The Study With The Refine-effect Of Human Umbilical Cord Mesenchymalstem Cells Exosomes On The Chronicsalpingitis Of SD Rats

Backgroud: Based on statistics by the World Health Organization(WHO),the rate of infertility among women is 10-15% [1].Pelvic factors account for about 35% of infertility cases;among those,ovarian anatomy and functional damage caused by chronic salpingitis are common causes of infertility.Traditional medications and surgical treatments may have some therapeutic effect on mild or early fallopian tube inflammation,but a large proportion of patients eventually rely on assisted reproductive techniques(ART).With the increase in the birth rate of IVF,ART is facing its complications: the risk of neonatal birth defects and adverse perinatal outcomes.Therefore,it is necessary to explore new ways to repair the fallopian tube inflammatory injury,restore the fallopian tube's reproductive function,and strive to make gamete(sperm/egg)combination,growth,and development under natural state.Mesenchymal stem cells(MSCs)are increasingly becoming the pearl of stem cell therapy because of their unique capacity for renewal and differentiation.The clinical advantages of mesenchymal stem cells derived from human umbilical cord(h UC-MSCs)are confirmed because of their unique low immunogenicity,extremely rich content and non-invasive access.h UC-MSCs transplantation treatment has unique anti-inflammatory and anti-fibrosis qualities that promote tissue repair and regeneration,and h UC-MSC is,therefore,the first choice for cell transplantation.However,the use of h UC-MSCs for clinical applications is limited due to their drawbacks,such as potential tumorigenicity.Recently,we found that MSCs' "paracrine" mechanism plays an important role in the treatment of diseases,so MSCs' secretion factor has become the focus of our research.Exosomes are a subcellular structure complex secreted by a membrane of small vesicles in living cells,and it can mediate information transfer between cells.The biological properties of exosomes are more clear than those of other vesicles.Exosomes have obvious advantages for clinical applications: 1)They can be used to repair damaged tissue;2)they have stable chemical properties;3)they have a high tissue selectivity;4)they feature targeted delivery;5)they are easy to store and facilitate transportation.It has been found that exocrine from MSCs may play a part in their biological functions and may be used to treat a variety of clinical diseases.In this study,h UC-MSCs-derived exosomes were used in the intervention of an SD rat model of chronic inflammatory injury in the fallopian tube.The changes in the structure of the fallopian tube,the secretion function,and the conception rate of MSCs in the human umbilical cord were evaluated.Our study explored new approaches to the treatment of salpingitis infertility using human umbilical cord mesenchymal stem cells exosomes(h UC-MSCs-Ex).Chap 1 The preparation and identification of hUC-MSCs derived exosomesObjective: To explore the preparation method of h UC-MSCs-Ex and to prepare freeze-dried powder for convenience.Methods: The hUC-MSCs were isolated by the tissue adherence method and the cells were identified by morphological identification.The specificity of cell surface-specific markers and cell differentiation were identified.The molecular weight and size of the protein were determined by SDS-PAGE gel electrophoresis.Western blot was used to identify the protein CD63 and CD81.The protein was identified by the freeze-thawing method.Results: Dry method hUC-MSCs derived from the secretions made of freeze-dried powder.The h UC-MSCs-Ex prepared by modified differential centrifugation method was used to observe the morphological structure under the transmission electron microscope,and the morphology of the h UC-MSCs was round or oval.The expression of the marker protein CD63 and CD81 was successfully obtained.The h UC-MSCs-Ex freeze-dried powder was obtained successfully.Conclusion: The hUC-MSCs derived from hUC-MSCs prepared by modified differential centrifugation have high purity and can be freeze-dried to powder,which provides a relatively stable and reliable source for subsequent in vitro and in vivo experiments.Chap 2 In vitro inflammation of h UC-MSCs derived exosomesObjective: To investigate the effect of hUC-MSCs-Ex on inflammation in vitro by the in vitro macrophage RAW264.7 tectonuric inflammatory model.Methods: LPS-stimulated macrophage RAW264.7 was used to construct the model of in vitro cell inflammation.The experiment was divided into 5 groups:(1)blank control group(NC): no drug treatment was added;(2)LPS model group(LPS): 0.1?g/ml LPS stimulation;(3)exosomes low-concentration group(Ex-L): 0.1?g/ml LPS stimulation after adding 15?g/ml exosomes;(4)exosomes medium-concentration group(Ex-M): 0.1?g/ml LPS stimulation after adding 45?g/ml exosomes;(5)exosomes high-concentration group(Ex-H): 0.1?g/ml LPS stimulation was followed by adding 135?g/ml of exosomes.The expression of tumor necrosis factor-? was detected by Western blot.Results: The h UC-MSCs-Ex down-regulated the