Effects Of Leptin On Proliferation And Apoptosis Of Porcine Ovarian Granulosacells And Its Mechanism

Leptin is a hormone secreted mainly by white adipose tissue.It can regulate the balance of energy and maintain the stability of body weight.In recent years,a growing body of evidence suggests that leptin can regulate the reproductive function by acting on the hypothalamus pituitary gonad axis.In this study,we investigated the proliferation and apoptosis of ovarian granulosa cells induced by leptin and the secretion of estradiol and progesterone in ovarian granulosa cells stimulated by leptin.The results of the study include the following parts:1.Primary culture and identification of porcine granulosa cells.Obtained granulosa cells with 1mL disposable syringes from follicular fluid in healthy sowabout 100 kg under aseptic condition,put that in 37 ? CO2 saturated humidity incubator,immunofluorescence of FSHR antibody was used to identify the purity of granule cells.Isolated granulosa cells were polygonal and stellate,later gradually transformed into fibrous cells;the purity of the isolated cells was identified by immunofluorescence assay(>98%);the results showed that the growth curve of the cells in the 7 days after the growth of cells in the adherent 48 h to 120 h is in the proliferation of the rising period,120 h growth was decreased.2.Effects of leptin on proliferation,apoptosis and secretion of granulosa cells.0?1 ? 10 ? 50 ? 100ng/mL leptin treatment of ovarian granulosa cells,different concentrations of leptin can stimulate proliferation of granule cells after 24h(P<0.05),only 100ng/mL could significantly promote after 48h(P<0.05);The apoptosis data showed that the early apoptosis of granulosa cells were significantly inhibited with 10 and 100ng/mL leptin(P<0.05),no effect on late apoptosis and total apoptosis;different concentrations of leptin secretion regulation of granulosa cells E2 and P4 had no significant effect.3.The expression of PI3 K,AKT1,AKT2 in ob-Rb and pi3k/akt signaling pathway and the expression of Bcl-2,Bax,Bad and Caspase-3 of apoptosis related factors mRNA and mRNA were detected by real-time fluorescence quantitative PCR.The results show that compared with the control group,treatment group 1,50ng/mL leptin is significantly increased ob-Rb expression of mRNA(P<0.05);10,50ng/mL leptin significantly up-regulated the expression of PI3 K in pi3k/akt signaling mRNA(P<0.05);10ng/mL leptin significantly upregulation of AKT1 expression of mRNA(P<0.05);1ng/mL leptin significantly;upregulated the expression of AKT2 mRNA(P<0.05).Leptin had no significant effect on the expression of Bcl-2 in granulosa cells,1ng/mL leptin can increase the expression of Bax in the cells of mRNA,the rest of the group were decreased Bax expression trend,but the differences were not significant(P>0.05);50ng/mL leptin can significantly increase the ratio of Bcl-2 and Bax(P<0.05);1ng/mL leptin significantly up-regulated the expression of Bad mRNA(P<0.05),10~100ng/mL leptin can significantly down regulate the mRNA expression of Bad(P<0.05).Compared with the control group,10-100ng/mL leptin significantly reduced the expression of Caspase-3 in mRNA(P<0.05).Conclusion: Leptin can stimulate the proliferation of granule cells,inhibit the cell apoptosis may be expressed by acting on pi3k/akt pathway regulates the expression of Bad and Caspase-3,increase the ratio of Bcl-2/Bax to achieve.