| BackgroundAlthough most causes of infertility can be treated with surgery or drugs,for patients with azoospermia or oligospermia,it cannot be cured by conventional treatment methods.In recent years,Mesenchymal stem cell(MSC)MSC has been widely used in regenerative medicine due to its pluripotency and low immunogenicity.MSC can be differentiated into advanced derivatives of all three germ layers,such as bone,cartilage,Fat,muscle,etc.,and some research results show that MSC has the potential to differentiate into germ cells(Germ cell,GC),which is expected to be used in the treatment of infertility.Therapies based on mesenchymal stem cells are expected to restore the fertility of infertile patients.In addition to the improvement of reproductive function by the paracrine effect of MSCs,the differentiation of MSCs into germ cell(GC)-like cells is still an effective way to repair the damaged reproductive system Methods.In addition,there is a time difference between MSC preparation and patient reinfusion,and in most cases the cells are in a microenvironment deprived of glucose and oxygen.Maintaining cell viability during the time difference is a powerful guarantee for the effectiveness of MSC treatment.Objectives1.To study the effect of bone morphogenetic protein 4(BMP4)on the differentiation of human umbilical cord mesenchymal stem cells(hUC-MSC)into germ-like cells;2.To study the effect of low temperature on the maintenance of menstrual blood-derived endometrial stem cells(MenSC)under the conditions of glucose and oxygen deprivation and the underlying mechanism.Methods1.Isolating hUC-MSC from umbilical cord tissue by tissue cutting method.After further culture and expansion,use the third-generation hUC-MSC to determine the non-hematopoietic stem cell surface markers CD73,CD90,CD73 and CD90 by flow cytometry.CD105 and hematopoietic stem cell surface markers CD34,CD45,CD11 b,CD19,HLA-DR were identified,and the multidirectional differentiation of hUC-MSC was identified through osteogenic differentiation and adipogenic differentiation.2.Using the third-generation hUC-MSCs to continuously induce culture with12.5ng/mL BMP4 for 21 days.Change the medium every 2 days and take pictures to record the morphological changes of the cells;detect germ cell-related changes by PCR experiments The expression levels of genes OCT4,Prdm1,Stella,etc.;the changes in their protein expression levels were detected by immunofluorescence.3.Extracting the total RNA from hUC-MSC and BMP4 in the control group for 21 days after treatment with hUC-MSC for transcriptome sequencing to screen the differential genes before and after induction and related signal pathways;then screen the transcriptome sequencing results for MSC surface marker genes before and after induction.The expression changes of related pluripotency genes,germ cell-related genes and methylation genes were used to analyze whether hUC-MSC differentiated into germ-like cells.4.Using the third-generation MenSC,resuspend in physiological saline,inhale a closed syringe and store at 4 ?,25 ? and 37 ? for 6 h,12 h and 24 h,respectively.Use CCK8 to detect cell viability and flow cytometry to detect cells Apoptosis,WB detects the expression of apoptosis-related proteins,and clarifies the changes in the biological activity of MenSC when stored at different temperatures for different times.5.Using the third-generation MenSC,set up the control group,the autophagy inhibition group and the autophagy enhancement group,and resuspend them in physiological saline respectively,and store them at 4 ? for 6 h and 12 h after inhaling a closed syringe;flow cytometry to detect cell apoptosis WB detects the expression changes of autophagy-related proteins to clarify the role of autophagy in maintaining MenSC activity at low temperature.Results1.The hUC-MSCs were successfully isolated from the umbilical cord.The third-generation hUC-MSCs showed a typical fibrous cell structure,and the cells were spindle-shaped.Flow cytometry showed that hUC-MSCs positively expressed MSC surface markers,such as CD73,CD90 and CD105 do not express hematopoietic stem cell markers CD34,CD45,CD11 b,CD19 and HLA-DR;in addition,the multi-directional differentiation potential test indicates that hUC-MSCs have adipogenic and osteoblastic differentiation potential.2.Twenty-one days after BMP4 induced the third generation of hUC-MSCs,compared with the control group,the hUC-MSCs morphology changed significantly after induction,showing a broad and flat morphology,and the density of hUC-MSCs decreased;RT-PCR results showed OCT4,Prdm1 The expression of,Ifitm3 and Stella genes were significantly reduced,and the immunofluorescence results showed that the expression level of Prdm1 protein decreased and the expression level of Prdm14 increased.3.Transcriptome sequencing of 663 differentially expressed genes,including 152up-regulated genes and 509 down-regulated genes.The GO enrichment analysis structure of the differential genes shows that the differential genes are mainly enriched in the metabolic process in biological processes,and the differential genes in the KEGG enrichment are mainly enriched in the autophagy signal pathway,ribosomal RNA and ribosomal protein signal pathway.Further analysis showed that compared with the control group,there were no significant differences in the expression levels of surface marker genes,pluripotency transcription factors,germ cell-related genes and DNA methylation genes of MSCs after induction.4.In the oxygen deprivation microenvironment,MenSC activity can be maintained for a long time at 4 ?,and the activity is inversely proportional to the prolongation of storage time;further studies have found that 4 ? storage can maintain MenSC activity through the occurrence of slow-release autophagy.Conclusions1.The process of MSC differentiation into germ cell-like cells is extremely complicated.Only under the action of BMP4,hUC-MSCs cannot be induced to differentiate into germ cell-like cells.2.Low temperature can slow down the occurrence of cell autophagy,thereby maintaining MenSC activity for a longer period of time. |
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