| With the opening of the third child in recent years with the adjustment of the fertility policy and the increase of the average age of childbirth,the demand for fertility has increased.Proper endometrial thickness is essential for embryo implantation,positioning,adhesion,implantation and successful pregnancy.With environmental changes,increasing number and frequency of abortions,and advancing age,the incidence of thin endometrium increases each year,and the resulting incidence of irregular menstruation,secondary infertility,and recurrent miscarriage gradually increases.It has been shown that endometrial thickness is an independent high-risk factor affecting pregnancy excluding embryonic chromosomal quality,immune factors and other factors,and a predictor of poor pregnancy outcome,and improving endometrial receptivity has become a major challenge that needs to be addressed.With the extensive development of regenerative medicine,regenerative therapies based on cell therapy and tissue repair offer hope for the treatment of this group of infertile patients.Various MSCs from different sources have been successfully reported in endometrial damage repair,validating the safety and efficacy of stem cell therapy,and paracrine secretion of stem cells is now considered as the main mediator of MSC-based therapy.Umbilical cord mesenchymal stem cells(UCMSC)and their exosomes play an important role in improving thin endometrial tolerance and increasing pregnancy rates by increasing endometrial thickness and subendometrial blood flow and inhibiting the expression of endometrial fibrosis-related factors.We used TGF-β1 to pretreat UCMSC to obtain high-quality umbilical cord mesenchymal stem cell exosomes(UCMSC-EXO);we innovated on the traditional method of applying anhydrous ethanol modelling by using modified ethanol foam to establish an animal model of thin endometrium in SD rats,which reduced the trauma to the endometrium from artificial surgery and better simulated the pathophysiological state of thin endometrium.pathophysiological state;in animal experiments,our study design was divided into four groups,namely the UCMSC group,the UCMSC-EXO group,the model group(negative control)and the sham-operated group.UCMSC and UCMSC-EXO were used to treat the thin endometrium of SD rats respectively.The obtained UCMSC-EXO was mixed with sodium hyaluronate to prepare an exosome-gel mixture(subsequently referred to as UCMSC-EXO group)with good biocompatibility,physical rheological properties and hyaluronic acid stabilized the structure of the exosomes and prolonged the action of the exosomes in the uterine cavity,which was also confirmed by in vivo tracing experiments.After the UCMSC and UCMSC-EXO interventions,we assessed the treatment effect in terms of endometrial thickness,number of glands,subendometrial vascular density,endometrial receptivity markers and pregnancy rate.In order to further verify the pro-angiogenic effects of UCMSC and UCMSC-EXO,it was found that UCMSC and UCMSC-EXO could significantly promote the proliferation and migration ability of human umbilical vein endothelial cells(HUVEC)by cell scoring,Transwell assay and EdU cell proliferation assay;the pro-angiogenic effects of UCMSC and UCMSC-EXO were verified by tubulogenesis assay and chick embryo chorioallantoic membrane angiogenesis assay The pro-angiogenic effect of UCMSC and UCMSC-EXO was verified by tubulogenesis assay and chicken embryonic chorioallantoic membrane angiogenesis assay,and the pro-angiogenic effect of UCMSC-EXO through AKT/VEGF was also verified by TGF-β1 pretreatment.In the in vivo experiment,we divided the experiment into three groups,namely UCMSC-co-culture group,UCMSC-EXO group and control group,acting on human endometrial mesenchymal cells(HESC)respectively.The cell scratch assay and EdU cell proliferation assay verified that both UCMSC and UCMSC-EXO significantly promoted the proliferation ability of HESC.Based on the previous experiments,we verified that TGF-β1 had the strongest inhibitory proliferation and pro-fibrotic effect on HESC at a concentration of 1Ong/ml.After co-cultivation of UCMSC with TGF-β1 for 48 hours,and the effect of medium containing UCMSC-EXO on HESC for 48 hours,we verified that both UCMSC and UCMSC-EXO significantly promoted the migration and proliferation ability of HESC and inhibited the fibrosis effect by TGF-β1/Smad 2/3 signalling pathway to achieve inhibition of HESC fibrosis.We found that UCMSC mainly achieves its