| Intrauterine adhesion(IUA)caused by endometrial injury is one of the important causes of infertility in women of reproductive age and requires advanced treatment strategies.In this regard,stem cell therapy using adipose-derived mesenchymal stem cells(ASCs)serves as an exciting candidates.With the in-depth research,increasing evidence suggests that the therapeutic effects of mesenchymal stem cells(MSCs)mainly depends on their capacity to secrete paracrine factors,and are mediated by MSCs derived exosomes.Compared with traditional cell therapy,exosomes are more stable in biological activity,easier to preserve,more standardized in extraction process,and free from the risk of tumorigenicity.Therefore,it is expected to be a new strategy for cell-free therapy.This study aimed to explore the therapeutic potential of exosomes derived from adipose-derived mesenchymal stem cells(ASCs-exo)in IUA rat models.Part one Isolation and identification of exosomes derived from adipose mesenchymal stem cellsObjective:To isolate and culture ASCs from rat adipose tissue,and to extract exosomes from the supernatant of the culture medium for subsequent experiments.Methods:1. The bilateral inguinal fat of rats was collected and ASCs was extracted by enzymatic digestion method under the condition of sterility.Flow cytometric analysis was used to identify cell surface markers of ASCs,including CD90,CD105,CD34 and CD45.Adipogenesis and osteogenesis were induced,and differentiation was identified by oil red O and alizarin red staining.2.Exosomes were extracted from ASCs culture supernatant by ultracentrifugation,protein concentration was determined by BCA.The exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis(NTA)and western blot.Results:1.ASCs grew adherently and exhibited fibroblast-like morphology.P3 ASCs growth curve presented:incubation period for the first2 days,entered into proliferation period at 3 days,then the phase of logarithmic growth at 3,4 and 5 days,at last the platform stage at 6 days.The cell surface antigen detected by flow cytometry revealed that the ASCs were positive for CD90 and CD105 and negative for CD34 and CD45.The expression rates were 98.39%,97.99%,0.91%,and 0.38%,respectively.Oil red-O staining for induced ASCs was positive,and presence of lipid droplets demonstrated that ASCs could differentiate into adipogenic lineage.Also,Alizarin red staining showed that a calcified extracellular matrix of induced ASCs was detected,which confirmed ASCs could differentiate into osteogenic cells.2.Morphological analysis of the ASCs-exo using transmission electron microscopy shows classic cup-shaped vesicles that are on average size30-200nm.The protein concentration was about 1.5mg/m L.Nanoparticle tracking analysis showed that the mean of particle size of ASCs-exo were109.5nm,presenting irregular Brownian motion.Western blot analysis of surface marker proteins found that CD63 and Alix protein were expressed in ASCs-exo.Conclusions:1 The enzyme digestion method can successfully isolate and cultureASCs from rat inguinal fat,and can be identified by flow cytometry and multi-directional differentiation.2 The exosomes derived from adipose mesenchymal stem cells can be successfully extracted by ultracentrifugation.The results of transmission electron microscopy,nanoparticle tracking analysis and western blot showed that the extract was consistent with the characteristics of exosomes and could be used in the next experiment.Part two Study on the stability of intrauterine adhesion modelObjective:The animal model of intrauterine adhesion(IUA)in SD rats was established by mechanical and infection double injury method and absolute alcohol chemical injury method.Through this study,we compared the effects of the two methods,hoping to provide an ideal and stable animal model for the study of this clinical problem.Methods:1. SD female rats(n=80)were randomly divided into 3 groups:normal group(n=20),double injury group(n=30)and alcohol injury group(n=30).Vaginal smear was used to observe the estrous cycle of rats.Normal group,rats had laparotomy without any treatment;double injury group,after curettage,the LPS cotton thread was kept in the uterine cavity and removed after 48 hours;alcohol injury group,absolute alcohol was injected into the uterine cavity for 4 minutes to damage the endometrium.2.Three animals were randomly selected from each group to collect uterine tissues:1,2,4,8 and 12 weeks after operation.HE staining and Masson staining were performed to calculate the endometrial thickness,number of glands and