Culture In Vitro And Identification Of Human Ovarian Granulosa Cells And A Preliminary Of The Effect Of Resveratrol On Its Growth

Part 1The separation,culture and identification of human ovarian granulosa cellsObjective: To establish a stable model of human ovarian granule cell culture in vitro.Method:Separation and purification by density gradient centrifugation of follicular granulosa cell wall layer,in DMEM/F12 culture completely,37? and 5% co2 incubator in the training.Granulosa cells were observed by inverted microscope.Cell immunohistochemical staining was used to identify the expression of follocular estrogen receptor(FSHR)on the surface of granulocytes,and calculate the purity of granulosa cells.The morphology of granulosa cells was obseverd under HE staining.CCK-8 method was used to test the OD value of the granulosa cells of24h-120 h in vitro and to plot the growth cuve.It was divided into two groups:autologous follicle fluid and unautologou follicular fluid,and the growth of each group was observed under a microscope.Results:(1)FSHR were stained in cytoplasm with the positive rate of 90%.(2)The proliferation of the granulosa cells resched the peak during 24 h to 72 h.(3)The growth state of granulosa cells in the experimental group was better than that in the contol group.Conclusion:The stable model of human ovarian granulosa cell culture was established,which laid a good foundation for the in vitro study of granulosa cells.Part 2The effcts of resveratrol on the cell proliferation activity and ATP production of human ovarian granulosa cells in vitroObjective: To explore the effects of resveratrol on the cell proliferation activity and ATP production of human ovarian granulosa cells in vitroMethod: Two age groups were investigated: the young group(<38 years old)and the old group(?38 years old).The mitochondrial ultrastructure of granulosa cell was observed by transmission electron microscopy.A luminometer was used to measure the ATP levels.The experiment was divided into a blank control group,a solvent control group(DMSO30?mol/L),and treatment group(Res 10,20 and 30?mol/L).Cell viability was measured by optical density(OD)values were measured at 450 nm after 24 h and 48 h.Cellular ATP levels of treatment group(Res 10?mol/L)and control group were measured using the ATP Assay Kit after 24 h..Results:(1)There were more abnormal,slender and vacuolate mitochondria in the old group than in the young group.(2)The intracellular ATP level in the young group was higher than that of the old group.(3)Compared with the control group,proliferation activity of the human follicular granulosa cells could be increased by using Res(10,20 and 30?mol/L)for 24 h.(4)ATP levels of treatment group(10?mol/L)were increased compared with control group after 24 h.Conclusion:Resveratrol can promote the proliferation activity and increase the ATP levels of human granulosa cells.