骨形成蛋白-2基因转染对成纤维细胞表型影响的实验研究

Experimental Study on the Phenotype of Human Embryonic Lung Fibroblasts Transfected with Ad-BMP-2

目的探讨腺病毒载体介导的骨形成蛋白-2(Ad-BMP-2)基因转染人胚肺成纤维细胞(HELFs)后,对HELFs的表型特别是矿化能力的影响。方法原代培养HELFs,并用Ad-BMP-2进行转染。观察转染前后HELFs的形态学变化;用四唑盐比色法、碱性磷酸酶染色、蛋白印迹以及体外矿化实验,检测转染BMP-2基因后HELFs增殖活性、碱性磷酸酶活性、BMP-2蛋白表达和矿化能力的变化。结果HELFs经Ad-BMP-2转染后,胞体变大,由单一细长梭形转变为多种形态,由单层生长变为复层生长;转染后HELFs增殖受到明显抑制;碱性磷酸酶染色见大部分细胞呈现阳性反应;蛋白印迹检测到明显的BMP-2蛋白条带;细胞经茜素红S染色后出现桔红色的矿化结节。结论经Ad-BMP-2转染后,HELFs向成骨细胞表型转变,具有了矿化能力。

Objective To investigate the effects on the phenotype, especially the mineralization ability of human embryonic lung fibroblasts（HELFs） by transfection with adenovims vector ecoding human bone morphogenetic protein-2 gene （Ad-BMP-2）. Methods The HELFs were primarily cultured, then transfected with Ad-BMP-2. The morphologic characteristics of the cells were observed. The cell proliferation, alkaline phosphatase activities, BMP-2 protein expression, and the mineralization ability were detected with the methods of MTT, ALP staining, Western blot, and alizarin red S staining, respectively. Results After transfection, the shape of HELFs changed from slim spindle to multifigure, the cells became bigger than before. The colonies changed from unilaminar into multilaminar. The proliferation of HELFs was severely inhibited after transfection. An obvious BMP-2 lane was shown in Western blotting. Most cells presented positive in ALP staining, and large number of nacarat mineralized nodes were observed after alizarin red S staining. Conclusion HELFs were capable of transforming into osteoblast-like phenotype, and were endowed with the ability of mineralization while being transfected with Ad-BMP-2.