| Chemotherapy is one of the main treatment of ovarian cancer, most ovarian cancer patients go through more cycles of chemotherapy, some patients have to undergo more chemotherapy. Chemotherapy in women with breast cancer, about 68% of patients in the treatment of ovarian function would be in danger of early failure. Premature ovarian failure caused great pain to the patient, how to prevent premature ovarian failure, recover ovarian function in patients, improve quality of life, is the key point that the domestic and foreign scholars and clinicians want to study and solve.Purpose The aim of this study is to make an objective evaluation of chemotherapyinduced ovarian damage in animal models of human umbilical cord mesenchymal stem cells(UC-MSCs) and rechargeable human amniotic mesenchymal stem cell(AMSCs) treatment in human amniotic intervention chemotherapy-induced ovarian damage in animal models,and hope to provide biology, morphology, biomechanics basis of UC-MSCs and AMSCs for clinical intervention with chemotherapy in patients with premature ovarian failure to provide.Methed Cultivation in human amniotic membrane and fetal UC-MSCs and AMSCs, extend the stabilized, by flow cytometry measurement testing two cells of the immune phenotype, growth curve and cell cycle. Cells through local injection method for the identification of after injection into the female Wistar rats ovaries, immunofluorescence tests confirmed UC-MSCs and AMSCs by local injection method can effective role in rat ovary. 200 healthy female Wistar inbred rats as old as 10 week are randomly divided into the control group of 50, chemotherapy damage ovarian animal model group of 50, chemotherapy damage ovarian animal model with UC-MSCs group of 50, and chemotherapy damage ovarian animal model with AMSCs group of 50. Use cyclophosphamide(CTX) intraperitoneal injection modeling. After modeling, for the rats of chemotherapy damage ovarian animal model with UC-MSCs group and AMSCs group, with 10% chloral hydrate intraperitoneal injection and anesthesia, incision of the abdomen, find and fix ovaries. Inject 106 UC-MSCs and 106 AMSCs respectively into the rat ovary of UC-MSCs group and AMSCs group, followed by abdominal closure and suture. Take tails blood of control group of diestrus as the basis of concentration control. Take 80 ml tails blood of other groups at l4d、30d、60d、90d after the modeling for the measurement of biology and biochemical indices of molecular; then the rats are killed, then the ovarian specimens of rats are dissected from the ovaries and are frozen and preserved in liquid nitrogen for the morphology observation, follicular count, stretch, stress relaxation and creep test. 1. Measure the E2, FSH, LH concentration of rats serum of every group with microplate reader by the means of radioimmunoassay(RIA). The set instrument sensitivity is 1.1 pg/m L, the intraassay coefficient of variation is ≤10%, the interbatch coefficient of variation is≤10% and the sensitivity of determination of T is 0.05 ng/m L. Sample determination adopts double tube parallel, obtains the reading through the instrument, and reads the corresponding concentration from standard curve. 2. Get the rat serum of each group, determinate insulin like growth factor 1(IGF-1) by enzyme immunoassay(ELISA) method. 3. Get the rat serum of each group and get VEGF concentration in the specimen from the standard curve with double antibody sandwich ABC-ELISA method. 4. Make the pathological specimens of ovarian tissue in rats of each group, observe the ovarian morphology under an inverted fluorescence microscope, and calculate ovarian primordial follicles, preantral follicles, antral oocytes, preovulatory follicles and atretic follicle numbers of rats of every group with CASTGRID software. 5. The rats in each group are fed for 6 months. Observe the fertility recovery of the rats with human umbilical cord mesenchymal stem cell intervention group, human amniotic mesenchymal stem cell intervention group and chemotherapy damage ovarian animal model group and observe the growth and development of young rats. 6. Chuck ovarian specimen clamping of the rats in each group respectively in electronic universal testing machine, conduct tensile experiments on rat ovarian specimens at a speed of 2 mm/min, and observe the maximum tensile stress, maximum strain, elastic limit strain, stress-strain curve and longitudinal tensile testing method. 7. Chemotherapy ovarian damage animal model stress relaxation experiment of rat ovarian specimens with 5%/s strain increase speed. Strain of the stress decreases continuously with the change of time and the computer automatically outputs the result after meeting the set experimental time. 8. Perform the creep experiments on rat ovarian specimens with 5% Mpa/s strain increase speed respectively. Strain of the stress increases continuously with the change of time and the computer automatically outputs the result after meet ing the set experimental time.Result Through the experiment of multi-directional differentiation potential can be calculated with the UC-MSCs and AMSCs, the cell expressing CD90, not between the expression of CD34, CD45 is AMSCs; The cell expressing CD29, CD105, no expression of CD34 is UC-MSCs. Two local injections of stem cells in rat ovary, DAPI staining shows two kinds of stem cells are still active in 7 days after the injection area gathered in ovarian injury. LH, FSH, E2 test results of the rats in each group show that after the modeling for 14 days, 30 days, 60 days and 90 days of the chemotherapy damage ovarian animal model group, LH and FSH of the serum are greater than the rats in the normal control group, and the serum estrogen E2 is less than that of the control group with a significant difference(p<0.05). It shows that chemotherapy results in the ovary function restrain of chemotherapy damage ovarian animal model. After the modeling of UC-MSCs group and AMSCs group of the chemotherapy