| Mammalian ovarian follicles are accompanied by a large number of follicular atresia during development. Follicular atresia is a complex regulatory process with a variety of factors involved in it. Further study for follicular atresia conduce to improve animal breeding rate. Granulosa cells and oocyte apoptosis are the essence of follicular atresia.The atresia of early antral follicles, late preantral follicles and mature follicles is mainly induced by the granulosa cells or membrane cell apoptosis. A large number of studies show that, apoptosis occurs during the each phase of follicle development,in which the granule cell apoptosis is important and direct cause of follicular atresia.The survival and development ability of the granule cell layer is the main determinants to decide the fate of of ovarian follicle.Follicular development and vascular is closely related. Each follicle can independently regulate the vasculogenesis.There is no vascular around small follicles. With the increase of the follicle diameter, peripheral vascular branches gradually increased, in order to supply sufficient nutrients to follicular. The more advanced vascular network provide preferred nutrient to dominant follicle,which is very important for dominant follicle development. The reduction of vascular endothelial cells can lead to the apoptosis of granulosa cells, further to led to the follicular atresia. VEGF is endothelial cell-specific mitogen, which is a strong angiogenic agent to induce vascular hyperplasia. At the same time, VEGF also increases vascular permeability, which is in favor of mass communication. Protein and mRNA of VEGF are mainly expressed in granulosa cells and theca cells of preovulatory follicles. As such, we decided to investigate the feasibility of overexpression VEGF gene in mice ovarian granulosa cells by lentivectors. We expect to provide some details for the in-depth study of VEGF function in mouse ovarian follicles during development. Through this strategy, we first designed different promoter plasmids containing CMV and CYP19. Through intracellular transient transfection in different types of cells, We determined tissue specificity of the CYP19promoter in ovarian granulosa cells. On this basis, we constructed a lentivector pL-CYP19-VEGF which can overexpress VEGF gene. It successfully promot the VEGF expression in granule cells, and the effect is very significant (P<0.01). Then we produced the lentivirus, which is detected the virus titer after concentrated, then the virus was used to infected cells. In this process, we screened out the most suitable amount of virus to infect cells in a tin plate. And we use the most appropriate amount of virus to transfect cells repeatedly. Real-time PCR revealed that the lentiviral can significantly promote the VEGF expression in granule cells (P<0.01). At the same time, we also examined the expression changes of some apoptotic genes in granule cells. We discoveryed that CASPASE-9, CASPASE-3, CASPASE-8expression was significantly decreased (P<0.05), Bcl-2/Bax ratio increased, these results show the resistance to apoptosis.In conclusion, we successfully use a lentiviral vector to delivery the VEGF gene CDS fragment to mouse ovarian granulosa cells, triggering the effective overexpression of VEGF gene, and simultaneously detecting the antiapoptotic phenomenon. Furthermore, the obtained over-expressing lentiviral vectors can be used to produce transgenic mouse, providing the experimental foundation of observing the VEGF effect on follicular developmen in vivo. |
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