pro-proliferative and fibrosis inhibitory effects through paracrine action,UCMSC-EXO is the main component of UCMSC paracrine secretion,and there may be relevant fibrosis inhibitory factors in UCMSC-EXO.The miR-199a-3p was found to be up-regulated and able to bind to the TGFB1 3’UTR after intersection with the target gene prediction databases starBase,Targetscan and RNA22.The relative fluorescence intensity of the TGFB1 3’UTR wild-type vector was significantly reduced by overexpression of miR-199a-3p,indicating that miR-199a-3p can bind directly to the TGFB1 3’UTR.This validates that UCMSC may downregulate TGFB1 via miR-199a-3p,thereby inhibiting the fibrosis process via the TGF-β1/smad2/3 signaling pathway.Part Ⅰ Umbilical cord mesenchymal stem cells and exosomes improve the receptivity of thin endometrium in ratsObjectiveTo establish an animal model of thin endometrium and investigate the effect of umbilical cord mesenchymal stem cells and their exosomes on improving the tolerance of thin endometrium in ratsMethodsA rat thin endometrium model was established by a modified method,umbilical cord MSCs were extracted and cultured by tissue apposition method,human umbilical cord MSCs were pretreated by TGF-β1,and optimized umbilical cord MSC exosomes were obtained by ultracentrifugation,which were used to treat the rat thin endometrium model,respectively,and their effects on the tolerance of thin endometrium were verified by H&E staining,Masson trichrome staining,immunohistochemistry,qRT-PCR,Western-Blot,in vivo tracing and fertilization assays,The effects of umbilical cord MSCs and their exosomes on the tolerance of thin endometrium were verified by H&E staining,Masson trichrome staining,immunohistochemistry,qRT-PCR,Western-Blot,in vivo tracing and fertilization assays.Results1.Changes in endometrial H&E and Masson’s trichrome stainingAfter modeling,the endometrial layer became thinner,the endometrial glands were reduced and the original intact structure was lost.In the other groups(UCMSC and UCMSC-exosome),the thickness of the endometrial layer was significantly increased and the number of glands was significantly increased.In addition,the structure of the endometrial layer in the UCMSC and UCMSC-exosome groups was closest to that of the sham-operated group.in the H&E stained images,it was seen that the endometrium of the model group was severely damaged,with sparse glands and blood vessels and severe thinning of the endometrium.Compared to the endometrial thickness and number of glands in the model group(25.263±0.501,5.25±2.630),the UCMSC group(131.605±22.528,27.000±6.218)and the UCMSC-exosome group(148.407±38.803,24.750±4.272)showed that the regenerated endometrium significantly thickened and the number of glands increased significantly(p<0.0001,p<0.001,respectively).To assess the collagen remodeling and inhibit fibrosis in the damaged endometrium after UCMSC and UCMSC-exosome treatment of thin endometrium in rats,Masson trichrome staining was performed and the collagen staining area was quantified.After modeling,the uterine cavity was narrowed and bruising occurred in the model group,whereas bruising was significantly reduced in the UCMSC and UCMSC-exosome groups,and collagen staining was significantly reduced compared with the model group.Histograms showed that collagen deposition was significantly reduced in the treatment group(UCMSC/UCMSC-exosome)compared to the model group(p<0.0001,respectively).2.Immunohistochemical staining of waveform proteins and cytokeratinsWaveform proteins and cytokeratins are expressed in the cytoplasm of endometrial epithelial and stromal cells,respectively.After endometrial injury,early and rapid re-epithelialization is essential for subsequent endometrial recovery.Waveform proteins and cytokeratins are markers of endometrial recovery.After treatment,the number of cells positively expressing waveform proteins and cytokeratins increased in the UCMSC group and UCMSC-exosome group.Compared to the model group,the UCMSC/UCMSC-exosome group showed a significant upregulation of the percentage of waveform protein(p<0.001,p<0.001,respectively)and cytokeratin(p<0.001,respectively)positive regions.3.Immunohistochemical staining of VEGF and CD34Promotion of microangiogenesis is an important part of tissue repair,therefore,the expression of vascular endothelial growth factor A(VEGF A),a specific vascular endothelial cell growth promoting factor,was assessed by immunohistochemical staining after