Li Ya, Chen Nan, Yu Hai-jin, Dong Xiao-pei, Huang Qiu-hua
Department of Nephrology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Zhonghua Yi Xue Za Zhi. 2006 Feb 28;86(8):544-8.
To investigate the effects of bone morphogenetic protein (BMP)-7 on the extracellular matrix (ECM) accumulation induced by transforming growth factor (TGF)-beta.
Mouse full length BMP-7 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 pcDNA3.1-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-beta, blank plasmid pcDNA3.1, blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta, pcDNA3.1-BMP-7, pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta, 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.
The recombinant plasmid pcDNA3.1-BMP-7 was successfully constructed. The cell mRNA expression levels of Col I and III and FN of the 5 ng/ml TGF-beta group and blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta 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-beta were all significantly lower than those of the TGF-beta group (all P < 0.05). The cell mRNA expression level of Col III of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta was lower, however, not significantly, than that of the TGF-beta group. The supernatant FN levels of the 5 ng/ml TGF-beta group and pcDNA3.1 + 5 ng/ml TGF-beta group were both significantly higher than that of the control group (both P < 0.05), and the supernatant FN level of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta group was significantly lower that that of the 5 ng/ml TGF-beta group (P < 0.05).
Over-expression of BMP-7 significantly inhibits the increased syntheses of collagen I and III, and fibronectin induced by TGF-beta. BMP-7 exerts its antifibrotic effect partially through blocking the TGF-beta-induced accumulation of extracellular matrix in human renal tubular epithelial cells.
研究骨形态发生蛋白（BMP）-7对转化生长因子（TGF）-β诱导的细胞外基质（ECM）积聚的影响。
将小鼠全长BMP-7 cDNA连接到真核表达载体pcDNA3.1中。采用限制性酶切分析和DNA测序来确认所构建的BMP-7表达质粒的准确性。将重组表达质粒pcDNA3.1-BMP-7通过脂质体介导转染到培养的人肾小管上皮细胞系HK-2中。筛选阳性克隆以获得稳定转染的人肾上皮细胞。将这些HK-2细胞培养并分为5组，分别用5 ng/ml TGF-β、空白质粒pcDNA3.1、空白质粒pcDNA3.1 + 5 ng/ml TGF-β、pcDNA3.1-BMP-7、pcDNA3.1-BMP-7 + 5 ng/ml TGF-β处理，另外收集细胞及细胞培养液的上清。通过蛋白质印迹法测定BMP-7蛋白的表达水平。采用逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）法测定不同组人肾小管上皮细胞及上清中Ⅰ型和Ⅲ型胶原（Col）以及纤连蛋白（FN）的mRNA和蛋白表达。
成功构建了重组质粒pcDNA3.1-BMP-7。5 ng/ml TGF-β组和空白质粒pcDNA3.1 + 5 ng/ml TGF-β组细胞中ColⅠ、Ⅲ及FN的mRNA表达水平均显著高于空白质粒pcDNA3.1组、pcDNA3.1-BMP-7组和对照组（均P < 0.05）。pcDNA3.1-BMP-7 + 5 ng/ml TGF-β组细胞中ColⅠ和FN的mRNA表达水平均显著低于TGF-β组（均P < 0.05）。pcDNA3.1-BMP-7 + 5 ng/ml TGF-β组细胞中ColⅢ的mRNA表达水平低于TGF-β组，但差异不显著。5 ng/ml TGF-β组和pcDNA3.1 + 5 ng/ml TGF-β组的上清FN水平均显著高于对照组（均P < 0.05），且pcDNA3.1-BMP-7 + 5 ng/ml TGF-β组的上清FN水平显著低于5 ng/ml TGF-β组（P < 0.05）。
BMP-7的过表达显著抑制TGF-β诱导的Ⅰ型和Ⅲ型胶原以及纤连蛋白合成增加。BMP-7部分通过阻断TGF-β诱导的人肾小管上皮细胞外基质积聚发挥抗纤维化作用。
