Improvement Of Methods For Isolation And Culture Of Ovarian Germline Stem Cells And Effects Of Oligochitosan On Its Proliferation And Senescence

Ovarian germinal stem cells(OGSCs)are bipotential stem cells(derived from the cortex of the ovary),which can produce a new stem cell through symmetrical division or differentiate into ovarian germ cells and primitive granulosa cells through asymmetric division.Ovarian germinal stem cells are circular under light microscope,about 15-20 um,the number is rare,which can be seen in a string or cluster after passage and proliferation,and its expression of MVH,OCT4 and other ovarian germinal stem cells markers are detected by PCR.Our laboratory has established a method for isolating,purifying and culturing ovarian germinal stem cells by improving the methods ofProfessor Ji Wu's laboratory from Shanghai Jiaotong University,which laying a solid foundation for the study of ovarian germinal stem cells.Our previous studies demonstrated that gavage of chitooligosaccharides could improve immune function and reproductive function in pathologically infertile mice.However,whether chitooligosaccharides directly promote the proliferation and differentiation of ovarian germline stem cells,or enhance the immunity of mice and then indirectly promote the proliferation and differentiation ofovarian germline stem cells to supplement the follicle pool,which is not yet clear;the mechanisms underlying the effects of chitooligosaccharides on proliferation and senescence of mouse germline stem cells remain unclear.This study first improved the method of isolation and culture of mouse ovarian germinal stem cells,and then tested whether chitooligosaccharides directly promote the proliferation of OGSCs at the cellular level,or indirectly promote OGSCs proliferation by enhancing the immune system,thereby delaying ovarian senescence and remodeling the ovary.Features.For the prevention and treatment of premature ovarian failure and infertility provide new ideas and methods and new drug interaction sites.1Improvement of methods for isolation and cultivation of OGSCs(1)Improvement of theOGSCs separation method: analysis and comparison of the advantages and disadvantages of the two enzyme methods(improved method)and classical two-step enzymatic method(classical method)and immunomagnetic beads method(magnetic beads method)for separation of OGSCs.Cellular purity was the highest in the magnetic beads method,followed by the modified method,and the classical method was the lowest;the cell proliferation curve was the best in the modified method,followed by the magnetic beads method,and the classical method was the worst.(2)Improvement of the OGSCs culture method: In this experiment,macrophages(RAW)were used for the first time instead of STO cells as a trophoblast.When mitomycin C was used to treat macrophages,treatment with 10mg/L for 2 hours had the best effect.The macrophage cell proliferation ability is basically inhibited and the cell state is best.After using macrophages as a trophoblast,the number of ovarian germ stem cells increased significantly.(3)Identification of OGSCs after isolation and culture: Ovarian germline stem cells were round and had a high karyoplasm ratio and were arranged in a bead-like pattern after morphological observation.Immunofluorescence double-labeling showed that the cells also express genomic markers MVH and The stem cell marker OCT4 also expresses the germline marker MVH and proliferation marker EDU at the same time;alkaline phosphatase staining is positive;and RT-PCR shows expression of MVH,OCT4,Fragilis,Dazl,Stella and other germ cell and stem cell markers.Conclusion: The OGSCs isolated by the improved two-step enzymatic separation method have good activity,few heterocytes and good cell proliferation,which lays an important foundation for the related research of OGSCs in the future.The improved culture method of the OGSCs uses macrophages as a nourishment layer to create a new road to OGSCs research.2 Preliminary exploration of the effects of chitooligosaccharides on the proliferation and senescence of OGSCsThe experiment was divided into four groups.The Control group(group 1)and the Cos group(group 2)investigated whether there was a direct effect.The RAW group(group 3)and the RAW+Cos group(group 4)explored whether there was an indirect effect.The data from the 1-4 group showed that the cell proliferation curve showed a different degree of increase;the alkaline phosphatase(AP)staining results showed a different degree of reduction;cell supernatants detected by cytokines,IL-2,TNF-The performance of delirium showed varying degrees of increase.IL-10 and TGF-? showed decreased levels of Cos-containing group compared with the corresponding control group;WB results showed that immune factors(IL-2,TNF-?)-related protein,ovarian reproduction The related proteins of stem cells(MVH and OCT4)and anti-aging genes(SIRT-1 and SIRT-3)all showed increased levels,while immune factors(IL-10,TGF-?)related proteins and aging genes(P21,P53)related proteins showed different degrees of reduction.CONCLUSION: Chitosan oligosaccharide may indirectly promote the proliferation of OGSCs and inhibit their senescence by regulating the secretion of cytokines from macrophages.