| With the incidence rate of malignant tumors increasing year by year,the age of onset gradually decreasing and the age of childbearing postponed,the demand for fertility preservation has increased dramatically.Compared with oocyte and embryo cryopreservation,ovarian tissue transplantation after cryopreservation can restore ovarian endocrine function and fertility,and allow multiple cycles of pregnancy.It is the most promising method of female fertility preservation,especially for unmarried and preadolescent women suffering from cancer.In 2004,the world’s first baby was born by cryopreservation and transplantation of ovarian tissue,which is a milestone in human reproductive medicine.After that,babies born after ovarian tissue transplantation in various places were reported in the past two years.So far,there are more than 130 babies born after cryopreservation and the transplantation of ovarian tissue in the world.Many scholars think that ovarian tissue transplantation can be used as an effective technology.There are two methods for ovarian tissue freezing: programmed freezing and vitrification.Programmed freezing has been used for more than 30 years.Whether vitrification is superior to programmed cryopreservation in ovarian tissue cryopreservation has not been determined yet.However,its superiority in egg and embryo cryopreservation has been proved.Because of the advantages of vitrification,such as no expensive instruments and no ice crystal formation in the process of freezing,it has a bright application prospect.In the past decade,many experiments have studied in different animal models,such as dogs,goats,rats,mice and so on.However,the results are quite different.Based on these experimental results,it is difficult to estimate the degree of ovarian tissue damage caused by vitrification.In addition to species differences,the size of the graft and the choice of cryoprotectants will have an impact on the transplantation effect.However,there is no detailed analysis of the reported results of lost follicles during cryopreservation and transplantation.It is reported in the literature that more follicles may be lost in the process of ovarian tissue transplantation than in the process of frozen ovarian tissue resuscitation.The main reason is ischemia and hypoxia before the blood supply is reestablished.However,the specific degree of follicle loss in the process of ovarian transplantation is still lack of detailed analysis.Compared with other fertility preservation methods,ovarian tissue transplantation has irreplaceable advantages,so it has a good prospect of clinical application.However,a large number of follicles lost in the process of ovarian transplantation,which seriously restricts the development of ovarian transplantation technology.Therefore,how to promote the formation of new blood vessels and restore the blood supply of the graft as early as possible is the key to the success of transplantation.The urinary bladder matrix(UBM)is an extracellular matrix,ECM)that has a complete three-dimensional ultrastructure of the extracellular matrix of living tissue,as well as growth factors,glycosaminoglycans,adhesins and other bioactive components,of which 42 are confirmed to adhere to tissue,promote binding,anti-apoptosis,and promote proliferation.At present,it has been used in clinical bed for skin,fat,muscle and other tissue repairs.Therefore,this study intends to use UBM in rat ovarian autotransplantation,observe the effect of UBM in ovarian transplantation,and explore the mechanism of UBM in improving the effect of ovarian transplantation.Stem cells are widely used in the treatment of ischemic and injurious diseases.Studies have shown that stem cells can promote angiogenesis and tissue repair.Adipose-derived mesenchymal stem cells(ADMSCs)have the advantages of convenient source,strong proliferation,non tumorigenicity and safe use.It has been reported that the survival rate of stem cells can be improved by using appropriate biological scaffolds.Some literature studies have shown that UBM can promote the proliferation and differentiation of stem cells.We tried to inoculate the stem cells on UBM and use them in the rat ovary autotransplantation.We tested whether the application of UBM can increase the effect of ADMSCs and further improve the effect of ovary transplantation.Part 1 The effect of vitrification and autotransplantation of ovary tissue in ratObjective: To observe the effects of vitrification and autotransplantation on ovarian tissue in rats.Methods: SD female rats were randomly divided into control group,vitrification group and autotransplantation group,(n=6 rats in each group).In the transplantation group,ovarian tissues were taken out from both sides,and one was transplanted into the same side of the renal dorsal membrane.In the vitrification group,ovarian tissues were vitrified and resuscitated,HE staining was used to observe the morphology of follicles and the number of follicles.TUNEL fluorescence staining was used to detect apoptosis.Masson staining was used to observe the apoptosis of cells and the ratio of VEGF positive areas in each group.Results: Compared with the control group,the total number of follicles at all levels in the vitrification group and the transplanted group decreased significantly(P < 0.05).The apoptosis rate of the vitrification group and autotransplantation group was significantly higher than that of control group(P < 0.05).VEGF was expressed in ovarian tissues of three groups,but the expression of VEGF in vitrification group and transplantation group was significantly lower than that in control group(P <0.05).Conclusion: Ovarian tissue vitrification or autotransplantation are accompanied by follicular loss and