damage ovarian animal model for 14 days, 30 days, 60 days and 90 days, E2 value of serum estrogen are greater than the model group with a significant difference(p<0.05). After the modeling of UC-MSCs group for 14 days, 30 days, 60 days and 90 days, LH and FSH of the serum are less than those in AMSCs group of chemotherapy damage ovarian animal model. E2 value of serum estrogen are greater than AMSCs group with a significant difference(p<0.05). The test results of vascular endothelial growth factor VEGF of rats in each group show that after the modeling for 14 days, 30 days, 60 days and 90 days of the chemotherapy damage ovarian animal model group, VEGF value of the serum is less than that in the normal control group with a significant difference(p<0.05). After the modeling of UC-MSCs group and AMSCs group of the chemotherapy damage ovarian animal model for 14 days, 30 days, 60 days and 90 days, VEGF value of serum are greater than the chemotherapy damage ovarian animal model group with a significant difference(p<0.05).After the modeling of UC-MSCs group of the chemotherapy damage ovarian animal model for 14 days, 30 days, 60 days and 90 days, VEGF value of serum are greater than AMSCs group of the chemotherapy damage ovarian animal model with a significant difference(p<0.05).The test results of IGF-1(Insulin growth factor) of rats in each group show that after the modeling for 14 days, 30 days, 60 days and 90 days of the chemotherapy damage ovarian animal model group, IGF-1 value of the serum is less than that in the normal control group with a significant difference(p<0.05).After the modeling of UC-MSCs group and AMSCs group of the chemotherapy damage ovarian animal model for 14 days, 30 days, 60 days and 90 days, IGF-1 value of serum is greater than the chemotherapy damage ovarian animal model group with a significant difference(p<0.05).After the modeling of UC-MSCs group of the chemotherapy damage ovarian animal model for 14 days, 30 days, 60 days and 90 days, IGF-1 value of serum is greater than AMSCs group of the chemotherapy damage ovarian animal model with a significant difference(p<0.05). The rat ovarian histology observation shows that after modeling for 14 days, chemotherapy damage ovarian animal model group has a large area of fibrosis, the visible follicle cells are hypertrophy and show the signs of atresia. The follicular morphology of UC-MSCs group and AMSCs group of chemotherapy damage ovarian animal model is better than that of chemotherapy damage ovarian animal model group in the same period and follicle cells are not seen hypertrophy. The follicle count results of every group show that after modeling for 14 days, the primordial follicles and primary follicles of chemotherapy damage ovarian animal model group, UC-MSCs group and AMSCs group of chemotherapy damage ovarian animal model decrease compared with the control group, which means that with the extension of time, the follicles gradually consume. The number of secondary follicles and antral follicles of UC-MSCs group and AMSCs group of chemotherapy damage ovarian animal model is higher than chemotherapy damage ovarian animal model group with a significant difference(p<0.05). The comparison of all-level follicle number of UC-MSCs group and AMSCs group has difference at each time point,(p<0.05). Fertility control group rats were normal. the model group rats during the whole observation fail to give birth to the young rats. UC-MSCs group and AMSCs group rats, significantly lower than the control group at conception rate, parturition rate and number of newborn rats, no significant difference between two groups. The weight difference of the newborn rats of each group is not significant.The weights of each group increase with the extension of time with a significant difference. The weights of rats in each group in different time points have no significant difference. After modeling for 30 days and 90 days of rats of each group, the ovarian tensile experimental results show that the maximum stress, maximum strain, elastic limit stress, elastic limit strain values of ovarian sample of UC-MSCs group and AMSCs group of chemotherapy ovarian damage animal model are greater than chemotherapy ovarian damage animal model group with a significant difference(p<0.05). The stress relaxation experiment results show that 7200 s stress relaxation quantities of modeling 30 days group and 90 days group of chemotherapy damage ovarian animal model group are less than those of the control group, UC-MSCs group and AMSCs group with a significant difference(p<0.05). The 7200 s stress relaxation quantities of AMSCs group are less than UC-MSCs group with a significant difference(p<0.05). The creep experimental result shows that the 7200 s creep quantity of modeling 30 days group and 90 days group of chemotherapy damage ovarian animal model group is less than the control group, UC-MSCs group and AMSCs group with a significant difference(p<0.05). The 7200 s creep quantities of modeling of AMSCs intervention group are less than UC-MSCs group with a significant difference(p<0.05).Conclusion The research results show that the molecular biology, biochemistry index, index of morphology, numbers of follicles, index, mechanical properties of tensile stress relaxation and creep properties of chemotherapy ovarian damage animal model group have changed and have lost the fertility. The molecular biology index, biochemistry index, ovarian histology, all-level ovarian follicles count, properties index of mechanical tensile, stress relaxation and creep properties in serum indicators of UC-MSCs group, AMSCs group of chemotherapy ovarian damage animal model group all have a certain recovery and the fertility is restored. By UC-MSCs, AMSCs intervention and treatment, the chemotherapy ovarian damage animal model has a certain effect, and the treatment effect of intervention with UC-MSCs is better. |
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