Effect Of Pilose Antler Stem Cells On The Repair Of Premature Ovarian Failure Induced By Cyclophosphamide In Rats

PART ONE Construction and verification experiment of premature ovarian failure model induced by cyclophosphamide in ratsObjective:To construct and verify the model of premature ovarian failure induced by cyclophosphamide in rats.Methods:40 healthy adult female SD rats(3-4 months old)were purchased from the Laboratory Animal Research Institute.The animals were housed in sterile polypropylene rat cages under a 12/12 hour light-dark cycle at an ambient temperature of 21±2°C.Feel free to provide food and water.The rats were randomly divided into two groups to establish a chemotherapy-induced premature ovarian failure rat model:control group(healthy adult female SD rats,intraperitoneal injection of the same amount of saline,n=20)and model group(healthy adult female SD rats,Intraperitoneal injection of cyclophosphamide induced a rat model of premature ovarian failure,n=20).All experimental animals were weighed and anesthetized by intraperitoneal injection of 10%chloral hydrate(350 mg/kg).After collecting blood samples for hormone determination,the rats were euthanized.Take out the ovaries and weigh them.Histopathological examination of rat ovary was performed by HE staining.The apoptosis rate of ovarian granulosa cells was detected by flow cytometry and apoptosis detection kit.Ten rats in each group were subjected to normal mating behavior in the 8th week,and the number of litters per litter was counted.Detect Follicle-stimulating Hormone(FSH),Luteinizing Hormone(LH),Estradiol(E2)and Anti-Muller hormone(AMH)levels by ELISA kit.RT-q PCR was used to detect the levels of germ cell marker DDX4,cell proliferation marker PCNA,bone morphogenetic protein(Bone Morphogenetic Protein,BMP)4 and FOXO3A,an inhibitor of primordial follicle activation,in the ovary.Biochemical analysis of the oxidants malondialdehyde(MDA)and antioxidants-glutathione(Glutathione,GSH),superoxide dismutase(SOD),and glutathione peroxide in rat ovaries Enzyme(Glutathione Peroxidase,GPx)and Catalase(Catalase,CAT)levels.Western blot analysis of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(phosphatidylinositol-3-kinase/protein kinase B/the mammalian target of Rapamycin,PI3K/Akt/m TOR)signaling pathway proteins.The level of phosphorylation.Results:There was no difference in the weight of rats in each group at baseline.Compared with the control group,the model group rat body weight,ovarian weight and ovarian surface area were all decreased(P<0.05).Normal histological appearance was observed in the control group:corpus luteum and secondary follicles,multilayer primary follicles,and primitive follicles.In the model group,we detected vascular congestion,hemorrhage around the corpus luteum and ovarian stroma,follicular atresia,and lower monocytes infiltrating the reproductive epithelium and ovarian stroma.Compared with the control group,the ovarian histopathological damage score of the model group was lower(P<0.05).Compared with the control group,the number of primordial follicles,primary follicles,secondary follicles and mature follicles in the model group decreased(P<0.05).Compared with the control group,the apoptosis rate of ovarian granulosa cells in the model group increased(P<0.05).Compared with the control group,the number of litters per litter in the model group decreased(P<0.05).Compared with the control group,the FSH and LH levels of the model group increased(P<0.05),and the E2 and AMH levels decreased(P<0.05).Compared with the control group,the DDX4,PCNA and BMP4 m RNA levels in the model group decreased(P<0.05),and the FOXO3A m RNA level increased(P<0.05).Compared with the control group,the MDA level of the model group increased(P<0.05),and the level of GSH,SOD,GPx and CAT decreased(P<0.05).Compared with control rats,the protein levels of p-PI3K,p-AKT and p-m TOR in the model group increased(P<0.05).Conclusion:Cyclophosphamide can increase the phosphorylation of PI3K,AKT,m TOR,regulate serum AMH,E2,FSH,LH levels,regulate oxidative stress,increase ovarian granulosa cell apoptosis,reduce primordial follicles,and affect the rat model of premature ovarian failure Fertility.PART TWO The molecular mechanism of deer antler stem cells affecting rat ovarian functionObjective:To explore the molecular mechanism of deer antler stem cells affecting rat