| It has been more than one century since the first description of intrauterine adhesions(IUAs).The major causes of IUAs are the traumatic injury to endometrium,infectious factors and the surgery for correction of congenital malformation.Following the development of anesthesia technique,the open of sex attitude and the weak awareness for contraception,uterine cavity operations increase sharply.IUAs may result in hypomenorrhea or amenorrhea,miscarriage,even infertility and recurrent abortion.IUAs has been acknowledged as one of the three unresolved clinical problems in the regenerative medicine: the failure to construct a functional endometrium with the correct morphology in patients with IUAs.Now,comprehensive therapy of IUAs include hysteroscopic adhesiolysis,prevention of adhesion reformation and improvement of endometrium regeneration.Even so,the rate of reformation of IUAs is high(3.1% to 23.5%),especially severe adhesions(20% to 60%).It really need to explore new treatments for regeneration endometrium and improvement of pregnancy outcomes.The endometrium repeat the cycle of proliferation,differentiation and shedding during women's whole reproductive age.The exports proposed that there were stem/progenitor cells in endometrial basal layer to support the scarless regeneration.Chan at al demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential,providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.Taylor HS demonstrated that endometrial cells can originate from donor-derived bone marrow cells and suggest that nonuterine stem cells contribute to the regeneration of endometrial tissue.All of these articles opened a new chapter in the regenerative medicine for endometrial damage.Nowadays,the cell sources focus on the bone marrow mesenchymal stemcells(BMSCs)and embryonic stem cells,or endometrial stem cells derived from menstrual blood.Many difficult issues are involved in the application,such as the ethical issues,complex collection process with high pollution rate and so on.While adipose-derived stem cells(ASCs)is better choice for regeneration of endometrium,as the abundant resource,less ethical arguments and low-immunogenicity.Therefor,the ASCs derived from green fluorescent protein transgenic rat(GFP-ASCs)were chosen.The present study focus on the proliferation,multilineage differentiation potential and the GFP expression of GFP-ASCs,establishment the rat IUAs model.The pathomorphology index and endometrial receptivity were detected after transplantation of GFP-ASCs with or without small intestinal submucosa(SIS)into rat IUAs model.The role of TGF-?1/Smad3 pathway in the pathogenesis of IUAs and the recovery of endometrium were explored for the foundation of new treatment of IUAs.Part one Isolation,culture and identification of GFP-ASCsObjective: to isolate,culture of GFP-ASCs,and to identify the differentiation and the GFP expression of GFP-ASCs.Methods:1 The GFP-ASCs were isolated from adipose tissue of GFP transgenic rat by type ? collagenase digestion under the condition of sterility.2 The expression of surface markers of the third passage ASCs-GFP,such as CD45,CD34,CD90,CD105,CD29,were detected by flow cytometry.The induced osteogenic and adipogenic differentiation was identified by alizarin red and oil red O staining.3 The green fluorescence labeling was observed by fluorescence microscope after DAPI staining in different passages of ASCs-GFP.Results:1 The primary culture of GFP-ASCs attached to the wall at 24 h,polygonal or spindle;the cells showed the long spindle,scattered in the cell colony formation nearly 72h;After 7~9 d of incubation,the cells reached80–90% confluence and a typical sphere-like colony was observed;the cellstend to be stable,a fibroblast like swirling since the second generation cells.2 The results showed that the ASCs-GFP obtained with our method positively expressed high levels of cell antigens of CD29,CD44,CD90,and CD105,but presented low levels of CD34,CD45 antigens.3 The primary,the 1st,the 2nd and the 3rd passage of GFP-ASCs in adherent cells were observed with clear morphology,clear cell outline green fluorescence under the fluorescence microscope.The fluorescence intensity was not attenuated with the increase of passage,the green fluorescence positive expression rate was nearly 100%.4 After culture in the induction medium,ASCs-GFP presented an expanded cell morphology and demonstrated positive staining of Oil red-O solution,indicating the lipid droplets formed on the cytoplasms of cells.After the induction of bone formation,the mineralized nodules were red after Alizarin red staining.5 The cytoactive of ASCs-GFP co-culture with SIS in the high concentration group was higher than that in the low concentration group in