The Efect And Mechanism Of Human Amnion Epithelial Cells On Repair Of Injured Endomertium In Rats

Intrauterine adhesions(IUA)is a common disease caused as a result of uterine endometrium basal layer injury(curettage、uterine operations infections,congenital malformations,etc).IUA induce endometrial fibrosis and absence of functional endometrium followed by complications such as oligomenorrhea、amenorrhea、repeated miscarriage and infertility.Recently,increasing number of uterine operations lead to the incidence of IUA rise rapidly and IUA has become one of the important reasons of female secondary infertility.Considering the damage of it,various methods have been applied for treatment.IUA treatment measures are mainly hysteroscopic adhesiolysis and then supplemented with measures such as intrauterine devices(IUD)placement and estrogen administration to prevent adhesion re-formation.While these methods addressed low cure rate,easy to relapse and low pregnant rate.The recurrence of adhesion after treatment is 20 % ~ 62.5 %,and it is difficult to solve the problems of infertility and endometrial regeneration.In recent years,many studies have reported that IUA might be related to the absence and damage of endometrial stem cells.Thus,more therapies of stem cells are urgently needed.Human amnion epithelial cells(hAECs)are obtained from the amnion and displayed the feature of multipotent stem cells,which could differentiate into all of the three germ layers(endoderm,ectoderm and mesoderm)in vitro and vivo,considering as a candidate and ideal source of cell therapy.The isolation from placenta poses few ethical concerns regarding the collection method and from which in excess of 100 million cells can be obtained;hAECs lack the telomerase activity and do not cause cancer.Additionally,hAECs secrete plenty of cytokines and anti-inflammatory factors due to paracrine activity.Previous studies reported that hAECs had already been applied in several fields,such as treatment of nervous system diseases,lung and liver fibrosis repair,the treatment of premature ovarian insufficiency and myocardial infarction.This study is to explore the effects and underlying mechanism of hAECs transplantation on repair of injured endometrium in rats and to provide theoretical basis for clinical treatment of IUA.PART ⅠSEPARATION,CULTURE,IDENTIFICATION AND LABEL OF HUMAN AMNION EPITHELIAL CELLSOBJECTIVE: To establish a method of the separation,extraction and culture of hAECs and investigate the phenotypic features of hAECs.PKH26 labeled hAECs was compared the effects on cell proliferation activity with unlabeled cells.METHODS:(1)hAECs were saparated and extracted from human amnion of term placentas and cultured with RPMI-1640 complete medium containing 12% fetal bovine serum(FBS).(2)The phenotypic features of hAECs was analyzed by flow cytometry and immunofluorescence to identify the primary cultured hAECs.(3)PKH26 was applied to label hAECs,the rate of PKH26-labeling cells was assessed by flow cytometry,and the cell proliferation of PKH26-labeled hAECs was detected by CCK-8 assay.RESULTS:(1)72 hours after inoculation of the primary cultured hAECs,60%-70% of the primary cultured hAECs were adhered and dot-shaped or fusiform-shaped.After that,the cells gradually fused into a piece and showed the cobblestone-like epithelial cell shape;After 9-12 d,the hAECs were harvested for the first passage.(2)The hAECs expressed OCT4,SSEA4,NANOG,CK18,CD324,CD29,and CD166,but did not express HLA-DR,HLA-DQ,CD146,CD34,and CD45,indicating that the cultured cells were hAECs(3)the rate of PKH26-labeling cells was 96.95±3.05%,which did not reduce after cell passaging.There was no significant difference in cell proliferation between PKH26-labeled and unlabeled hAECs.CONCLUSION: Viable hAECs with stable phenotype can be obtained from amnion;PKH26 is a stable method to tracing the migration,distribution and colonization of hAECs in vivo with high labeling rate,and not affect the proliferation activity of hAECs.PART Ⅱ THE ROLE OF HUMAN AMNION EPITHELIAL CELLS ON REPAIR OF INJURED ENDOMERTIUM IN RATSOBJECTIVE: To investigate the effect of h AECs transplantation in vivo on damaged endometrium in rats,and to provide a theoretical basis for clinical treatment of IUA.METHODS:(1)12 female Sprague Dawley rats(8-10 weeks)with regular estrous were selected to establish the intrauterine adhesion model by mechanical injury method in this study.12 female Sprague Dawley rats were randomly grouped into two:(1)Normal control group(n=6);(2)Model group(n=6):using a 5-mm endometrial curette to scrape the upper two-third of the uterus until the uterine wall became rough.7 days after modeling,rats were scarified and bilateral uterines were collected.Morphological changes of the endometrium were observed by HE staining and glandular counts were observed among groups under microscope.The degree of endometrial fibrosis was observed by Masson staining.Fibrosis histological feature were evaluated by the fibrosis rate(excluding the uterine cavity).(2)96 female Sprague Dawley rats(8-10 weeks)with regular estrous were selected in this study.96 female Sprague Dawley rats were randomly grouped into four:(1)Normal control group(n=24);(2)Model group(n=24): only received mechanical injury;(3)h AECs intrauterine injection group(n=24):7 days after mechanical injury,the uterine was exposed and h AECs(5x106 cell In 300μ l)suspensions were injected inside each one,then suturing immediately;(4)h AECs vein injection group(n=24):the animals received h AECs(5x106 cell In 300μ l)via the tail vein one week after mechanical injury.At 2 and 4 weeks after h AECs transplantation,rats were scarified and relevant experimental samples were collected.Trace the migration,distribution and colonization of h AECs in vivo.Morphological changes of the endometrium were obseved by HE staining,vessel numbers and glandular counts were observed among groups under microscope,and endometrial thickness was measured by the vertical distances between serous membranes and luminal surfaces.The degree of endometrial fibrosis was observed by Masson staining.Fibrosis histological feature were evaluated by the fibrosis rate(excluding the uterine cavity)of four randomly HPF.At the same time,the other rats in each group were caged at the corresponding time point to analyze the effect of h AECs transplantation on the fertility of the rat IUA models.RESULTS:(1)Rat IUA models were established by mechanical damage.7 days after modeling,the uterus of medel group showed discontinuous uterine cavity surface and the uterine cavity was narrow.Compared with the normal control group,the number of endometrial glands was significantly reduced and the fibrotic area was significantly increased in model group.These results suggested that mechanical injury leads to a successful establishment of a rat model of intrauterine adhsions.