Ghosh-Choudhury N, Windle J J, Koop B A, Harris M A, Guerrero D L, Wozney J M, Mundy G R, Harris S E
Department of Medicine, University of Texas Health Science Center, San Antonio 78284, USA.
Endocrinology. 1996 Jan;137(1):331-9. doi: 10.1210/endo.137.1.8536632.
Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.
能够在体外形成骨组织的成骨细胞系将为理解骨细胞分化过程中的基因表达提供有用的模型。为此，利用一个2.9千碱基的骨形态发生蛋白2（BMP - 2）启动子片段构建转基因小鼠，该启动子驱动猿猴病毒40 T抗原作为转基因。由BMP - 2启动子驱动的猿猴病毒40 T抗原的表达使细胞永生化。从转基因小鼠的颅骨中分离并克隆出了多个成骨细胞系。其中一个克隆的成骨细胞系，称为2T3，已被鉴定并显示能产生矿化骨结节。重组人BMP - 2（rhBMP - 2）可加速这些矿化骨结节的形成。在早期结节形成及随后的矿化过程中，2T3细胞表达碱性磷酸酶、I型胶原、骨钙素和内源性BMP - 2信使核糖核酸（mRNA）的时间顺序与正常新鲜分离的胎鼠颅骨细胞相似。如生长曲线和透射电子显微镜所示，2T3细胞在分化过程中还表现出广泛的生长和多层化。与新鲜分离的胎鼠颅骨细胞一样，1,25 - 二羟维生素D3抑制碱性磷酸酶活性和碱性磷酸酶mRNA表达，但刺激骨钙素mRNA表达。rhBMP - 2还加速了2T3细胞中碱性磷酸酶活性和mRNA、骨钙素mRNA以及BMP - 2 mRNA的表达，同时形成更大且矿化程度更高的骨结节。2T3细胞在mRNA和转录水平表现出自我调节。2T3成骨细胞系为研究成骨细胞分化过程中BMP - 2基因的自我调节及下游基因表达提供了一个系统。2T3细胞可再次克隆并保持其分化能力。