treatment.the number of VEGFA positive cells was significantly higher in the UCMSC/UCMSC-exosome group than in the model group.Positive expression of VEGFA was significantly higher in the treatment group(UCMSC/UCMSC-exosome)than in the model group(p<0.001 and p<0.05,respectively).To investigate the effect of UCMSC/UCMSC-exosome on promoting subintimal microvascular regeneration,we performed immunohistochemical staining for CD34,which has high sensitivity and moderate specificity and is the most widely used immunohistochemical marker in angiogenesis and microvessel density studies,and was positive on paraffin sections,ice blocks and protein blotting techniques.CD34 expression is commonly used to assess micro vessel densities(MVD),and MVD counts are based on the WEIDNER method(isolated brownish-yellow vascular endothelial cells or clusters of cells represent a single vessel,and the area with the highest yellowish-brown density is now searched for under low magnification,and the number of vessels in it is counted against a high background,and the number of microvessels in three fields of view is recorded and averaged).mean value),the results showed that the microvessel density(MVD)in the UCMSC/UCMSC-exosome group was significantly higher than that in the model group(p<0.01,respectively).4.LIF immunofluorescence stainingLIF is essential for maternal meconium formation,which is produced by endometrial glands that act on the endometrial epithelium to accept blastocyst attachment and on the stroma to meconiumize it for implantation and placental development.immunofluorescence staining for LIF(red fluorescence)showed that its protein expression was significantly higher in the UCMSC/UCMSC-exosome group,similar to the sham-operated group,but almost no expression in the model group.5.Fertilization experiment in rats with thin endometriumWe further evaluated whether UCMSC/UCMSC-exosome could improve pregnancy outcomes in a thin endometrial rat model.The pregnancy rate of rats in the sham-operated group was 100%,while the pregnancy rates in the UCMSC/UCMSC-exosome group were 60%and 80%,respectively,which were significantly higher than those in the model group(p<0.0001).The sham-operated group had the highest number of embryos implanted(n=5)with 13,17,15,15 and 13 embryos,respectively,and no embryos were implanted in the model group.The number of implanted embryos was significantly higher in the UCMSC/UCMSC-exosome group compared to the model group,with the number of embryos implanted at 4,9,7,0,0,and 16,18,13,5,0,respectively(p<0.0001).more embryos were implanted in the UCMSC-exosome group than in the UCMSC group,but the difference was not statistically significant.6.PCR and westernblotting to detect the expression of molecular markers related to endometrial fibrosis and tolerogenicityPCR was used to detect the expression of mRNA related to endometrial fibrosis and tolerability after treatment.Endometrial fibrosis is considered to be an important pathological change in thin endometrium.Compared with the model group,the mRNA expression of fibrosis-related factors,such as TGF-β1,CTGF,PAI-1 and Collage-I,was significantly reduced in three groups,the UCMSC group,the UCMSC-exosome group and the sham-operated group,after treatment.In contrast,mRNA expression of factors related to endometrial tolerance,such as VEGF and LIF,was significantly higher in the UCMSC and UCMSC-exosome groups than in the model group.western blotting experiments verified that expression markers of fibrosis-related factors,such as TGF-βl,CTGF,PAI-1 and Collage-I,were expressed in UCMSC and UCMSC-exosome groups were significantly reduced,and the trend was consistent with the trend of PCR results.Similarly,the protein expression levels of waveform proteins,cytokeratin,integrin β3,LIF and VEGF associated with endometrial tolerance were significantly higher in the UCMSC and UCMSC-exosome groups than in the model group,consistent with the PCR validation results.7.In vivo tracing of intrauterine injection of UCMSC and UCMSC-exosome hydrogelTo verify the time and site of UCMSC and UCMSC-exosome retention in vivo,UCMSC and UCMSC-exosome were successfully labeled with PKH26 according to the method described in a previous study,thus tracking the time and site of UCMSC-exosome retention in vivo.We investigated the retention and distribution of UCMSC and UCMSC-exosome hydrogels at different time points(24h,48h and 72h)using in vivo fluorescence imaging.The results showed that in