Quanititative Determination And Its Siginificance Of Core-banding Factor A1 In Osteosarcoma And Aseptic Necrosis Of Femoral Head

Skeletal development is a complex process, which includes the formation of mesenchymal cell condensation, formation ,growth and remodeling of bones and cartilages. Many orthopedic diseases have some relation with bone formation, such as osteosarcoma, aseptic necrosis of femoral head, ankylosing spondylitis,ossification of posterior longitudinal ligament and osteoporosis. Cbfa1(core binding factora 1), a member of the runt family of transcription factors, is an essential transcription factor in osteoblasts differentiation and bone formation which also plays an important role in chondrocytes maturation and endochondrial ossification.We designed a set of Cbfa1-specific primers and Taqman probe according to the published human Cbfa1 gene sequence. Total DNA was extracted from human peripheral blood lymphocytes and used as template. By PCR , a 200bp target gene was amplified and cloned into EcoR V site of pMD18-T vector. Sequence determination showed that the inserts fragment was the same as the reported Cbfa1 gene fragment. Then we build up a standard curve using the vector as a calibration. Total RNA was extracted from samples of osteosarcoma and aseptic necrosis of femoral head. After RT-PCR, Cbfa1-specific fragment was amplified using cDNA of the samples as templates by LightCycler II(Roche) to examined the expression level of Cbfa1. We found that Cbfa1 mRNA was detected in all samples of osteosarcoma and aseptic necrosis of femoral head. Cbfa1 transcription level of osteosarcoma was 4 times more than in normal lymphocytes of blood which are reported to express 20-fold less Cbfa1 mRNA than normal osteoblast, and transcription level of different kind of osteosarcoma were variable,Cbfa1 expression level of Common central type is higher than that of minicell type. Transcription level of destructcartilages of aseptic necrosis of femoral head was 37 times more than that of normal cartilages. In all, we established a standard curve of Cbfa1, which we utilized to examined the expression level of Cbfa1 in collection of osteosarcoma and aseptic necrosis of femoral head. It concluded that Cbfa1 expression level might have some relation to the level of differentiation and typing of osteosarcoma. In the course of aseptic necrosis of femoral head, Cbfa1 expresses in destruct cartilages might be relevant to osteoarthropathy, which take place after aseptic necrosis of femoral head.