| Objective:1.The ovaries of aging macaque monkeys after HA-MSC treatment were analyzed by 10X Genomics single nuclear transcriptome sequencing.The cellular and molecular changes of the ovaries of aging macaque monkeys before and after the treatment were analyzed by bioinformatics method.The cellular and molecular mechanisms of the effects of HA-MSC on the ovaries of aging macaque monkeys were revealed by comparative analysis.2.Human granular cells（hGC）aging model was established by induction of H2O2with appropriate concentration in vitro,and HA-MSC and hGC were cultured indirectly in Transwell chamber.HGC after HA-MSC action was obtained by observing its cell morphology,structure and function,which provided follow-up verification for the mechanism of HA-MSC action in single cell sequencing results.Methods:1.Single nuclear transcriptome sequencingAfter the macaques were euthanized,the ovarian tissues were collected,marked,and sent to Guangzhou Gidio Biotechnology Co.,Ltd.for 10X Genomics single nuclear transcriptome sequencing according to the sample delivery requirements.2.Analysis of single-cell transcriptome sequencing data（1）Visualize the results of cell subgroup classification;（2）The cells were analyzed for differences between groups,distribution,enrichment and other personalized analysis;（3）To predict the differentiation trajectory of cells during development by pseudo-time analysis;（4）Construct cell communication networks,and analyze the regulatory potential of ligands on target genes and the activity of ligands.3.Establishment of hGC aging modelHuman hGC strains were inoculated into 6-well culture plates,and when the cell fusion degree reached 70-80%,100,200,300,400 and 500μmol/L H2O2were added to hGC for 24,48 and 72h,respectively.According to the growth morphology,proliferation and cell cycle of hGC.The suitable induction conditions of hGC senescence model were analyzed.4.Co-culture of HA-MSC and aging hGCThe proliferation and growth morphology of aging hGC were observed under optical microscope after co-culture with P3 generation HA-MSC for 48h.The ultrastructure of hGC was observed under transmission electron microscope.β-galactosidase staining was performed to observe the changes of hGCβ-galactosidase gene expression.The expression levels of hGC P16 and P21 genes were detected by immunohistochemical staining.The migration ability of co-cultured cells was detected by cell migration assay.The genes screened by single nucleus transcriptome sequencing were verified in vitro by q-pcr.Results:1.Analysis results of single-cell transcriptome sequencing data（1）The cells were divided into 8 subgroups:stromal cells,endothelial cells,granulosa cells,follicular membrane cells,T cells,epithelial cells,smooth muscle cells and monocytes.（2）The up-regulated genes were mainly distributed in endothelial cells,granulosa cells and monocytes.The proportion and expression of anti-aging genes were higher in the HA-MSC treatment group,while the proportion and expression of age-related genes were lower in the HA-MSC treatment group.（3）The genetic differences were significant in stromal cells,endothelial cells,granulosa cells,epithelial cells,smooth muscle cells and monocytes,but not in follicular membrane cells and T cells.Among the genes with significant differences,the genes highly expressed after HA-MSC treatment were mainly GPSM,PAPPA2,FGF,which were related to growth and development and protein synthesis regulation,while the genes highly expressed in the aging model group were mainly FOSB,HS3ST2,FSTL4,which were related to tumor,cancer and other diseases.（4）The time-like analysis showed that GC cells in the HA-MSC treatment group and the senescence model group were in different developmental stages.Almost all GC cells in the senescence model group were in the period of regeneration but non-division,proliferation and apoptosis,while almost all GC cells in the HA-MSC treatment group were in the state of division and differentiation.（5）The analysis of intercellular communication showed that compared with the aging model group,the number of cell receptor pairs in each subgroup of HA-MSC treatment group increased overall.（6）GO annotation analysis showed that differential genes were mainly concentrated in cell components and molecular functions,and organelles were mainly affected in cell components.The main influence in molecular function is binding,including the binding of enzymes,proteins,nucleic acids and various compounds.（7）Significant enrichment of Pathway showed that:The differentially expressed proteins were significantly enriched in the Ribosme pathway and MAPK pathway,and the size subunits with changes in the Ribosme pathway were all upregulated.In MAPK,the ERK1/2 pathway in the four classical pathways was activated mainly by upregulating GF,RTK,Raf1,MEK1 and RSK2 genes.After being activated by Raf,ERK1/2 can continue to phosphorylate downstream transcription factors and induce gene expression related to cell cycle and proliferation.In addition,activated ERK1/2 can also phosphorylate a variety of intracellular kinases and play a role in cell proliferation and adhesion.2.Establishment of hGC aging model（1）There was no significant change in the growth of hGC under 100μmol/L H2O2co-culture conditions compared with the normal control group,but the proliferation capacity of hGC under 200μmol/L and 300μmol/L H2O2treatment was significantly weakened compared with the normal control group,and the cells were flattened and enlarged.Almost all hGC treated with 400 and 500μmol/L H2O2died.（2）Flow cytometry showed that the proportion of hGC cells in G0/G1 phase was the highest when 200μmol/LH2O2was treated（p<0.01）;（3）β-galactosidase staining was performed at 200μmol/L H2O2co-cultured with hGC for 24h,48h and 72h,and the blue-staining rate was the highest at 48h（p<0.01）.Finally,treatment with200μmol/LH2O2for 48h was selected as the appropriate condition for induction of hGC senescence,and the hGC senescence model was obtained by batch induction under this condition for further experiments.3.Effect of HA-MSC on aging hGC（1）Optical microscopy showed that the cells were spspindle or polygon after co-culture,and the number of cells increased,indicating that the proliferation activity of aging hGC was restored.（2）The results of transmission electron microscopy showed that there was heterochromatin aggregation,patchy distribution,mitochondrial swelling,a small amount of facial endoplasmic reticulum dilatation,and no obvious autophagosomes in the cells of aging hGC.The chromatin distribution of cocultured hGC cells was uniform,mitochondria were still swollen,rough endoplasmic reticulum was slightly expanded,and a small number of autophagosomes could be seen inside the cells.（3）β-galactosidase staining of the three states of hGC showed that the blue-staining rate decreased after co-culture（p<0.01）;（4）Immunohistochemical staining of p16 and p21 gene expression showed that the proportion of positive cells decreased after co-culture（p<0.01）;（5）The results of cell migration experiment showed that the absorbance value measured under the enzyme label was higher in the co-culture group than in the aging group（p<0.01）;（6）q-pcr results showed that the expression levels of GF,Raf,MEK1 and RSK2 genes in MAPK signaling pathway were higher in co-culture group than in aging group（p<0.01）.These results indicate that after non-contact HA-MSC co-culture with senescent hGC,HA-MSC can reverse the cell structure,proliferation ability,senescence condition,expression level of senescent genes and expression levels of key genes regulating senescence pathway in the direction of normal hGC.Conclusions:1.Through 10X Genomics single nuclear transcriptome sequencing,we investigated the mechanism of HA-MSC treatment on ovarian senescence at cellular and molecular levels,and confirmed that HA-MSC treatment can jointly play a role in the resistance to ovarian senescence through multiple cellular and molecular mechanisms,and improve the tissue structure and secretion function of the ovary.2.Validation experiments were carried out in vitro to further support the results of single cell sequencing.These results provide a reference scheme for the cellular and molecular mechanism of HA-MSC products in the treatment of ovarian aging,a new choice for stem cell treatment of ovarian aging,and a new technical method and scientific basis for the clinical treatment of ovarian aging. |
