose- dependent manner （r = 0.844）. When bFGF in culture reached 20ng/ml, the number of colonies was 4.2 times higher than that of the controls. Similar results were obtained from MTT assay. On methyl blue staining, the colonies showed larger diameter and contained higher cell density after bFGF stimulation. A part of the cells also had vacuolation change. Western blot demonstrated that bFGF （1.25 to 20 ng/ml） treatment for 96 hours caused the decrease of BMP-7 in a dose-dependent manner, and that 20ng/ml bFGF stimulation resulted in the decrease of BMP- 7 by 〉90%. bFGF treatment for 96 hours also inhibited α-SMA expression, but without significant change in vimentin expression. Conclusion Our data suggest that bFGF induces podocyte proliferation and vacuolation, and down-regulates BMP-7 and α-SMA expressions in podocytes. Based on the renal protective function of BMP-7, the down-regulation of BMP-7 in podocytes by bFGF stimulation may be an important mechanism in mediating bFGFinduced renal injury.

关 键 词：纤维母细胞生长因了 足细胞 细胞骨架蛋白 骨形态蛋白-7 

分 类 号：R692[医药卫生—泌尿科学]