骨形态发生蛋白3基因转染成纤维细胞后细胞形态及生物学变化

Morphological and biological changes of fibroblasts after bone morphogenetic protein 3 transfection in vitro

目的 通过基因转染方法 ,用骨形态发生蛋白 3(BMP3 )基因转染成纤维细胞 (NIH 3T3细胞 ) ,观察转染不同时期被转染细胞的形态学改变及细胞内碱性磷酸酶含量的变化 ,初步探讨骨组织工程种子细胞来源的另一途径。方法 定向克隆法将BMP3 基因构建于PcDNA3表达载体 ,脂质体介导法转染NIH 3T3细胞 ,G418筛选获得稳定转化体 ,观察不同时期被转染细胞的形态学变化 ,钙钴法染色转染细胞不同时相的碱性磷酸酶并作图像分析。结果 转染细胞培养筛选 6周提取的总RNA用Dotblot可以检出有明显的BMP3 mRNA表达。转染细胞第 1周细胞形态由长梭形变为杆状 ,转染后 2周细胞由长梭形变为多角形 ,转染后 4周细胞变为多边形 ,类似成骨细胞 ,钙钴法碱性磷酸酶染色示转染组细胞胞浆内有大量染成黑色的碱性磷酸酶颗粒 ,及图像分析示转染细胞内碱性磷酸酶明显高于同期对照组 ,两组间有显著性差异 (P <0 .0 1)。结论 通过向NIH 3T3细胞内转染BMP3 基因 ,可以使NIH 3T3细胞形态向成骨细胞形态发生转化 ,被转染细胞内碱性磷酸酶水平明显增高 ,已具有成骨细胞的某些特性。

Objective To explore the morphology, phenotype, biological characters and alkaline phosphatase (ALP) level of fibroblast cell line NIH-3T3 transfected with bone morphogenetic protein 3 (BMP 3) gene. Method A recombined BMP 3-PcDNA3 cDNA was obtained by using gene recombination technique to clone the BMP 3 cDNA into PcDNA3 vector directly, and then introduced into NIH-3T3 cells by means of lipofectamine transfer. Cell morphology was observed with microscopy and biological characters with Gomori-Takamatsu method to check ALP activity in the cells. Results BMP 3 mRNA expression was found in the transfected NIH-3T3 cells 6 weeks after transfection with the cell shape changed from fusiform to bacilliform in the first week, bacilliform to multiangular in the second week, and multiangular to polygonal, as osteoblast, in the fourth week. ALP levels was significantly higher in NIH-3T3 cells with BMP 3 transfection than normal NIH-3T3 cells cultured in the same periods ( P <0.01). Conclusion The phenotype of NIH-3T3 cells change to osteoblasts gradually after BMP 3 gene transfection with some osteoblast characters and higher ALP Levels than the cells without transfection.