The Study Of The Regulation Of Tumor Necrosis Factor Alpha On Growth And Bone Morphogenetic Protein Receptors Of Osteoblasts

Tumor necrosis factor alpha(TNF-α)is a potent stimulator of bone resorption.It is an important factor of Senile Osteoporosis and Alveolar Bone Loss.However,the mechanisms which TNF-αregulates osteoblasts growth are not yet known.This study has examined the regulation of TNF-αon growth and bone morphogenetic protein receptors(BMPRs)of rat osteoblasts to find the mechanisms which TNF-αregulate osteoblasts growth.Objective:Examine the regulation of TNF-αon growth and BMPRs of rat osteoblasts to find the mechanisms which TNF-αregulate osteoblasts growth.Methods:1.Isolation and culture of rat osteoblasts.Osteoblasts were obtained from the calvaria of 7d born neonatal Wistar rat by using primary culture.2.The cell culture.The isolated cells were cultured in DMEM medium until cells were lined up in one layer.The cell were trypsinized and removed from the flask and collected for next step.3.Identification for osteoblasts.Cell morphology on phase-contrast microscope;ALP staining(Gomori ca-co method);bone nodule staining.4.Regulation of TNF-αon growth of rat osteoblasts:To assay osteoblasts proliferation by MTT;to assay ALP activity of osteoblasts by PNPP.5.Regulation of TNF-αon BMPRs of rat osteoblasts.Examined the effects of TNF-αon BMPR-IA,BMPR-IB and BMPR-II expression by RT-PCR,and normalized to that of GAPDH.Results:1.Isolation of osteoblasts.We obtained rat osteoblasts cultured in vitro.And we have identification the Purity of osteoblasts.2.Ragulation of TNF-αon growth of rat osteoblasts.1)To assay osteoblasts proliferation by MYT.Compared with control group,the osteblasts proliferation was decreased in treatment groups.The significantly lower levels of proliferation was observed in above 50ng/ml groups(P＜0.05).2)To assay ALP activity of osteoblasts by PNPP.Compared with control groups,the ALP activity was decreased in treatment groups.The significantly lower levels of ALP activity was observed in aboved 5ng/ml groups(P＜0.05).3)Regulation of TNF-αon BMPRs of rat osteoblasts:The results showed that TNF-αdown-regulated BMPR-IA and BMPR-II transcripts(P＜0.05),it increased the level of BMPR-IB mRNA(P＜0.05).Conclusion:The study shown that TNF-αdown-regulated osteoblasts proliferation and differentiation.And it is possible that they may be negatively controlled by inhibiting the expression of BMPRs.