骨形成蛋白-7基因转染对人肾小管上皮细胞外基质分泌的影响  

Transfection of recombinant bone morphogenentic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-β

作　　者：李娅[1] 陈楠[1] 俞海瑾[1] 董晓蓓[1] 黄秋花[2] 

机构地区：[1]上海第二医科大学附属瑞金医院肾脏科,200025 [2]上海血液学研究所

出　　处：《中华医学杂志》2006年第8期544-548,共5页National Medical Journal of China

基　　金：上海市科委重大项目基金资助(03JC14084);上海市卫生局领先专业重点学科资助项目(983009)

摘　　要：目的构建骨形成蛋白-7(BMP-7)全长基因表达质粒,观察BMP-7过表达对转化生长因子(TGF)-β诱导的人肾小管上皮细胞细胞外基质分泌(ECM)的作用。方法将BMP-7全长cDNA连接进入真核细胞表达质粒pcDNA3·1中,以脂质体Superfect介导的方法将重组表达质粒pcDNA3·1-BMP-7转染入人肾小管上皮细胞,挑选阳性克隆,以获得稳定转染的人肾小管上皮细胞株。采用Western印迹方法检测BMP-7在肾小管上皮细胞培养上清液中的表达。给予TGF-β(5ng/ml)进行处理,应用RT-PCR和ELISA的方法,观察BMP-7过表达对纤维粘连蛋白(FN)、胶原Ⅰ、Ⅲ(ColⅠ、Ⅲ)等细胞外基质mRNA和蛋白质表达的作用。结果EcoR I限制性酶切鉴定以及DNA双向测序结果均说明所构建的重组质粒为BMP-7全长基因表达质粒。Western印迹方法检测稳定转染人肾小管上皮细胞蛋白表达量明显高于空载体转染组(P<0·05)。TGF-β处理24、48、72h后,5ng/ml TGF-β处理组、空白质粒转染(pcDNA3·1)+5ng/ml TGF-β组ColⅠ、Ⅲ、FN mRNA的表达量明显高于正常对照组,空白质粒转染组(pcDNA3·1)[ColⅠ(A值):0·897±0·100、1·054±0·090vs0·286±0·010、0·319±0·080;ColⅢ(A值):1·114±0·040、0·961±0·090vs0·354±0·020、0·403±0·040;FN(A值):1·257±0·090、1·188±0·060vs0·413±0·020、0·454±0·060,均P<0·05];Col I、FN mRNA表达量pcDNA3·1-BMP-7转染组+5ng/ml TGF-β组明显低于TGF-β处理组(0·591±0·007、0·687±0·020,P<0·05),ColⅢmRNA表达有降低趋势(0·809±0·090),但差异无统计学意义。细胞培养上清液FN含量5ng/ml TGF-β处理组、空白质粒转染(pcDNA3·1)+5ng/mlTGF-β组明显高于正常对照组(P<0·05),而pcDNA3·1-BMP-7转染组+5ng/ml TGF-β组表达明显低于TGF-β处理组(P<0·05)。结论BMP-7过表达可以显著减少TGF-β所致的人肾小管上皮细胞FN、ColⅠ、ⅢmRNA和细胞培养上清液中的FN的表达。表明BMP-7减少小管间质炎症反应和纤维化的发生,改善�Objective To investigate the effects of bone morphogenetic protein （BMP）-7 on the extracellular matrix （ECM） accumulation induced by transforming growth factor （TGF）-β. Methods Mouse full length BMPo7 cDNA was ligated into a eukaryotic expression vector pcDNA3. 1. Restriction enzymatic analyses and DNA sequencing were used to confirm the accuracy of the BMP-7 expressing plasmid thus constructed. The recombinant expression plasmid pcDNA 3.1-BMP-7 was transfected into cultured human renal tubular epithelial cells of the line HK-2 mediated by liposome. Positive clones were selected so as to obtain the human renal epithelial cells with stable transfection. These HK-2 cells were cultured and divided into 5 groups to be treated with 5 ng/ml TGF-β, blank plasmid pcDNA3.1, blank plasmid pcDNA3. 1 ＋ 5 ng/ml TGF-β, pcDNA3.1-BMP-7, peDNA3.1-BMP-7 ＋ 5 ng/ml TGF-β, and an additional grin the cells and the supernatant of the cell culture fluid were collected. The expression level of BMP-7 protein was determined by Western blotting. RT-PCR and ELISA were used to determine the mRNA and protein expression of collagen （Col） Ⅰ and Ⅲ, and fibronectin （FN） in the human renal tubular epithelial cells and supernatant of different groups. Results The recombinant plasmid pcDNA3.1-BMP-7 was successfully constructed. The cell mRNA expression levels of Col Ⅰ and Ⅲ and Ⅲ of the 5 ng/ml TGF-β group and blank plasmid pcDNA3. 1 ＋ 5 ng/ml TGF-β group were all significantly higher than those of the blank plasmid pcDNA3.1 group, pcDNA3.1-BMP-7 group, and control group （ all P 〈 0.05 ）. The cell mRNA expression levels of Col Ⅰ and FN of the pcDNA3.1-BMP-7 ＋ 5 ng/ml TGF-β were all significantly lower than those of the TGF-β group （ all P 〈0.05 ）. The cell mRNA expression level of Col Ⅲ of the pcDNA3.1- BMP-7 ＋ 5 ng/ml TGF-β was lower, however, not significantly, than that of the TGF-β group The supernatant FN levels of the 5 ng/ml TGF-β group and pcDNA3. 1 ＋ 5 ng/ml TGF-β group we

关 键 词：肾小管 上皮细胞 骨形态发生蛋白质类 转染 

分 类 号：R692[医药卫生—泌尿科学]