fibrotic area,and immunohistochemical staining was performed to observe CD31 expression on endometrium.Results:1. The endometrial thickness of the two injury groups was significantly thinner than that in normal group(P<0.05)after 2 weeks of modeling and tended to be stable.The thickness of endometrium in alcohol injury group was significantly thinner than that in double injury group(P<0.05).2. The number of endometrial glands in the two injury groups was significantly less than that in the normal group(P<0.05)after 1 weeks of modeling,and the number of endometrial glands in the alcohol injury group was significantly less than that in the double injury group(P<0.05).3.The fibrotic area percent of endometrium in the two injury groups was higher than that in the normal group(P<0.05)after 1 week of modeling,but the fibrotic area percent in the alcohol injury group decreased after 8 weeks of modeling(P<0.05).4.The expression of CD31 in endometrial stroma of the two injury groups was significantly lower than that in the normal group(P<0.05)after 1week of modeling,and the alcohol injury group was significantly lower than that in the double injury group(P<0.05).Conclusions:1.Both double injury and alcohol chemical injury can lead to endometrial injury and intrauterine adhesion.2. The double injury model is close to the clinical injury mode,and the fibrosis degree of the model is more stable,which can meet the research needs related to intrauterine adhesion.3. The method of chemical damage with absolute alcohol is simple and economical,but the stability of fibrosis is poor.The endometrial thickness,number of glands and vascular changes are consistent with the pathological changes of thin endometrium,which is more suitable for the research related to thin endometrium diseases.Part three Evaluation of efficiency of ASCs-exo transplantation for the treatment of IUAObjective:The purpose of this study is to investigate the efficacy of ASCs-exo in an established intrauterine adhesion rat model.Methods:1.The dual injury method with more stable fibrosis degree was selected to construct the model,and different treatment were given after 2 weeks of modeling.2.Forty-eight rats were subdivided into 4 groups,12 rats in each group:1)normal control group,rats had laparotomy without any treatment during the modeling and treatment;2)IUA group,after 2 weeks of modeling,0.2 m L PBS was given in each side of uterine horn;3)ASCs group,after 2 weeks of modeling,0.2m L cell suspension(including 1×10~6ASCs)was given in each side of uterine horn;4)ASCs-exo group,after 2 weeks of modeling,0.2m L ASCs-exo suspension(100?g)was given in each side of uterine horn.Four weeks after treatments,24 rats(n=6 per group)were sacrificed at their estrous phase,and the uterine tissues were harvested for the next experiment.3. The uterine tissues were collected and the morphological changes and fibrosis were monitored after treatment by HE/Masson staining.IPP6.0 was used to calculate endometrial thickness,number of glands and the mean area percentage of collagen fiber.Immunohistochemical staining was used to observe the recovery of endometrial vessels(CD31)and epithelium(CK7)in the endometrium.4. Receptivity factors,such as Integrin?3,LIF and VEGF were detected by Western blot after extraction protein from uterine tissue.5. Functional test:Four weeks after treatment,female rats from 4experimental groups(n=6 per group)and fertile males were cohabitated in a1:1 ratio for a period of 8 weeks.The time to conceive,the number of pregnant rats and implanted embryos were recorded.Results:1.Changes of pathological morphology indexes1)The endometrial thickness:the endometrial thickness of the IUA group was significantly thinner than that in the normal group(526.90±79.68?m vs738.29±18.16?m,P=0.009).After 4 weeks of treatment,the thickness of the ASCs group and ASCs-exo group was significantly thicker than that in the IUA group(797.38±45.26?m vs 526.90±79.68?m,P=0.001;782.45±43.40?m vs 526.90±79.68?m,P=0.002);and there was no significant difference in endometrial thickness between ASCs group and ASCs-exo group(797.38±45.26?m vs 782.45±43.40?m,P=0.840).2)The number of endometrial glands:the number of endometrial glands in the IUA group was significantly smaller than that in the normal group(4.96±1.25 vs 8.17±0.47,P=103),but the difference was not statistically significant.After 4 weeks of treatment,the number of endometrial glands in the ASCs and ASCs-exo groups was significantly larger than that in the IUA group(13.13±1.55 vs 4.96±1.25,P=0.000;12.04±1.69 vs 