live rats,the UCMSC-exosome hydrogel group retained significantly enhanced fluorescence intensity in the endometrial injury zone compared with the UCMSC group(P<0.01,respectively).Conclusion1.A modified approach to modeling thin endometrium in rats reduces artificial damage to the endometrium and reduces the occurrence of hydrocele,abdominal adhesions and necrosis.2.Both UCMSC and UCMSC-exosome significantly inhibited the expression of fibrosis-related factors;significantly improved the tolerance of thin endometrium and increased the pregnancy rate in rats.3.Higher concentration of UCMSC-exosome obtained after TGF-β1 pretreatment,which better promotes the proliferation of endometrial mesenchymal cells.4.Sodium hyaluronate gel mixed with exosomes can stabilize the structure of exosomes and prolong the action of exosomes in the body.Part II Mechanism of umbilical cord stem cells and their secretions promoting angiogenesis through AKT/VEGF pathwayObjectiveThe previous study found that endometrial VEGF and CD31 expression were significantly decreased after endometrial injury in rats,and the endometrium was thinner;after treatment with umbilical cord mesenchymal stem cells and their exosomes(UCMSC/UCMSC-EXO),the damaged endometrium not only had a significant increase in VEGF and CD31 expression,but also the endometrium was better repaired;this indicates that umbilical cord mesenchymal stem cells and their This suggests that the umbilical cord MSCs and their exosomes may repair the damaged endometrium and improve the fertility of the damaged endometrium by promoting endometrial vascularization.However,the mechanism by which umbilical cord MSCs and their exosomes promote angiogenesis is not clear.In this section,we would like to further explore the ability of umbilical cord stem cells and their exosomes to promote angiogenesis(including endothelial cell proliferation,apoptosis,migration,and hematopoiesis)by paracrine action,and to explore the possible signaling pathways involved,so as to provide a more adequate theoretical basis for the application of umbilical cord stem cells and their exosomes in endometrial injury repair.To provide a more adequate theoretical basis for the application of umbilical cord stem cells and their exosomes in endometrial injury repair.MethodsThe pro-angiogenic mechanism of umbilical cord stem cells and their exosomes was mainly verified by in vitro experiments.The previous experiments have fully verified that stem cells improve the tolerance of thin endometrium mainly through the paracrine effect,by promoting the angiogenesis and subendothelial blood flow and inhibiting the fibrosis of damaged endometrial cells,and have verified that the effect of exosomes is similar or even better than that of stem cells.We have verified through animal experiments at the histological,mRNA and protein levels that umbilical cord MSCs improve the tolerance of thin endometrium in rats mainly through paracrine effect,and that the effect of exosomes is similar to or even better than that of stem cell therapy,and we have optimized the extraction and preparation process of exosomes,and the quality of exosomes optimized with TGF-β1 is higher and the therapeutic effect is more stable.)and TGF-β1-optimized exosomes(EXO-tgfβ)on the proliferation,migration ability and angiogenesis of human umbilical vein endothelial cells,and further examine the therapeutic effect and optimal concentration of exosomes.This part mainly verifies the molecular mechanism of pro-angiogenesis of stem cell exosomes and provides a theoretical basis for stem cell regenerative medicine research.Results1.Umbilical Cord Mesenchymal Stem Cell Exosomes Promote Scratch Repair and Migration Capacity of HUVECWe optimized the extraction and preparation process of exosomes,and the content of exosomes optimized using TGF-β1 was higher and more purified.We measured the concentrations of conventionally extracted exosomes(EXO group)and optimized exosomes(EXO-tgf β group)by BCA method,and then dissolved each in DMEM medium containing 2%FBS at the same concentration,and incubated the HUVEC in six-well plates for scratching experiments.The results showed that both EXO and EXO-tgf β groups significantly increased the proliferation ability of HUVEC,and the effect of EXO-tgf β group was better than that of EXO group.We conducted transwell migration assay and found that both EXO group and EXO-tgfβ group significantly increased the migration ability of HUVEC,and the