Study On Mechanism Of VEGF On The Proliferation On Granulosa Cells

Vascular endothelial growth factor (VEGF) was effect on endothecial cells, promoting endothelial cell proliferation, angiogenesis and changing vascular permeability as a cytokines. A large number of studies showed that the development of ovarian follicle was closely related to angiogenesis around ovarian follicle. Whether VEGF promoted directly or indirectly granulose cell proliferation and differentiation through autocrine or paracrine was still unkown. Therefore, after treatment with different concentrations of VEGF in sheep granulosa cells cultured, the study was known VEGF was effect on granulosa cell proliferation and differentiation, further revealed the mechanism of VEGF on follicular granulosa cells. The optimized culture condition in vitro to sheep granulosa cells was obtained. And it was known that the regulation of proliferation and differentiation on granulosa cells after treatment with different concentrations of VEGF. It was obtained the expression of FSHR and ER after treatment with different concentrations of VEGF in sheep granulosa cells by Western-blot and RT-PCR. The mechanism of VEGF on the proliferation and differentiation on granulosa cells had a systematic study by the granulosa cell proliferation and differentiation and the linear relationship between the expression of FSHR gene act and VEGF.The results showed:1. Granulosa cells were collected from the healthy sheep's follicles, and the primary granulosa cells were cultured and subcultured. The best cultured condition in vitro to Sheep granulosa cells was established:with 5% PAA M199 medium,37.5℃,5% CO2.2. To find out effects of vascular endothelial growth factor on the proliferation activity of sheep granulosa cells, its proliferation were mesured by MTT colorimetric assay, ALPase activity was measured by enzyme kinetic methods. The treatment of vascular endothelial growth factor on sheep granulosa cells had a significantly affect on the proliferation activity by MTT colorimetric assay.In generally, the treatment was lOng/ml and more that could promote the proliferation of granulosa cells. When the treatment was 20ng/ml or more, the change of ALPase activity was not significant by enzyme kinetic methods.3. The expression of FSHR and E2R after treatment with different concentrations of VEGF in sheep granulosa cells was measured by Western-blot and RT-PCR. Vascular endothelial growth factor could promote the expression of FSHR on sheep granulosa cells in vitro from the relationship between VEGF and the expression of FSHR on sheep granulose cells. The increase was significant compared with the control group (P<0.05). When the treatment reached 5ng/ml, the expression of FSHR was significantly higher than the control group. But continue to add to VEGF, the expression of FSHR had little effect. At the same time vascular endothelial growth factor could promote the expression of E2R on sheep granulosa cells in vitro according to the relationship between VEGF and the expression of E2R on sheep granulose cells. The increase was significant compared with the control group (P<0.05). When the treatment reached 20ng/ml, the expression of E2R was significantly higher than the control group, the group of 5ng/ml,10ng/ml,15ng/ml.