The Effect Of Different Concentrations Of Estrogen On VEGF Expression In Rat Osteoblasts

Destination:Osteoporosis(OP) Is a metabolic diseases of bone which is characterized by osteopenia and changes of bone microarchitecture. estrogen (ESG) and cell growth factor is very important In the metabolic process of bone tissue. Vascular endothelial growth factor (VEGF) is not only angiogenesis factor but also bone growth factor. The role of VEGF is to regulate the Activity of osteoblast and osteoclast and to adjust their generation and differentiation. The progress of this experiment is as follows:first,osteoblast culture in vitro; second,24 hour excitation of different concentration ESG; Third, to observe the expression of osteoblast VEGF in the way of immunohistochemistry and RT-PCR; At last, approaching the effection of ESG to the expression of osteoblast VEGF.Method:selecting 6 newborn rats aged 48h, removing their craniums, we can get the osteoblast from enzymolysis of these craniums,then,use the osteoblast for culture in vitro. By alkaline phosphatase staining (ALP staining) and mineralized nodules staining we can complete identification of osteoblasts. The 4th generational osteoblasts were randomly divided into 5 groups:one group were treated with 17β-E2 0 mol/L,the second group were treated with 17β-E2 10-10 mol/L,the third group were 10-8 mol/L,the fourth group 10-6mol/L, the fifth group 10-4 mol/L.24h later, we can observe the expression of osteoblast VEGF in the way of immunohistochemistry and RT-PCR.Consequence:1. observing cell shape and identifying cell:we can found from inverted phase contrast microscope that all cells were cultured By 24h after cell inoculation when adherent cell colonies formed, adherent cells display Fusiform-like, triangle or polygon. By alkaline phosphatase staining, Most of the cellular membrane and cytoplasm are visible black-brown granules.when the cells begin calcification, it is the time to stain alizarin Bordeaux,then,we can found the red nodus.2. RT-PCR Testing shows:VEGF express stably in the original generation osteoblast; semiquantitative analysis confirm that different doses of 17β-E2 groups show regular expression;with the dose of 17β-E2 increased, the expression of VEGF gradually increase, the VEGF reach the highest expression when 17β-E2 is 10-6mol/L.Exceeding this dose,the expression of VEGF decreased. Conclusion:VEGF express stably in the original generation osteoblast; ESG can promote the VEGF expression in osteoblast, and this accommodation of expression is dose dependent.