骨成形蛋白7对人肾小管上皮细胞增殖和转分化的影响  

BMP-7 counteracts TGF0-β1-induced tubular epithelial-myofibroblast transdifferentiation of cultured renal tubular epithelial cells

作　　者：陈楠[1] 李娅[1] 刘峰[1] 严富洪[1] 

机构地区：[1]上海第二医科大学附属瑞金医院肾内科,200025

出　　处：《中华肾脏病杂志》2004年第5期343-346,共4页Chinese Journal of Nephrology

基　　金：上海市科学委员会重大项目基金(03JC14084);上海市卫生局领先专业重点学科资助项目(983009)

摘　　要：目的观察骨成形蛋白7(BMP-7)对TGF-β1诱导的人肾小管上皮细胞转分化的影响,以探讨其延缓小管间质纤维化病变的作用及其可能机制。方法将体外培养的人肾小管上皮细胞分为空白对照组、TGF-β1组、TGF-β1+50ng/mlBMP-7组、TGF-β1+100ng/mlBMP-7组。应用MTT比色法测定BMP-7对人肾小管上皮细胞增殖的作用;间接免疫荧光法测定人肾小管上皮细胞α-SMA、角蛋白、波形蛋白的表达;流式细胞仪检测表达α-SMA阳性的HK-2细胞百分率;RT-PCR检测细胞中α-SMAmRNA的表达。结果与空白对照组相比,不同浓度的BMP-7处理HK-2细胞24、48h后,HK-2细胞均明显增殖(P<0.05)。免疫荧光检测结果显示,经TGF-β1刺激后,角蛋白表达减弱,α-SMA和波形蛋白表达增强;加入50ng/mlBMP-7处理24、48h后,细胞中α-SMA和波形蛋白表达减弱。流式细胞仪检测发现,加入50ng/mlBMP-7处理24、48h后,表达α-SMA的HK-2细胞阳性率逐渐下降,与TGF-β1处理组相比差异有显著性意义(P<0.05)。RT-PCR结果显示,空白对照组无α-SMAmRNA表达,5ng/mlTGF-β1组与空白对照组相比,α-SMAmRNA表达明显上调(P<0.05),而TGF-β1+50ng/mlBMP-7组、TGF-β1+100ng/mlBMP-7组与TGF-β1组相比,α-SMAmRNA表达有下降趋势,但无显著性差异(P>0.05)。Objective To study the possible role of bone morphogenetic protein 7(BMP 7) in the TGF β1 induced tubular epithelial myofibroblast transdifferentiation (TEMT) of cultured renal tubular epithelial cells. Methods The normal rat kidney tubular epithelial cell line (HK 2) was cultured for three days on plastic plates in the presence or absence of recombinant TGF β1 and BMP 7. The alterations in the phenotype were assessed by phase contrast microscopy. Transdifferentiation of tubular cells into myofibroblasts was assessed by immunofluorescence, with monoantibodies to alpha smooth muscle actin (α SMA), vimentin and cytokeratin respectively. The expression of α SMA of HK 2 cells was measured by flowcytometry. The expression of α SMA mRNA of HK 2 cells was assessed with reverse transcriptase polymerase chain reaction (RT PCR). Results Treatment of HK 2 cells with BMP 7(50 and 100 ng/ml) for 24～48 hours increased cellular proliferation. The culture of HK 2 cells in the presence of TGF β1 induced a clear fibroblast like morphology, a loss of the epithelial marker cytokeratin and de novo expression of α SMA and vimentin. Immunofluorescence staining showed the addition of various concentrations of BMP 7 to subconfluent cells for 24 and 48 hours, and the expression of α SMA and vimentin was decreased. There was an increase in the percentage of cells expressing α SMA with TGF β1, which was completed inhibited by an addition of BMP 7(P< 0 05).Conclusions TGF β1 is a key mediator that regulates the transdifferentiation of tubular epithelial cells into α SMA(+) myofibroblast. The ability of BMP 7 to block TGF β1 induced TEMT indicates that BMP 7 treatments may lead to the design of a new therapeutic strategy for renal fibrosis.

关 键 词：TGF-β1 BMP-7 Α-SMA 肾小管上皮细胞 表达 转分化 骨成形蛋白 增殖 波形蛋白 RNA 

分 类 号：R692.6[医药卫生—泌尿科学]