Effects Of Soy Isoflavones On The Proliferation And Apoptosis Of Yak Ovary Granulosa Cells

Soy isoflavones are phytoestrogens,their chemical structure is similar to mammalian estrogen 17-? estradiol,combined with estrogen receptor(ER),exerts estrogen-like activity,can scavenge free radicals and inhibit peroxidative damage And has a neuroprotective function.Studies have shown that soy isoflavones can promote cell proliferation.In order to further study the effect of soybean isoflavones on the proliferation and apoptosis of yak granulosa cells,this experiment isolated and cultured yak ovary granulosa cells,added different concentrations of daidzein,genistein,17-? estradiol and progesterone,and detected the cells by MTT method Vitality,Elisa method was used to detect the concentration of estrogen and progesterone in granular cell culture fluid,real-time fluorescence quantitative PCR method was used to detect proliferation-related genes AKT1 and apoptosis-related genes Bcl-2,Bax,Bad,p53,Fas,FasL and Caspase in granular cells-3 mRNA expression,the research content is as follows:1.Isolation,culture and identification of yak ovary granular cells.The ovary of healthy female yak was collected,the follicular fluid was aspirated,and the cell suspension was cultured in a constant temperature incubator at 37 ° C and 5% CO2 saturation humidity.FSHR was detected by immunocytochemical methods.Results: The granulocytes isolated and cultured showed a single layer of epithelial growth and adherence.The cell morphology was intact,mostly long spindle-shaped and polygonal.The growth curve showed an "S" shape.Cells grew slowly and were in a latent growth phase until 24 h.Cells developed vigorously and proliferated rapidly from 24 h to 48 h and entered an exponential growth phase.Cells proliferated steadily from 48 h to 120 h and were at a flat top.After 120 h,the density of living cells began to decline,and the cells entered a degenerate phase.2.The effects of daidzein,genistein,17-? estradiol and progesterone on the proliferation and secretion function of granulosa cells.In vitro cultured granulosa cells were added different concentrations of daidzein,genistein,17-? estradiol(concentrations are 1000,2000,3000,4000,5000 pg / mL)and progesterone(concentrations 100,200,400,800,1000 ng / mL)After 48 hours of culture,the cell viability was detected by MTT method.Results: Hormones of different concentrations can increase the viability of granulosa cells.The concentration of daidzein,genistein and 17-? estradiol significantly increased cell viability at 3000 pg / mL.Different concentrations(100,200,400,and 800 ng / mL)of progesterone significantly increased the viability of granulosa cells(P <0.001).The granulosa cells were added with daidzein,genistein and 17-? estradiol(concentration of 3000 pg / mL)and progesterone(100 ng / mL)respectively,and cultured for 48 hours.Cell culture fluid was collected to detect estradiol and Progesterone concentration.Results: Genistein and daidzein had no significant effect on the secretion of estradiol from granulosa cells,and progesterone significantly increased the secretion of estrogen(P <0.05).Genistein significantly reduced the secretion of progesterone by granulosa cells(P <0.05),and estradiol significantly promoted the secretion of progesterone by granulosa cells(P <0.05).3.RT-qPCR method was used to detect the expression of granule cell proliferation and apoptosis related genes.As a result,compared with the control group,genistein significantly promoted the expression of AKT1,Bcl-2,Bax,Fas,and p53,significantly increased the ratio of Bcl/Bax,and significantly reduced the expression of Bad and Caspase-3(P<0.01).Daidzein significantly increased the expression of AKT1,Bcl-2,Bax,Fas,p53,and Caspase-3,significantly increased the ratio of Bcl/Bax,and significantly reduced the expression of Bad.Estradiol significantly increased the expression of p53 and Caspase-3,and significantly reduced the expression of AKT1,Bcl-2,Bax,Bad and Fas(P <0.01).Progesterone significantly increased the expression of Bax and p53,significantly reduced the expression of AKT1,Bcl-2,Bad,Fas and Caspase-3,and progesterone significantly reduced the ratio of Bcl-2/Bax(P<0.01).The above results show that soy isoflavones can promote the proliferation and inhibit apoptosis of yak granule cells cultured in vitro;proliferation is achieved by increasing the expression of the AKT1 gene in the PI3K/AKT signaling pathway.Inhibition of granulocyte apoptosis may be achieved by up-regulating the expression of the anti-apoptotic gene Bcl-2 or down-regulating the expression of the pro-apoptotic genes Bad,Caspase-3 and FasL.3000 pg/mL of estradiol,daidzein,genistein,and 100 ng/mL of progesterone all increase granulocyte viability,so soybean isoflavones have estrogen-like activity.