(2)The location of transplanted PKH26-labeled h AECs in endometrium was traced after cell transplantation.The results showed that PKH26-labeled h AECs(the red fluorescent signal)were mainly located to the basal layer of rat endometrium after transplantation in h AECs uterine injection group and vein injection group.In control group,the luminal surface of endometrium had abundant endometrial glands.And endometrial fibrotic areas were rarely observed.In model group,decreased endometrial glands and increased ECM collagen depositions were observed in the endometrial cavity.Endometrial thickness,endometrial vessel number,the number of endometrial glands,fibrosis area rate presented a significant difference in rats with h AECs transplantation compared with that of model group.HE staining revealed that endometrial thicknesses,vessel number,and glands number were significantly higher in h AECs uterine injection group and vein injection group compared with that in model group after transplantation.Masson staining validated that fibrotic areas after transplantation were significantly decreased compared with that of model group.(3)Female rats were bred after h AECs transplantation.Fetuses within the uteri were found at gestation day 15.The pregnant rate in both uterine injection group(83.33%)and tail vein injection group(83.33%)was significantly higher than that in model group(50%),while all of the rats in control group were pregnant。Besides,the average number of embryos in uterine injection group(9.5),tail vein injection group(9)and control group(12.5)was significantly higher than that in model group(2.8).CONCLUSION: Mechanical injury method can establish a stable and effective rat IUA animal models;h AECs transplantation via uterine and tail vein had an important role in the regeneration of injured endometrium to a certain extent and could significantly promoted the implantation of embryos.PART Ⅲ THE MECHANISM OF HUMAN AMNION EPITHELIAL CELLS ON REPAIR OF INJURED ENDOMERTIUM IN RATSOBJECTIVE: To explore the possible mechanism of h AECs transplantation in vivo on repair of damaged endometrium in rats,and to provide a theoretical basis for clinical treatment of IUA.METHODS:(1)72 female Sprague Dawley rats(8-10 weeks)with regular estrous were selected in this study.72 female Sprague Dawley rats were randomly grouped into four:(1)Normal control group(n=18);(2)Model group(n=18): only received mechanical injury;(3)h AECs intrauterine injection group(n=18):7 days after mechanical injury,the uterine was exposed and h AECs(5x106 cell In 300μ l)suspensions were injected inside each one,then suturing immediately;(4)h AECs vein injection group(n=18):the animals received h AECs(5x106 cell In 300μ l)via the tail vein one week after mechanical injury.At 2 weeks after h AECs transplantation,rats were scarified and relevant experimental samples were collected.(2)The expression level of basic fibroblast growth factor(b FGF),vascular endothelial growth factor(VEGF),insulin-like growth factor-1(IGF-1),collagen type I alpha 1(COL1A1),tissue inhibitor of metalloproteinase-1(TIMP-1)and transforming growth factor-β(TGFβ)was analyzed by IHC analysis and RT-PCR analysis.Differential m RNA analysis was performed on rat uterine tissues using RNA sequencing technology,and the differentially expressed genes were selected and verified by RT-PCR analysis.RESULTS:(1)The IHC and RT-PCR result showed that the expression of growth factors(b FGF,VEGF and IGF)in h AECs uterine injection group and vein injection group was higher than that of model group and normal control group.As for ECM deposition related cytokines(COL1A1,TIMP and TGFβ),its expression levels increased apparently in model group compared with control group.Moreover,transplanted h AECs significantly down-regulated the expression of these cytokines compared with model group.(2)Totally,the expression of 1035 m RNAs were found to be different between h AECs uterine injection group(h AECs uterine transplantation group)and model group,including 600 genes that were upregulated and 435 genes that were downregulated.The differentially expressed genes between h AECs treatment group and model group were mainly enriched for PI3K-Akt,Wnt,Chemokine,c AMP,protein processing in endoplasmic reticulum and cytokine-mediated signaling pathways.Three differentially expressed genes(PDGF-C,THBS1,and CTGF)of PI3K-Akt signaling pathway which is involved in regulating tissue fibrosis,two differentially expressed genes(Wnt5a and Snai2)of Wnt signaling pathway which is involved in regulating embryo development and tissue regeneration,and two differentially expressed genes(Rho A and Rock)of Chemokine signaling pathway which is involved in cellular migration processes during embryonic development were selected to be analyzed in h AECs-regulated restoration of IUAs.In our study,we examined the expression of PDGF-C,THBS1,CTGF,Wnt5 a,Snai2,Rock and Rho A at the m RNA level by RT-PCR.The results showed that h AECs transplantation significantly inhibited PDGF-C,THBS1 and CTGF level while Wnt5 a,Snai2,Rock and Rho A were significantly increased after h AECs transplantation.These results were consistent with our microarray analysis.CONCLUSION: h AECs transplantation may promote the repair of damaged endometrial tissues by increasing the expression of b FGF,VEGF and IGF-1;it may inhibite ECM deposition by down-regulating the expression of COL1A1,TIMP-1,TGF-β,PDGF-C,THBS1 and CTGF;may promote embryo implantation by promoting the secretion of Wnt5 a,Snai2,Rock and Rho A.