migration ability of HUVEC in EXO-tgfβ group was stronger in the same time.2.Umbilical cord mesenchymal stem cells and their exosomes Promoting the proliferative capacity of HUVECTo further confirm the effect of umbilical cord MSCs and their exosomes on HUVEC proliferation,HUVEC were co-cultured with UCMSC as well as UCMSC-EXO-containing medium for 24 h.DNA replication(green fluorescence)was detected using 5-ethynyl-2’-deoxyuridine(EdU).in the UCMSC group and UCMSC-EXO group DNA replication of HUVEC was significantly greater than that of the control group.3.Pro-angiogenic effect of umbilical cord MSC exosomesIn addition,we evaluated the potential pro-angiogenic effect of umbilical cord stem cell exosomes using stromal gel angiogenesis assay.New capillary networks with good lumen structure were observed in the EXO and EXO-tgf β groups 5 hours after inoculation of HUVEC onto stromal gel,and the formation of vascular-like network structures was significantly increased in the EXO-tgf β group.Quantitative analysis of the number of rings,tube length and area of vessels showed that umbilical cord stem cell exosomes promoted HUVEC vessel formation(P<0.05).4.Pro-angiogenic effect of umbilical cord mesenchymal stem cells and their exosomes in the membrane of chick embryonic chorionic vesicleThrough the chick embryo chorionic villus allantoic membrane experiment,we found that more angiogenesis was observed in both the umbilical cord MSC group and the exosome group compared to the control group,respectively,with more graded small vessels.And the optimized exosome group had significantly more capillaries of grade IV and grade V capillaries.5.Umbilical cord mesenchymal stem cell exosomes promote HUVEC microvasculogenesis and development by regulating the expression of angiogenesis-related factorsThe exosomes of umbilical cord MSCs were applied to HUVEC,and after 24h,48h and 72h of incubation,the RNA of each group of cells was extracted and PCR assay was performed.We found that the expression of VEGFa and VEGFc,factors related to the promotion of angiogenesis,was significantly increased,and VEGFb was decreased at 24-48h of action and started to increase after 72h of action,which is a slow regulator;while VEGF receptors 1 and 2 increased significantly after 24h and decreased rapidly after 48h;the expression of ANG1,an angiogenesis-inhibiting factor,decreased significantly,while ANG2,eNOS and TIE2 increased after 24h and decreased significantly after 48h.The expression of pro-angiogenic factors such as VEGFa,VEGFb,VEGFc,and VEGFR1 was significantly increased in the EXO-tgf β group,and was stable in the EXO-tgf βgroup.The expression of VEGFR2 and angiogenesis-inhibiting factors ANG1 and TIE2 were decreased in both groups.In contrast,ANG2 expression was significantly higher in the EXO-tgf β group and significantly lower in the EXO group.6.Umbilical cord mesenchymal stem cell exosomes increase HUVEC downstream target gene expression through activation of AKT pathwayAKT signaling pathway plays an important role in angiogenesis,and in the present study,we also found that AKT phosphorylation levels in HUVEC were significantly increased in expression after the action of umbilical cord mesenchymal stem cell exosome(UCMSC-EXO).And the expression of its downstream target gene,VEGF,was also significantly increased.In addition,we applied qRT-PCR to determine the transcript levels of AKT downstream target genes,including VEGF family genes,Ang family genes and eNOS,all of which are associated with angiogenesis.The results showed that the expression of Tie2,eNOS,VEGFa,VEGFb,VEGFc and VEGFR1 was significantly increased in all EXO groups and EXO-tgfβ group compared to the control group.These data suggest that exosomes can induce overexpression of angiogenesis-related factors,consistent with the finding that exosomes promote endothelial cell growth,migration and angiogenesis.Conclusion1.UCMSC and UCMSC-exosome can promote the proliferation and migration of HUVEC2.The pro-angiogenic effects of UCMSC and UCMSC-exosome were verified by tubule formation and chick embryo chorioallantoic membrane angiogenesis experiments3.The pro-angiogenic effect of UCMSC-exosome through AKT/P-AKT/VEGF was verified by mRNA and protein levelsPart Ⅲ Umbilical cord mesenchymal stem cells and their secretions pass through TGF-β/Study on the mechanism of Smad2/3 pathway inhibiting endometrial fibrosisObjectiveTo better understand the pathogenesis of endometrial fibrosis in patients