decreased VEGF expression in ovarian tissue.Increasing VEGF expression in the transplanted ovarian tissue may play an active role in promoting angiogenesis and improving the efficiency of ovarian transplantation.Part 2 The effect of UBM in ovarian autotransplantation in ratObjective: To verify that UBM promote neovascularization of transplanted ovary,reduce the loss of follicle number,improve the effect of transplantation,and explore its mechanism of promoting angiogenesis.Methods: 1.The SD rats were divided into five groups: control group,autotransplantation group,autotransplantation + UBM group,autotransplantation + VEGF group and castration group.The control group was used for follicular count comparison,while the castration group was only used for serum hormone level comparison.On the 28 th day after autotransplantation,HE staining was performed to observe cell morphology and follicle count;serum FSH and AMH levels were measured by ELISA;CD31 staining was performed on the 7th and 28 th day after transplantation to observe angiogenesis;on the 7th day after autotransplantation,Tunel and Masson staining were used to observe apoptosis and fibrosis.2.F3 HUVECs were divided into control group and UBM group.UBM group was seeded on the bottom of the six pore plate in advance to observe the cell proliferation.CCK8 experiment was carried out on the 1st,3rd and 5th day after inoculation to detect the cell proliferation.Western blot and RT-PCR were used to detect the mechanism of UBM promoting angiogenesis in vitro.3.The SD rats were divided into two groups: auto-transplantated group and autotransplanted + UBM group(UBM group)according to the different treatment methods during the operation.7 days after the auto-transplantation of ovary,the vascular cast was used to observe the angiogenesis of the two groups.The ovarian tissue was taken out and detected by Western blot and RT-PCR to explore the role of UBM in promoting angiogenesis in vivo System.Results: 1.Compared with the transplantation group,the number of primordial follicles and growth follicles in UBM group increased significantly(P<0.05);The serum FSH level of autransplantated group,UBM group and VEGF group was lower than that of castration group(P < 0.05).FSH in UBM group was significantly lower than that in autograft group and VEGF group(P < 0.05).Compared with the transplantation group and VEGF group,the positive expression rate of CD31 in UBM group increased significantly(P < 0.05);Tunel staining showed that the apoptotic cells in UBM group and VEGF group were decreased compared with the transplantation group(P < 0.05);Masson staining results: compared with transplantation only,the fibrosis area of UBM group was small,and the difference was statistically significant(P < 0.05).2.CCK8 experiment: HUVEC also proliferated well on UBM,and there was no significant difference compared with the control group(P > 0.05);Western blot and RT-PCR results suggested that the expression of KDR,Notch 1 and Ang2 in human umbilical vein endothelial cells increased in UBM group.3.The results of Western blot and RT-PCR showed that the expression of KDR,Notch 1 and Ang2 increased in UBM group.Both in vivo and in vitro experiments showed that UBM upregulated the expression of KDR,Notch 1 and Ang2,and promoted angiogenesis.Conclusion: UBM enhance the expression of KDR,Notch 1 and Ang2,activate the signal pathway of angiogenesis,and improve the effect of ovarian transplantation by promoting angiogenesis.UBM has a good application prospect in ovarian transplantation,which is worth further research and promotion.Part3 The effect of adipose mesenchymal stem cells combined with UBM on ovarian autotransplantation in ratObjective: To verify that ADMSCs can promote neovascularization and improve the effect of ovarian transplantation,and to prove that ADMSCs combined with UBM can further improve the effect of ovarian transplantation.Methods: 1.ADMSCs were isolated and prepared.Their ability of multiple differentiation was identified by Lipogenic and osteogenic experiments,and their surface antigens were determined by flow cytometry.2.The 8-week-old SD rats were divided into 5 groups according to the different treatment methods: control group,autotransplantation group,autotransplantation + ADMSCs group,autotransplantation + ADMSCs + UBM group,castration group.On the 28 th day after autotransplantation,HE staining was performed to observe the cell morphology and follicle count;on the 28 th day after operation,serum FSH and AMH levels were measured by ELISA;CD31 staining was performed on the 7th and 28 th day after transplantation respectively to observe the angiogenesis;on the 7th day after autotransplantation,Masson staining was used to observe the fibrosis.Results: 1.ADMSCs were successfully prepared.2.Compared with the ADMSCs group,the number of primordial follicles and growth follicles in the ADMSCs group increased significantly;Compared with autotransplantation group,FSH level of ADMSCs group and combination group decreased significantly(P < 0.05);CD31 results showed that the number of neovascularization in ADMSCs group and combination group increased compared with that in autotransplanted group;Masson staining results: compared with that in simple autotransplanted group,the fibrovascular area in combination group decreased significantly,the difference was statistically significant.Conclusion: The application of ADMSCs can improve the effect of ovarian transplantation,UBM can promote the attachment and colonization of adipose mesenchymal stem cells in the transplantation site,admsc with UBM can significantly increase the blood supply of the transplanted ovarian tissue. |
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