expression of tumor necrosis factor-? and inhibited the inflammatory response induced by LPS in vitro.With the increase of exosome concentration,h UC-MSCs-Ex showed the anti-inflammation effect.Conclusion: The h UC-MSCs-Ex can effectively inhibit LPS-induced in vitro inflammatory response.Chap 3 Establishment of Chronic Fallopian Infant Model in SD Female RatsObjective: To establish an animal model of chronic inflammation of the fallopian tube by injecting 3×10~8/ml of Escherichia coli(single bacteria)into the bilateral oviduct of SD female rats.Methods: Fifty SD rats were randomly divided into two groups: control group(n= 10)and Escherichia coli(n= 40).Surgery was performed to open the peritoneal cavity to inject the Escherichia coli into the bilateral oviduct of SD female rats.On the 5th,10 th,15th,and 20 th day after the operation,the formation of tubal inflammation was assessed.Results: The rats in the experimental group had developed chronic inflammation of the fallopian tube by the 15 th day after the operation.Conclusion: Escherichia coli(single bacteria)can produce inflammation in the fallopian tube of SD female rats and be used to establish a model of chronic inflammation.Chap 4 Intervention and evaluation of h UC-MSCs derived exosomes in rats with chronic salpingitis injuryObjective: To evaluate the therapeutic effect of h UC-MSCs-Ex on chronic salpingitis in infertile rats by intraperitoneal injection of h UC-MSCs-Ex to treat chronic oviduct inflammation in SD female rats;to compare the changes in the structure and function of the heart,liver,and kidney in the normal group and in the treatment group,and to understand the safety of h UC-MSCs-Ex on SD rats in the present therapeutic dose.Methods: 3×10~8/ml Escherichia coli(single bacteria)suspension was injected into the bilateral oviduct of SD female rats to establish the animal model of chronic inflammation of the fallopian tube.The experiment was divided into 4 groups(n= 20 in each group):(1)Sham group(sham operation-model control group),no drug treatment;(2)group(model control group),after 2 weeks of modeling,intraperitoneal injection of 0.9% sodium chloride injection,0.5ml//only/day for 2 weeks;(3)Ex-A group(model group),after one day of modeling with h UC-MSCs-Ex lyophilized powder diluted with 0.9% sodium chloride injection,concentration of 120?g/ml,dose of 0.5ml/ only/day,intraperitoneal injection for 2 w eeks;(4)Ex-B group(post-modeling administration group),after 2 weeks of modeling with 0.9% sodium chloride injection diluted h UC-MSCs-Ex freeze-dried powder,concentration of 120?g/ml,dose 0.5ml/only/day,intraperitoneal injection for 2 weeks.After two weeks of treatment,half of the rats were euthanized(i.e.,ten rats in each group).Changes in tumor necrosis factor-? and interferon-? in the serum of each group were observed.Changes of DPF-1 and its mRNA in tubal tissue were detected by a Western blot and real-time quantitative PCR.Ovarian fibrosis was observed by staining.The remaining ten rats in each group were compared with the male rats,and so was the size of their stool.At the same time,the biochemical indexes of the rats were compared,and the structure of the heart,liver,and kidney were studied under a light microscope.Results: After the transplantation of h UC-MSCs-Ex in Ex-A and Ex-B groups,the structure of the fallopian tubes recovered(P<0.05).The levels of DPF-1 and mRNA in groups Ex-A and Ex-B were significantly higher than those in group M(P<0.05).The proliferation of tubal collagen in Ex-A and Ex-B was significantly lower than in group M(P<0.05).The concentration of tumor necrosis factor-? in Ex-A was significantly lower than in group M(P<0.05).The tumor necrosis factor-? concentration in Ex-B group was lower than that in M group,but the difference was not statistically significant(P>0.05).The concentration of interferon-? in the serum of Ex-A group was significantly lower than that of M group(P<0.05).The concentration of interferon-? in Ex-B group was lower than that in group M(P<0.05).Compared with Ex-B group,the concentration of interferon-? was slightly lower in Ex-A group,but the difference was not statistically significant(P>0.05).The average litter size of Ex-A group was significantly higher than that of M group(P<0.05),and the difference was statistically significant(P<0.05)Sham group compared with the number of litter size and the difference was statistically significant(P<0.05).Compared with the Sham group,the litter size of the Ex-B group was 100%,and the litter size of the Ex-B group was significantly higher than that of the M group(P<0.05);the account wooden dwor was statistically significant(P<0.05).The number of litter size in Ex-A group was slightly higher than that in Ex-B group(P <0.05).The biochemical indexes of each group were normal,and the heart,liver,and kidney of each group were normal.Conclusion: The h UC-MSCs-Ex can restore the reproductive capacity of chronic salpingitis SD female rats to a certain extent.The experimental dose h UC-MSCs-Ex was not found to change the structure and function of the heart,liver,and kidney of SD rats.