中胚叶叉头-1在骨成形蛋白-2诱导肌胚细胞C2C12转化成骨细胞过程中的作用

为了研究中胚叶叉头-1(MFH-1)基因在骨骼形成和细胞分化中的作用，利用基因重组、杂交瘤技术制作MFH-1单克隆抗体, 利用蛋白质印迹和RNA印迹分析观察了骨成形蛋白-2 (BMP-2)诱导小鼠肌胚细胞C2C12表达MFH-1、产生碱性磷酸酶和骨钙蛋白.小鼠肌胚细胞C2C12低水平地表达内源性MFH-1蛋白以及导入小鼠MFH-1 cDNA的人膀胱癌细胞HTB9也表达小鼠MFH-1蛋白，这种蛋白质定位于细胞核中.用BMP-2处理后, MFH-1蛋白和mRNA在C2C12细胞中的表达显著地增加.用反义MFH-1序列转染小鼠肌胚细胞C2C12可降低内源性MFH-1水平, BMP-2不能诱导导入反义MFH-1序列的肌胚细胞C2C12产生MFH-1蛋白，也不能诱导碱性磷酸酶(ALP)活性和骨钙蛋白量的增加.结果表明, BMP-2诱导的MFH-1蛋白在调节肌胚细胞C2C12向成骨细胞分化方面起关键作用.

The Effects of MFH-1 on BMP-2-induced Osteoblastic Differentiation of C2C12 Myoblasts

In order to investigate the possible role of mesenchyme forkhead-1 (MFH-1) in osteogenesis and osteoblast differentiation, the gene-recombination and hybridization methods are used to produce anti-mouse MFH-1 monoclonal antibody. The expression of MFH-1 induced by bone morphogenetic protein-2 (BMP-2) in myoblasts C2C12 was examined by Western blot and Northern blot analysis. The alkaline phosphatase (ALP) activity and osteocalcin were used as the markers of the osteoblasts lineage, and also were measured. The results showed that the anti-mouse MFH-1 monoclonal antibody was able to identify specifically the mouse MFH-1 protein which was expressed in human bladder carcinoma HTB 9 cell transfected with CX-MFH-1 plasmid by Western blot analysis. The myoblasts C2C12 could express the endogenous MFH-1 protein in its nucleus. MFH-1 protein and MFH-1 mRNA both increased markedly in C2C12 cells after treatment with BMP-2; after lowering the endogenous MFH-1 level by stably transfecting C2C12 cells with antisense MFH-1 sequence, the alkaline phosphatase(ALP) activity and production of osteocalcin induced by BMP-2 were significantly lowered in antisense MFH-1 cell lines than in control cell lines. It can be concluded that the results suggest that the BMP-2-induced MFH-1 protein may play an essential role in regulating the osteoblastic differentiation of myoblasts C2C12.