4.96±1.25,P=0.001);There was no significant difference in the number of endometrial glands between ASCs group and ASCs-exo group(13.13±1.55 vs 12.04±1.69,P=0.570).3)The percent of fibrosis area:the percent of fibrosis area in the IUA group was significantly larger than that in the normal group(50.23%±1.99%vs 34.65%±3.55%,P=0.000).After 4 weeks of treatment,the percent of fibrosis area in ASCs-exo group was significantly smaller than that in the IUA group(36.90%±1.03%vs 50.23%±1.99%,P=0.001);the percent of fibrosis area in ASCs group was smaller than that in IUA group(48.56%±2.41%vs50.23%±1.99%,P=0.630),the difference was not statistically significant;the percent of fibrosis area in ASCs-exo group was significantly smaller than that in ASCs group(36.90%±1.03%vs 48.56%±2.41%,P=0.003).2.Immunohistochemistry1)CD31 expression:CD31,which was a sign of neovascularization,was decreased in the IUA group.After 4 weeks of treatment,CD31 expression in the ASCs group and ASCs-exo group were higher than that in the IUA group,and the ASCs-exo group was slightly more than the ASCs group.2)CK7 expression:only a small amount of CK7 was expressed in the IUA group.After 4 weeks of treatment,CK7 expression in the ASCs and ASCs-exo groups were significantly higher than the IUA group,and the ASCs-exo group was slightly more than the ASCs group,which was a sign of neoepithelialization.3.Western blot analysis1)The expression level of Integrin?3 protein:the expression of integrin?3 in IUA group was significantly lower than that in normal group(P=0.000).After 4 weeks of treatment,the expression of integrin?3 in ASCs group and ASCs-exo group was significantly higher than that in IUA group(P=0.000;P=0.009);and the expression of integrin?3 in ASCs group was significantly higher than that in ASCs-exo group(P=0.015).2)The expression level of LIF protein:the expression of LIF in IUA group was significantly lower than that in normal group(P=0.021).After 4weeks of treatment,the expression of LIF in ASCs group and ASCs-exo group was significantly higher than that in IUA group(P=0.014;P=0.026);there was no significant difference between ASCs group and ASCs-exo group(P=0.783).3)The expression level of VEGF protein:the expression of VEGF in IUA group was significantly lower than that in normal group(P=0.005).After 4weeks of treatment,the expression of VEGF in ASCs-exo group was significantly higher than that in IUA group(P=0.004);and higher than that in ASCs group(P=0.018).4.Functional test1)Pregnancy rate:After 4 weeks of caging,the pregnancy rate in the IUA group was significantly lower than that in the normal group(16.67%vs100.00%,P<0.05).ASCs group and ASCs-exo group were higher than IUA group(50.00%vs 16.67%,P>0.05;83.33%vs 16.67%,P>0.05),and ASCs-exo group was higher than ASCs group,but the differences were not statistically significant.After 8 weeks of caging,the pregnancy rate in the IUA group was still lower than that in the normal group(50.00%vs 100.00%,P>0.05),ASCs group and ASCs-exo group were higher than IUA group(66.67%vs 50.00%,P>0.05;83.33%vs 50.00%,P>0.05),and ASCs-exo group was higher than ASCs group,but the differences were not statistically significant.2)The number of implanted embryos:After 8 weeks of caging,the number of implanted embryos in the IUA group was significantly lower than that in the normal group(1.00±0.52 vs 9.83±0.79,P<0.05);The ASCs group and ASCs-exo group were significantly more than the IUA group(6.67±2.22vs 1.00±0.52,P<0.05;5.00±1.10 vs 1.00±0.52,P<0.05)and the difference was statistically significant;The ASCs group was slightly more than the ASCs-exo group(6.67±2.22 vs 5.00±1.10,P>0.05),but the difference was not statistically significant.3)The time to conceive:After 8 weeks of caging,the time to conceive in the IUA group was significantly longer than that in the normal group(42.00±8.79 vs 2.00±0.52,P<0.05).ASCs group and ASCs-exo group were shorter than IUA group(31.50±9.43 vs 42.00±8.79,P>0.05;13.83±8.95 vs42.00±8.79,P>0.05),and ASCs-exo group was shorter than ASCs group(13.83±8.95 vs 31.50±9.43,P>0.05),but the difference was not statistically significant.Conclusions:Both exosomes and ASCs transplantation can improve the morphology of damaged endometrium,reduce the area of interstitial fibrosis,increase endometrial receptivity and improve fertility.However,ASCs-exo seems to be superior to ASCs in terms of receptivity related proteins such as VEGF expression,improvement of fibrosis degree,pregnancy rate and the time to conceive after caging. |
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