Study Of Umbilical Cord Mesenchymal Stem Cells Combined With Soybean Isoflavone In The Treatment Of Premature Ovarian Failure In Mice

BackgroundThe traditional treatment of premature ovarian failure mainly include hormone replacement,accept donated eggs,autologous ovarian tissue and monozygotic twin ovarian tissue transplantation.However,these treatment options can only improve ovarian function to a certain extent.Stem cells have been widely used in tissue repair and regeneration due to its important roles.Recent studies have shown that transplantation of Mesenchymal Stem Cells（h UC-MSCs）may be a new option for the treatment of ovarian damage caused by chemotherapy.Soy isoflavones are the most studied phytoestrogens for reducing menopausal symptoms: Soy isoflavones can regulate the growth and early development of ovarian primordial follicles,promote the development of dominant follicles,promote oocyte maturation,and inhibit the formation of atretic follicles;For adolescence and perimenopause,it can delay the deterioration of ovarian function and prolong the reproductive years.Objectives1.Studying the effects of a single injection of Cyclophosphamide（CTX）and Busulfan（BUS）on the establishment of a mouse model of Premature ovarian failure（POF）;Further use this model to study the treatment effect of h UC-MSCs combined with soy isoflavones on the recovery of ovarian function in POF mice.2.Screening genes related to ovarian function recovery by transcriptomics to further explore their role in ovarian function recovery in POF mice.Methods1.First,warton’s gum was separated from human umbilical cord;Second,h UC-MSCs were isolated and cultured by tissue block culture method.The second generation h UC-MSCs were used to identify surface markers（CD73,CD90,CD105,CD34,CD45,CD11 b,CD19 and HLA-DR）by flow cytometry.2.h UC-MSCs,soy isoflavones and the combination of the h UC-MSCs and soy isoflavones were used to treat the POF mice,respectively.Subsequently,the serum of these POF mice was used to determine the content of hormones（E2,AMH and FSH）at 14 d,21 d,28 d and 60 d,respectively,and the POF mouse ovary was observed by HE staining.3.To analyze the molecular mechanism of h UC-MSCs restoring ovarian function in POF mice,transcriptome sequencing was performed using the mice ovary（the model group,the h UC-MSCs treatment group for one week and the h UC-MSCs treatment group for four week）,and the differentially expressed genes（fold change>2 or <0.5,p<0.05）were used for GO（Geneontology）and KEGG（Kyoto Encyclopedia of Genes and Genomes）analysis.Results1.h UC-MSCs were isolated from umbilical cord tissue and were successfully cultured.P2 cells were mainly spindle shaped,translucent,clear in outline and good in refraction.Flow cytometry showed that 99% of isolated cells highly accumulated CD63,CD90 and CD105,and only a few cells（<0.5%）accumulated CD34/CD45/CD11b/CD19/HLA-DR,suggesting we isolate high-purity h UC-MSCs.2.After h UC-MSCs,soy isoflavones and their combined treatment,the number of follicles in h UC-MSCs treatment group and combined treatment group all increased significantly,while the number of follicles in soy isoflavone treatment group did not increase significantly.Transplantation of h UC-MSCs is able to improve the ovarian endocrine function of CTX injury,increase E2/AMH and decrease FSH levels in serum;soy isoflavone treatment can also improve the ovarian endocrine function of CTX injury,and reduce serum FSH level.3.The results of transcriptome sequencing showed that: at 1 week of treatment,the expression level of 290 genes was significantly changed in the treatment group 147up-regulated and 143 down-regulated);at 4 weeks of treatment,the expression level of 343 genes was significantly changed in the treatment group（156 up-regulated and 187down-regulated）.GO enrichment analysis showed that these differentially expressed genes were mainly involved in the negative regulation of peptidase activity,acute phase response,response to cytokines,cyclooxygenase pathway,and immune system processes at 1 week of treatment;and are mainly involved in inflammatory response,immune response,adaptive immune response,immune system process,positive regulation of angiogenesis,etc at 4 weeks of treatment.KEGG enrichment analysis showed that these differentially expressed genes were mainly involved in the complement and coagulation cascade,allograft rejection,autoimmune thyroid disease,IL-17 signaling pathway,vascular endothelial growth factor and other signaling pathways at 1 week of treatment;and are mainly involved in signaling pathways such as rheumatoid arthritis,allograft rejection,systemic lupus erythematosus,Th1 and Th2 cell differentiation,and Th17 cell differentiation at 4 weeks of treatment.Conclusions1.Soy isoflavones alone have no significant effect on the recovery of ovarian function in POF mice.2.Treatment of h UC-MSCs and h UC-MSCs combined with soy isoflavones can significantly restore ovarian function in POF mice.3.h UC-MSCs treatment improved the recovery of endocrine function and ovarian function in POF mice by regulating the expression of PKHD1L1,CFHR2,GPR75,STOX1,USP49,ADAMTS-1,BRMS1,ALDH1A2,ACAP1,IL33,PTGS1 and other genes.