ovarian function.Methods:After constructing a POF rat model,ASCs were injected intravenously,and POF ovaries were co-cultured with ASCs and/or GANT61.FGSC was pretreated with busulfan and ASCs and/or GANT61,and co-cultured with M1 macrophages pretreated with ASCs.Measure the weight of mice and their ovaries and the number of follicles to evaluate ovarian function,anti-oxidative stress,inflammation,and FGSC survival rate.Results:ASCs significantly increased the weight of POF rats and their ovaries and the number of follicles,and at the same time reduced the atresia rate of follicles.After ASCs were treated in vivo and in vitro,higher levels of Mvh,Oct4,SOD2,GPx and CAT were detected.ASCs treatment resulted in a significant decrease in the concentration of TNF-αand IL-6 in the ovary,and a significant increase in the concentration of IL-10.In FGSC,the concentration of Mvh,Oct4 and SOD2 in the treatment group was higher,while the concentration of TNF-α,IL-6 and MDA was lower.ASCs reversed ovarian damage by blocking the Hh signaling pathway.Conclusion:ASCs can effectively improve the ovarian function and FGSC production capacity of the POF model by reducing oxidative stress and inflammation and the mechanisms involved in the Hh signaling pathway.This indicates that ASCs treatment is a potential anti-POF therapy.PART THREE The regulatory mechanism of velvet stem cell-derived microvesicles and cytokines on ovarian granulosa cellsObjective:To explore the regulatory mechanism of velvet antler stem cell-derived microvesicles and cytokines on ovarian granulosa cells.Method:150-200 g female SD rats(3 to 4 weeks old)were used in the experiment.The rats were reared at room temperature of 23±2℃,humidity of 45-55%and light for 12 hours,and separated from the ovaries Rat primary ovarian granulosa cells,velvet stem cells and granulosa cells were co-cultured at 37°C and 5%CO2 inα-MEM containing 10%fetal bovine serum.According to the experimental requirements,the cultured cells were divided into a control group and a joint culture group.The cell viability and apoptosis were detected by CCK-8 and flow cytometry.Western blot analysis of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione reductase(GR)and other enzymes Protein.Western blot analysis of the protein expression of transforming growth factor-β(TGF-β),fibroblast growth factor(FGF)and bone morphogenetic protein(BMP).Real-time RT-PCR analysis of the m RNA expression of IL-6,IL-1β,Tumor necrosis factor(TNF)-α,C-reactive protein(CRP),SIRT-1 and ERK1/2.Results:Compared with the control group,the cell viability of the combined culture group was increased(P<0.05),and the apoptosis rate and the number of apoptotic cells were decreased(P<0.05).There was no difference in cell proliferation at the 12th hour(P>0.05),and the cell proliferation in the 24h,36h,48h and 72h co-culture group was higher than that of the control group(P<0.05).The expression of MDA enzyme protein in the co-culture group was lower than that in the control group(P<0.05),and the expression of SOD,CAT and GR protein in the co-culture group was higher than that in the control group(P<0.05).Compared with the control group,the protein expression of TGF-β,FGF and BMP in the combined culture group was higher(P<0.05).The m RNA expression of IL-6,IL-1β,TNF-αand CRP in the combined culture group was lower than that in the control group(P<0.05).Compared with the control group,the protein expression of Bax and caspase-3 in the combined culture group was lower(P<0.05),and the protein expression of Bcl-2 was higher(P<0.05).Compared with the control group,the expression of SIRT-1 and ERK1/2m RNA was higher in the co-culture group(P<0.05).Conclusion:Deer antler stem cell-derived microvesicles and cytokines can promote the proliferation and differentiation of ovarian granulosa cells,reduce the occurrence of apoptosis,and protect cells from degeneration.They can be considered as potential therapeutic agents for ovarian diseases.PART FOUR Study onthe signal pathway of antler ste mcell tail veintransplantation onthe repair of ovarianfunctionin rats with pre mature ovarianfailureObjective:To explore the signal pathway of antler stem cell tail vein transplantation on ovarian function repair in a rat model of premature ovarian failure.Method:Purchase 48 SPF female SD rats(age 12 weeks;no birth;average weight 216.88±12.56g).The experiment