| BackgroundPremature ovarian insufficiency(POI)is a manifestation of ovarian dysfunction in women before the age of 40,with oligomenorrhea or menopause,accompanied by of high gonadotropins and low estrogens clinical features.Chromosomal and genetic defects,autoimmune ovarian damage,infectious factors,iatrogenic factors(mainly surgery,chemotherapy,and radiotherapy,etc.)can cause POI,and there are still some unclear causes called idiopathic POI.Patients with POI have an increased risk of osteoporosis,cardiovascular disease,cognitive dysfunction,genitourinary syndrome,and decreased fertility.The current treatment is mainly to improve lifestyle,hormone supplements etc.,but there is no definite method to improve fertility.For young women with malignant tumors,oocytes,ovarian tissue or embryos can be frozen before radiotherapy to preserve fertility,but there is a risk of carrying tumor cells.Human amnion-derived mesenchymal stem cells(hAD-MSC)are derived from human amniotic membrane and have the ability of three-way differentiation in vitro.They have low immunogenicity and secrete various factors.Our group and other studies have confirmed that intravenous injection of hAD-MSC could improve the ovarian function of POI animals,but after the cells were transplanted into the body,the homing rate to the target organs was low.Therefore,this study intends to treat the POI of rat by orthotopic transplantation of hAD-MSC,increase the number of hAD-MSC homing to the ovary,and conduct a preliminary study on the possible mechanism of improving POI function.Methods(1)Isolation of hAD-MSC from human placental amnion membrane and culture it.Observe the morphological characteristics of the cells and carry out multi-directional differentiation culture.(2)Screening the POI model of SD rats:160 rats weighing 240-260g and having a normal estrous cycle were randomly divided into 10 groups.,and four commonly used drug regimens(cisplatin,cyclophosphamide,busulfan and 4-vinylcyclohexene diepoxide(VCD)to establish animal POI were selected,and a comparative study of different modeling schemes was conducted.1)Cisplatin-induced POI model scheme:32 rats were randomly divided into two groups.The cisplatin group was intraperitoneal injected of cisplatin(2 mg/kg for 6 consecutive days),and the cisplatin control group was intraperitoneal injected with equal volume of normal saline.2)Busulfan combined with cyclophosphamide-induced POI model scheme:48 rats were randomly divided into three groups.A single intraperitoneal injection of cyclophosphamide(83.52 mg/kg)and busulfan(20.88 mg/kg)was given to busulfan combined with cyclophosphamide group.The rats in the control group(normal saline group and sesame oil group)were injected abdominal cavity respectively equal volume of normal saline and sesame oil.3)POI model scheme caused by cyclophosphamide alone:32 rats were randomly divided into two groups,and the first dose of cyclophosphamide(50 mg/kg)was given to the cyclophosphamide experimental group and continued at 8 mg/(kg·d)for 14 days,and the control group was intraperitoneally injected with an equal volume of normal saline every day.4)VCD-induced POI model scheme:48 rats were randomly divided into three groups.The VCD experimental group was intraperitoneally injected with VCD(80 mg/kg)for 14 consecutive days,and the control group(saline group and sesame oil group)was intraperitoneally injected with an equal volume of normal saline and sesame oil.After treatment,the general condition,estrous cycle and body weight of the rats were observed.The sex hormone levels of the rats were measured 4 weeks after the start of the treatment,and 10 rats in each group were sacrificed.The number of follicles in the ovarian pathology and counts were observed,and the heart and liver,spleen,lung,kidney,brain organ index and pathological changes were observed.Another 6female rats and male rats were subjected to a cage test at 3:2 to observe the pregnancy condition at 6 months.(3)In vivo test of hAD-MSC transplantation for POI in SD rats:48SD rats weighing 240-260 g and having normal estrous cycle were divided into 4 groups,namely normal control group,POI group and hAD-MSC(tail vein)group and hAD-MSC(in situ)group.The POI model was established in the POI group,the hAD-MSC(tail vein)group and the hAD-MSC(in situ)group,respectively,and the control group was intraperitoneally injected with the same amount of normal saline and sesame oil.24 hours after the model was established,0.5 ml PBS cell suspension(containing 4×10~6 hAD-MSC)was injected into the hAD-MSC(tail vein)group via the tail vein,and 0.05 ml(0.025 ml per side ovary)PBS was injected into the ovary.In the hAD-MSC(in situ)group,0.5 ml PBS was injected through the tail vein,and the ovary was injected with 0.05 ml PBS cell suspension(containing 4×10~6 hAD-MSC,each side ovary 0.025 ml);the model group and the control group.The group was injected with 0.5 ml of PBS through the tail vein,while the ovaries were injected with 0.05 ml of PBS(0.025 ml per side ovary).After hAD-MSC transplantation,the general condition,weighing and vaginal secretion smear of the rats were observed.After 4 weeks of transplantation,the rats were tested for sex hormone levels,and all rats were sacrificed.The fluorescence signal was used to track the homing status of hAD-MSC in rats,the number follicles at various levels were counted.(4)Mechanism of orthotopic transplantation of hAD-MSC in the treatment of POI:The ovary of POI group and hAD-MSC(in situ)group was detected by protein chip,and the change of ovarian local protein expression spectrum was detected.The first three proteins were verified by western blot.Result(1)hAD-MSC was successfully isolated and cultured from human amniotic membrane,adherently grown,spirally arranged,and have capable of three-way differentiation and subculture and keep cell morphology consistent.(2)Rat POI model in rat was established by cisplatin,cyclophosphamide combined with busulfan,cyclophosphamide and VCD.All the regimens reduced normal estrous cycle,AMH level,the number of ovarian primordial follicle,primary follicle,secondary follicle and mature follicles.Cyclophosphamide combined with busulfan also reduced E2 levels,increased FSH levels,and decreased fertility better than the other experiment protocal.There were no abnormalities in the appearance and pathology of other organs in this group.(3)The proportion of estrous cycle in hAD-MSC in situ and tail vein transplantation group increased,body weight,AMH level and the number of ovarian follicles increased.The distribution of ovarian fluorescence signals in the hAD-MSC(in situ)group was stronger than that of hAD-MSC(tail vein),and the E2 level was significantly higher than that in the model group.(4)After transplantation(in situ)of hAD-MSC,the expression of JNK2,p38,Serpin E1,Akt,MKK3/6,RSK1,MMP and other proteins in ovarian tissue increased.Conclusions(1)Human amniotic mesenchymal stem cells were successfully isolated and cultured from human amniotic membrane,and have the ability of three-way differentiation and be mesenchymal stem cells.(2)POI model in rat was established by cyclophosphamide combined with busulfan,VCD,cisplatin and cyclophosphamide.The POI model of SD rats was successfully established by all four protocols.Cyclophosphamide combined with busulfan can better induce the POI model and the side effects were small in the four schemes.Finally,the POI model scheme was selected for the next step.(3)hAD-MSC ovary transplantation in situ and tail vein transplantation can improve the ovarian function of POI rats.After transplantation in situ,MSCs reach more ovarian tissue cells,and the E2level of POI rats is better.(4)hAD-MSC secretes cytokines,which may up-regulate JNK2,p38,Serpin E1,Akt,MKK3/6,RSK1,MMP and other proteins in the ovary through MAPK signaling pathway and angiogenesis pathway,and promote cell proliferation and gene expression to repair ovarian function. |
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