Isolation And Identification Of Exosomes From Porcine Follicular Fluid And Their Effect On Granulosa Cell Proliferation

In order to improve the reproductive performance of sows,there was a need to fully grasp the physiological mechanisms underlying their follicular development.Exosomes and ovarian granulosa cells play crucial roles in follicle development,but little research has been done on the regulatory mechanisms of exosome-mediated ovarian granulosa cell proliferation.We isolated follicular fluid exosomes by ultra-high speed centrifugation,observed the morphology of exosomes by transmission electron microscopy,identified the diameter size and concentration of particles by nanoparticle tracer technique and detected the expression of marker proteins of exosomes by western bolt;added the isolated exosomes into ovarian granulosa cells and explored the proliferative effect on granulosa cells by using CCK8 to detect cell viability;exosomes,Small RNA transcriptome sequencing(Small RNAseq)was performed in the exosome and granulosa cell co-culture groups to screen for miR-200 b,a key miR-200 b affecting granulosa cell proliferation;miR-200 b was overexpressed and miR-200 b expression was inhibited in granulosa cells,and experimental assays by CCK8,Ed U,and q RT-PCR were used to explore whether miR-200 b could promote proliferation of ovarian granulosa cells.The results are as follows:1.The isolated follicular fluid exosomes were round cups.The diameter is about 106 nm;Exosome marker proteins CD9 and CD63 were expressed.2.The addition of exosomes can promote the proliferation of porcine ovarian granulosa cells.The cell viability of the exosome concentration of 120 μg/m L group was significantly increased compared to the blank group(P<0.01).3.Through analysis of sequencing results,49 differentially expressed miRNAs were found in the Granulosa cell group,the co-culture group of exosome and granulosa cells,among which 41 miRNAs were up-regulated and 8 miRNAs were down-regulated.Target genes of 49 miRNAs were predicted,and 6033 target genes were obtained.GO and KEGG annotation of target genes showed that 272 GO items and 57 pathways were significantly enriched.Among them,the signal pathways related to granulosa cell proliferation and apoptosis included c AMP signal pathway,MAPK signal pathway,m TOR signal pathway,Ras signal pathway,etc.The candidate miRNAs were identified as miR-200 b,miR-202-3p,miR-371-5p and miR-141.4.The cell viability of the NC group was significantly enhanced by constructing mimics vector and overexpressing miR-200b(P<0.01);the cell viability of the NC group was significantly decreased by constructing inhibitor vector and inhibiting miR-200 b expression(P<0.01);the relative expression of cell cycle-related genes CCND1 was significantly increased relative to the control group(P< 0.01);the relative expression of steroid hormone synthesis-related genes HSD3B1 and CYP11A1 were significantly increased relative to the control group(P<0.01).