Jiang Xin-quan, Zhang Zhi-yuan, Chang Qing, Chen Jian-guo, Zhou Xiao-jian, Chen Wan-tao
Department of Oral and Maxillofacial Surgery, Ninth People's Hospital, Shanghai Second Medical University, Shanghai 200011, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2003 Feb;25(1):13-6.
Bone marrow stromal cells (MSCs) were transfected with human bone morphogenetic protein-4 (hBMP-4) gene in vitro to provide BMP gene modified cells for tissue-engineered bone.
MSCs were cultured and transfected with pEGFP-hBMP4, pEGFP plasmids respectively or left uninfected as control. Transcription of BMP-4 gene as well as gene transfection efficiency was tested. Morphological and growth feature of the transfected cells were valued. Alkaline phosphatase (ALP), von Kossa, and Osteocalcin (OC) were tested to determine the phenotypes of osteoblast.
The gene transfection efficiency was 20%-30%, based on GFP expression. RT-PCR showed that MSCs had low transcription of BMP-4 that was enhanced by the gene transfer. Morphological feature of MSCs transfected with pEGFP-hBMP-4 changed but growth curves did not show much difference among the groups. In pEGFP-hBMP-4 group, ALP positive stain area and the number of calcium nodules were increased, as well as the expression of OC.
A high transfer efficiency of MSCs was achieved under optimized conditions. The gene transfer technique strengthened the transcription of BMP-4 and promoted differentiation from MSCs to osteoblasts. hBMP-4 transferred MSCs may serve as an ideal cell source for tissue-engineered bone.
体外将人骨形态发生蛋白-4（hBMP-4）基因转染至骨髓基质细胞（MSCs），为组织工程骨提供BMP基因修饰细胞。
分别用pEGFP-hBMP4、pEGFP质粒转染培养的MSCs或不感染作为对照。检测BMP-4基因转录及基因转染效率。评估转染细胞的形态和生长特性。检测碱性磷酸酶（ALP）、冯库萨染色及骨钙素（OC）以确定成骨细胞表型。
基于绿色荧光蛋白（GFP）表达，基因转染效率为20%-30%。逆转录聚合酶链反应（RT-PCR）显示MSCs中BMP-4转录水平低，基因转染可增强其转录。转染pEGFP-hBMP-4的MSCs形态特征改变，但各组生长曲线差异不大。在pEGFP-hBMP-4组中，ALP阳性染色面积、钙结节数量以及OC表达均增加。
在优化条件下实现了MSCs的高转染效率。基因转染技术增强了BMP-4转录，促进了MSCs向成骨细胞的分化。转染hBMP-4的MSCs可能成为组织工程骨的理想细胞来源。
