| Exosome is a kind of vesicle secreted by a variety of cells with a diameter of about 40 ~ 150 nm,the surface is rich in lipid substances,which contains a variety of mRNA,miRNA,DNA,protein,cytokines and other organisms Active substance.At first,it was considered to be cell debris,so people's understanding of exosomes is insufficient,but now exosomes are increasingly regarded as important carriers of intercellular communication and circulating biomarkers,which can be used for disease diagnosis and prognosis.Great clinical potential.Exosomes are found in various body fluids,including blood,emulsion,urine,etc.However,there are few studies on follicular fluid exosomes.The impact of exosomes on granulosa cells has not been reported.This study will To explore this,the main content and results are as follows:1.Isolation and identification of porcine follicular fluid exosomesIn this experiment,the ultracentrifugation method was used to separate the exosomes of follicular fluid.The 170,000 g ultracentrifugation successfully separated the exosomes.The morphology,size and marker proteins of the isolated exosomes were identified by scanning electron microscope,nanoparticle tracing technology and Western blot.The results showed that the exosomes isolated by ultracentrifugation were in the shape of a pie with a particle size range of30-100 nm.TSG101;CD63;CD9 protein were all expressed.2.Uptake and suppression of porcine follicular fluid exosomesIn this experiment,confocal laser microscopy was used to observe the phagocytosis of exosomes by granulosa cells.Exosomes were labeled with PKH26 for red fluorescence,FITC-phalloidin labeled cell microfilaments for green fluorescence,and cell nuclei were labeled with DAPI for blue fluorescence.The results showed that red fluorescent exosomes entered the cell and were distributed around the blue nuclei.The granulosa cells were treated with clathrin-dependent endocytosis inhibitor(chlorpromazine)and exosomes.Observing the distribution of exosomes in the cells,compared with the exosomes alone treatment group,the results showed that after chlorpromazine treated granulosa cells,exosome phagocytosis was significantly inhibited.The results indicate that exosomes enter granulosa cells through clathrin-dependent endocytosis.3.The effect of exosomes on the proliferation and progesterone secretion of porcine follicle granulosa cells through clathrin-dependent endocytosisIn this experiment,chlorpromazine was used to inhibit clathrin-dependent endocytosis.Exosomes and chlorpromazine were treated separately and at the same time to treat granulosa cells,and then the proliferation of granulosa cells and the secretion of progesterone were observed.The experimental results show that,compared with the exosome-treated group,the cell viability andthe proportion of S-phase cells in the exosome + chlorpromazine-treated group were significantly reduced(p<0.01),and the amount of progesterone secretion was also very significant Decrease(p<0.01).The experiment further verified the changes in gene and protein expression levels.Comparison of the results of each group showed that exosomes promoted the proliferation of porcine follicular granulosa cells and the secretion of progesterone by clathrin-dependent endocytosis.4.The effect of exosomes on the proliferation and progesterone secretion of porcine follicular granulosa cells through the MAPK/ERK1/2 signaling pathwayIn this experiment,exosomes were treated with granulosa cells at six time points of 0,5,15,30,60 and 120 min,and then Western blot was used to detect the phosphorylation level of ERK1/2.The experimental results showed that the phosphorylation level of ERK1/2 increased significantly at 5 and 15 minutes(p<0.01).Granulocytes were treated with PD98059 and chlorpromazine together with exosomes,and the phosphorylation level of ERK1/2 was detected after 15 minutes of treatment.The experimental results show that,compared with the exosome-treated group,the ERK1/2 phosphorylation level in the exosome+chlorpromazine-treated group and the exosome+PD98059-treated group was significantly reduced(p<0.01).After treating granulosa cells with PD98059 and exosomes individually and together,the proliferation of granulosa cells and the secretion of progesterone were detected.The experimental results show that,compared with the exosome treatment group,the cell viability of the exosome + PD98059 treatment group was significantly reduced(p<0.05),and the proportion of S-phase cells was significantly reduced(p<0.01).The amount of ketone secretion decreased significantly(p<0.05).The detection of gene and protein expression levels in each group showed that exosomes promoted the proliferation of porcine follicular granulosa cells and the secretion of progesterone through the MAPK/ERK1/2signaling pathway.5.The effect of exosomes on the proliferation and progesterone secretion of porcine follicular granulosa cells through the PI3K/AKT signaling pathwayIn this experiment,exosomes were treated with granulosa cells at six time points of 0,5,15,30,60 and 120 min,and then Western blot was used to detect the phosphorylation level of ERK1/2.The experimental results showed that the phosphorylation level of AKT increased significantly at15 minutes(p<0.01).Granular cells were treated with LY294002 and chlorpromazine together with exosomes,and the phosphorylation level of AKT was detected after 15 minutes of treatment.The experimental results showed that,compared with the exosome-treated group,the AKT phosphorylation level in the exosome+chlorpromazine-treated group and the exosome+LY294002-treated group was significantly reduced(p<0.01).After treating granulosa cells with LY294002 alone and together with exosomes,granulosa cell proliferation and progesterone secretion were detected.The experimental results show that,compared with the exosome-treated group,the cell viability of the exosome+LY294002-treated group was significantly reduced(p<0.05),and the proportion of S-phase cells was significantly reduced(p<0.01).The amount of ketone secretion decreased significantly(p<0.05).Examination of the gene and protein levels of each group found that exosomes promoted the proliferation of porcine follicular granule cells and the secretion of progesterone through the PI3K/AKT signaling pathway.In summary,the results of this study indicate that exosomes exist in the follicular fluid of pigs.Exosomes derived from follicular fluid can promote the proliferation of granulosa cells and the secretion of progesterone in swine follicles.Further research found that exosomes enter granulosa cells through clathrin-dependent endocytosis to activate the MAPK-ERK1/2 and PI3K-AKT signaling pathways,promote the expression of proliferation-related genes and proteins(PCNA,CCND1),and then promote The proliferation of porcine follicular granulosa cells;at the same time,these two signaling pathways also promote the expression of steroid hormone synthesis related genes and proteins(HSD3B1,CYP11,and StAR),which in turn promotes the secretion of progesterone from porcine follicular granulosa cells.This experiment provides a new basic theoretical support for exploring the role of follicular fluid-derived exosomes in follicular growth and development. |
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