was divided into three groups:control group(age-matched female C57BL/6 rats without induction and treatment,n=16),model group(female C57BL/6 rats with cyclophosphamide induction,60μL DMSO injected into the tail vein,n=16)and velvet stem cell group(female C57BL/6 rats induced by cyclophosphamide,injected 60μL of the 5th passage 2×10~6velvet stem cells,n=16).The antler tissue digested complex was cultured in DMEM medium containing10%fetal bovine serum,100μg/ml streptomycin and 100 units/ml penicillin.The weight of each rat was recorded weekly.Follicle-stimulating Hormone(FSH),Estradiol(E2),Gonadotropin releasing hormone(Gn RH)and Anti-Muller hormone(Anti-Muller hormone)were checked by ELISA kits.AMH)level.The estrus cycle was assessed by the Papanikola staining method.The number of follicles in the three groups was evaluated by HE staining.The expression levels of TNF-α,IL-1βand IL-6m RNA were checked by RT-q PCR.The sterile rats after tail vein cell transplantation were screened by real-time imaging to identify GFP-positive cells in vivo.Tracking antler stem cells by immunofluorescence staining.Apoptosis was detected by TUNEL staining.Caspase-3,Caspase-9,nerve growth factor(Nerve Growth Factor,NGF),high-affinity nerve growth factor receptor(tropomyosin-related kinase,Trk A)and follicle stimulating hormone receptor(Follicle Stimulating Hormone Receptor,FSHR)protein expression level.Statistics of the pregnancy rate and the number of embryos of each group of rats.Results:Compared with the control group,the model group lost weight at 3 and6 weeks(P<0.05).Compared with the model group,the antler stem cell group gained weight at 3 and 6 weeks(P<0.05).Compared with the control group,the FSH level of the model group increased,and the E2,Gn RH and AMH levels decreased(P<0.05).Compared with the model group,the FSH level of the deer antler stem cell group decreased,and the E2,Gn RH and AMH levels increased(P<0.05).Four weeks after transplantation,the animals treated with antler stem cells and the untreated control animals observed similar pre-estrus,estrus,late estrus and rest periods.However,the periodicity of the animals in the model group was irregular,the estrus cycle was longer or shorter,and the animals were in the rest period throughout the experiment.Compared with the control group,the number of primordial follicles,primary follicles,secondary follicles and mature follicles in the model group decreased(P<0.05).Compared with the model group,the number of primordial follicles,primary follicles,secondary follicles and mature follicles in the antler stem cell group increased(P<0.05).Compared with the control group,the expression levels of TNF-α,IL-1βand IL-6 m RNA in the model group increased(P<0.05).Compared with the model group,the expression levels of TNF-α,IL-1βand IL-6 m RNA in the antler stem cell group were reduced(P<0.05).GFP(+)cells first enter the pelvic organs 6 to 12 hours after transplantation,and migrate to the chest 24 hours after transplantation.Two months after transplantation,GFP stained cells were found in the ovarian tissue matrix.GFP(+)staining and human antinuclear staining co-localized in the ovarian matrix.Compared with the control group,the number of TUNEL positive cells in the model group increased at 3 and 6 weeks(P<0.05).Compared with the model group,the number of TUNEL positive cells in the antler stem cell group at 3 and 6 weeks decreased(P<0.05).Compared with the control group,the expression levels of Caspase-3,Caspase-9 and FSHR proteins in the model group increased,and the expression levels of NGF and Trk A proteins decreased(P<0.05).Compared with the model group,the expression levels of Caspase-3,Caspase-9 and FSHR proteins in the antler stem cell group decreased,and the expression levels of NGF and Trk A proteins increased(P<0.05).Compared with the control group,the pregnancy rate and the number of embryos in the model group decreased(P<0.05).Compared with the model group,the pregnancy rate and the number of embryos in the velvet stem cell group increased(P<0.05).Conclusion:By regulating the NGF/Trk A signal pathway,deer antler stem cells can inhibit inflammation and reduce cell apoptosis,which can repair ovarian damage,stimulate regeneration and improve ovarian function.Deer antler stem cell transplantation may provide an effective and novel method for the treatment of premature ovarian failure.