with uterine adhesions and to find effective treatments,this study was conducted to verify the role of TGF-β1 in promoting the transformation of endometrial cells into myofibroblasts using in vitro experiments,and to determine whether transmural stem cells reduce endometrial fibrosis and promote endometrial repair by reversing the pro-fibrotic effect of TGF&.The results of this study are as followsMethodsThe differentiation of UCMSC to HESC was verified by isolating endometrial mesenchymal cells from human endometrium and performing primary culture,and the differentiation of UCMSC to endometrial mesenchymal cell-like cells was verified by UCMSC-Transwell co-culture group and chemical induction;the proliferation of UCMSC and its exosomes was verified by scratch assay and EdU proliferation assay on The repair and pro-proliferative effects of UCMSC and its exosomes on HESC were verified by scratch assay and EdU proliferation assay;the pro-fibrotic effect of TGF-β1 on HESC was verified by applying UCMSC and its exosomes to TGF-β1 pretreated HESC for 48 hours,and the expression of fibrosis-related molecules was verified by mRNA and protein levels.In order to explore the upstream post-transcriptional regulation mechanism of TGFB1 in thin endometrium,we sequenced exosomal miRNAs and found the top 10 miRNAs differentially expressed,then combined with the target gene prediction databases starBase,Targetscan and RNA22 online prediction,we found a total of 13 miRNAs might bind to TGFB1 After intersecting the above three databases with the sequencing results,we found that only miR-199a-3p was up-regulated and could bind to the TGFB1 3’UTR,so we selected miR-199a-3p as the target for further study.We verified whether miR-199a-3p could directly bind to TGFB1 3’UTR by double luciferase reporter gene assay.Results1.Endometrial mesenchymal cells isolation,primary culture and identification resultsEndometrial mesenchymal cells were isolated and primary cultured,and the isolated endometrial mesenchymal cells grew against the wall after 24 hours and spread across the bottom of the dish after 3-4 days(see Figure 3-1 A).Cellular immunofluorescence identified mesenchymal cells positive for vimentin(see Figure 3-1C),and flow cytometry assay analysis revealed typical endometrial mesenchymal cells(E-cadherinlow Vimentinhigh)2.Results of UCMSC differentiation to endometrial mesenchymal cell-like cellsAfter 3 weeks of culture in the control group,UCMSC-Transwell co-culture group and chemical induction group,the cell morphology of the three groups was observed under the light microscope,and it can be seen in Figure 3-2 A.Compared with the control group,the cells in the co-culture group with HESC were still long spindle-forming fibroblast-like cells,with a smaller number of cells,but they were still arranged in a swirling pattern,and features similar to the distribution of endometrial mesenchymal cells in clusters could be observed;the chemical In the chemically induced group,most of the cells were long spindle-forming fibroblast-like cells,and fewer endometrial mesenchymal cells were observed,with atypical distribution in clusters.The three groups were examined by cellular immunofluorescence for waveform protein expression(Figure 3-2B).From the quantitative fluorescence analysis,it was seen that the expression of waveform protein was more obvious in the group of endometrial mesenchymal cells co-cultured with HESC.3.TGF-β1 inhibits endometrial mesenchymal cell proliferation and promotes fibrosisIn order to clarify the optimal action time and concentration of TGF-β1 to inhibit the growth of endometrial mesenchymal stromal cells in vitro,TGF-β1 was formulated at six concentrations of 0,1.5.10 and 15 ng/ml,added to DMED/F12 culture medium of endometrial mesenchymal cells,and incubated for 24,48 and 72 hours,respectively.The results are shown in Figure 3,with the increase of the concentration of TGF-β1,its effect of inhibiting the proliferation of endometrial mesenchymal stromal cells was more significant,and at the three time points of 24,48 and 72 hours,it showed that the inhibitory effect of TGF-β1 was significantly stronger at the concentration of 10 ng/ml than at the concentrations of 0,1,5 and 15 ng/ml(p<0.05)(Figure 3-3B).The inhibitory effect of TGF-β1 was also enhanced gradually with the extension of the action time,and the inhibitory effect was significant after 48 hours of action(Figure 3-3A).In order to achieve the optimal effect of