Study On The Apoptosis Of Human Ovarian Luteinized Granulosa Cells And KGN Cell Lines By Dehydroepiandrosterone

Objective: To study the effect of different concentrations of Dehydroepiandrosterone on the apoptosis of human ovarian luteinizing granulosa cells and KGN cell line. Intervention in primary human ovarian luteinizing granulose cells, the expression of mitochondrial apoptotic protein BCL-2,BAX and its mRNA were observed. Verification of the apoptosis pathway through different concentrations of DHEA intervene KGN cell line. Explore the influence of androgen on oocytes.Methods:1. Culture the ovarian luteinizing granulosa cells from normal ovarian reserve function in patients and identifit the cells: Preovulatory granulose cells were collected from follicular fluid obtained from patients of 25-30 years old with normal ovarian reserve accepted undergoing in vitro fertilization-embryo transfer or intracytoplasmic sperm injection from October 2014 to December 2014 in Chengdu University of Tradional Chinese Medicine.The Second Affiliated Hospital Reproductive Center. The granulose cells were isolated and purified by 40% Percoll and identified by FSHR.2. Study the different concentrations of Dehydroepiandrosterone on the apoptosis of human ovarian luteinizing granulosa cells:Granulose cells were cultured for 48 hours with different concentrations of DHEA medium, divided into five groups:negative control group, DHEA0.01μM group, DHEA0.1μM group, DHEA1μM group and DHEA10μM, All groups intervented for 24 hours, The expression of BCL-2.BAX, P-ERK,ERK were detected with Western blot, The expression of theirs mRNAs were detected with QPCR.3. Study the different concentrations of Dehydroepiandrosterone on the apoptosis of KGN:The growth inhibition of DHEA on KGN cell lines by CCK-8. The apoptosis rates were detected by flow cytometry. the detection of the protein expression of ERK,P-ERK,BCL-2,BAX,AR were with Western blot,and the expression of theirs mRNAs with QPCR.Results:1.40% Percoll was used isolate and purify cells to obtained high purity granulosa cells, the newly collected granulosa cells were round, containing coarse particulate matter. Cultured for 24 hours, granule cells have adhered, and presented fusiform.in 48 hours, the cells reached a peak of proliferation with abundant, intracellular particulate matter pseudopodia tight junctions.2. The human ovarian luteinzing granulose cells were cultured in the presence of different concentrations of DHEA. Compared with negative control group the expression of BCL-2 protein has a gradual downward trend. mRNA expression of BCL-2 has a downward trend, but the difference was not statistically significant; BAX protein and mRNA expression presented no significant difference in the control group and intervention group. the expression of ERK was no difference in the intervention group and the control group, The expression of P-ERK in all intervention group were significantly lower than the control group.3. Compared with negative control group, different concentrations of DHEA on KGN cell lines growth inhibition were statistically significant(P<0.05), in which DHEA 10μM inhibition KGN cell lines with the most significant(P<0.01). Apoptosis rate was detected by flow cytometry, the results with increasing concentrations of DHEA,the apoptosis rate increased. There was a significant difference between the DHEA 10μM and the negative control group(P<0.05). The expression of BCL-2 protein and mRNA gradually decreased with the increasing concentration of DHEA. Differences between DHEA10μM group and the negative control group was statistically significang(P<0.05), while the expressions of the protein of BAX, AR and the theirs mRNAs were not statistically significant between the control group and the intervention groups(P>0.05). Compared with the negative control group, the expression of P-ERK in all intervention groups decreased, the difference was statistically significant between the DHEA10μM group and negative control group (P<0.05).Conclusion:1. In this study, the granulose cells in follicular fluid were isolated and purified to obtain high purity granulose cells, it was proved that the methods of separation and purification were feasible.2. The expression of BCL-2/BAX of the DHEA intervention groups reduced, which suggested that DHEA could induce apoptosis of granulose cell through the mitochondrial pathway.3. Different concentrations of DHEA inhibited KGN growthand accelerated KGN apoptosis.these effects may be associated with P-ERK-BCL-2 apoptotic pathway, but not associated with AR. This study was to investigate the effect of DHEA on the granulosa cells with the paitents of normal ovarian reserve function, lay the foundation for the study of preliminary tests in patients with poor ovarian response, provide an experimental basis for clinical application in patients with DHEA.