TGF-β1 with the minimum concentration and the shortest duration of action,we chose the concentration of 10 ng/ml as the optimal concentration of TGF-β1 and 48 hours as the optimal duration of action.After pretreatment of endometrial mesenchymal cells with TGF-β1 for 48 hours,the fibrosis-related factors PAI-1,CTGF,SMA and Collagen-1 were significantly increased by PCR(Figure 3-3C).4.Transwell co-culture and UCMSC conditioned medium enhance the proliferation of damaged HESCEndometrial mesenchymal cells were co-cultured with UCMSC or UCMSC-conditioned medium(UCMSC-CM)at a concentration of 10 ng/ml of TGF-β1 for 48 hours,and the proliferation ability of damaged endometrial mesenchymal cells was observed and tested.The results showed that UCMSC significantly improved the proliferative ability of damaged endometrial mesenchymal cells(P<0.05),and the proliferative ability of damaged mesenchymal cells was restored more significantly as the co-culture time increased.5.UCMSC and UCMSC-EXO reduce endometrial fibrosis by regulating TGF-β1 to inhibit Smad2/3 phosphorylation and promote HESC migration and proliferationTo verify the effect of UCMSC and UCMSC-EXO on HESC,cell migration was analyzed by cell scratch assay.The results showed that the migration potential of the UCMSC and UCMSC-EXO groups was greater than that of the control group after 24 h(p<0.0001 and p<0.0001.respectively)(Figure 3-9A).To further confirm the effect of proliferation on HESC,DNA replication(green fluorescence)was detected using 5-ethynyl-2’-deoxyuridine(EdU)after 24 h of incubation in UCMSC-EXO medium.DNA replication of HESC was significantly greater in the UCMSC and UCMSC-EXO groups than in the control group(p<0.05 and p<0.05)(Figure 3-9B).TGF-β1 is considered to be one of the most important regulators of endometrial fibrosis.To investigate whether UCMSC and UCMSC-EXO attenuate endometrial fibrosis by inhibiting the TGF-β1/smad2/3 signaling pathway,we examined the expression of TGF-β1,smad2/3 and phosphorylated smad2/3(p-smad2/3)in HESC by western blotting(Figure 3-10).The protein expression levels of TGF-β1.smad2/3 and p-smad2/3 were significantly lower in the UCMSC-EXO and UCMSC groups compared to the control group(p<0.01,p<0.001 and p<0.0001,respectively).In addition,the expression of other fibrotic factors(PAI-1,CTGF and col-1)was also significantly reduced compared to the control group.In conclusion,the results suggest that UCMSC-EXO and UCMSC groups inhibited the TGF-β1/Smad2/3 signaling pathway in fibrosis of HESCs.In addition,the UCMSC-EXO group showed stronger inhibition of fibrosis-related proteins such as p-samd3,CTGF and TGF-β1.6.Screening and validation of regulatory molecules at the post-transcriptional level upstream of TGFB1 UCMSC-exosome regulates TGFB1 inhibition of fibrosis via miRNA199a-3pIn order to explore the upstream post-transcriptional regulation mechanism of TGFB1 in thin endometrium,we sequenced exosomal miRNAs and found the top 10 miRNAs differentially expressed,then combined with the target gene prediction databases starBase,Targetscan and RNA22 online prediction,we found that a total of 13 miRNAs might bind to TGFB1 After intersecting the above three databases with the sequencing results,we found that only miR-199a-3p was up-regulated and could bind to the TGFB1 3’UTR(Figure 3-16A),so we selected miR-199a-3p as the target for further study.miR-199a-3p was found to bind to the TGFB1 mRNA,and miR-199a-3p was found to bind to the TGFB1 mRNA.The binding site of miR-199a-3p to TGFB1 mRNA was located at the position of 290-296 bases of TGFB13’UTR(Figure 3-16B).We constructed TGFB1 3’UTR wild-type vectors and mutant vectors with deletion mutations at the binding site(Figure 3-16C).The results of the dual-luciferase reporter gene assay showed that overexpression of miR-199a-3p significantly reduced the relative fluorescence intensity of the TGFB1 3’UTR wild-type vector,but not that of the mutant vector(Figure 3-16D).It indicates that miR-199a-3p can directly bind to the TGFB1 3’UTR.Conclusion1.UCMSC can differentiate into endometrial mesenchymal cell-like cells,but mainly acts through paracrine secretion2.UCMSC-exosome reduces endometrial fibrosis by inhibiting the TGF-β/smad2/3 signaling pathway and decreasing the expression of its downstream fibrosis-related factors3.UCMSC-exosome suppresses TGFB1 gene expression by upregulating miRNA-199a-3p and thus attenuates fibrosis... |
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