the whole process.The trend of the two groups was basically the same,and the highest was in the composite at 24 h,then decreased,and then increased at the beginning of 4 d.Conclusions:GFP-ASCs has a good ability of proliferation and differentiation,and the expression of green fluorescence is stable and not attenuated.It can be used as the source of stem cells transplantation.High concentration of GFP-ASCs co-culture with SIS for 24 h were chosen as the criteria for stem cell transplantation.Part two Establishment of rat IUAs model and evaluation the stability of modelObjective: to establish a stable and successful rat IUAs model,which pathological changes were similar with the clinical change.Methods:1 Forty-eight female SD rats were involved in present study,which werein estrous cycle determined by vaginal smear method.The experimental rats were randomly divided into three groups:(1)dual injury group: side uterine horns were curette with micro scratch and placed a soaked lipopolysaccharide(LPS)cotton,which were pulled out after 48 h;(2)chemical injury group:phenol mucilage 0.03 ml was injected into uterine horns;(3)the normal control group.2 Four rats were sacrificed of each group at different time points after operation: 2 d,7 d,14 d,21 d.The thickness of endometrium,the number of glands and fibrosis area were observed and calculated by HE and Masson staining,for comprehensive evaluation of intrauterine adhesions.3 The expression of keratin(AE1/AE3)was detected by immunohistochemistry.Results:1 The changes of endometrium HE staining:(1)dual injury group: 2 d after injury,the integrity of the visible endometrial epithelial line was destroyed,generation of the epithelial cells was barely visible.A large number of intrauterine tissue debris shedding,fibrin exudation,inflammatory cell infiltration were in uterine cavity.Interstitial edema,slight collagen fibers were observed in the stroma.7 d after injury,some highly fibrous adhesion was observed in uterine cavity,the cavity of uterus was shred and scarce.On the 14 th day,the uterine cavity was further reduced and nonfunctional flattened epithelial cells covered the areas without adhesions.Then 21 days after the injury,the endothelial morphology had failed to reconstruct and had formed a scar,which resembled that seen after 14 days.(2)chemical injury group: 2 d after injury,cell nuclei were disappeared,rupture and lost the normal morphology;the lining were burned necrosis,and infiltration of inflammatory cells were observed in endometrial layer and muscle layer junction;7 d after injury,fibrous hyperplasia adhesion was formed,epithelial cells covered the residual endometrial parts.There was little coverage,new interstitial glands;14 d and 21 d after injury,uterine cavity slightly restored,columnar epithelial cells covered the endometrial cavity,interstitial glandincreased,no inflammatory cells infiltrated.2 Evaluation of the morphological changes:(1)The number of endometrial glands in two injury groups were obviously less than that in the normal control group at 2 d,7 d and 14 d after injury(P<0.05);the number in dual injury group was still less than that in normal control group(P<0.05),while there was no difference in chemical injury group and the normal group(P>0.05);(2)The endometrial thickness in two injury groups were less than that in normal group at 2 d,14 d and 21 d after injury(P<0.05).(3)The fibrosis area percent of dual injury group was higher than that of normal group(P<0.05)at 7 d,14 d and 21 d after injury,but there was no significant difference in the fibrosis area percent between chemical injury group and normal group(P>0.05).3 The expression of AE1/AE3 in two injury group were significantly lower than that in normal control group 2 d,7 d and 14 d after injury(P<0.05).The level of AE1/AE3 was still less in dual injury group than that in normal control group(P<0.05),while there was no difference between chemical injury group and normal group(P>0.05).Conclusions:The chemical injury and the curette infectious injury can cause endometrial damage and form intrauterine adhesions.The model of dual injury is stable and successful,and it is the ideal animal model for the further stem cells transplantation research.Part three Evaluation of efficiency of GFP-ASCs transplantation for the treatment of IUAsObjective: To study whether small intestine submucosa(SIS)could be potential material for IUAs,to evaluate the efficacy of ASC-GFP in situ injection or composite SIS transplantation in the treatment of IUAs.Methods:1 Different treatments were given to the rats at 7d after establishment dual injury model.2 The experimental groups:(1)Model group: the rats only underwenton-off abdomen without any treatment.(2)Gel group: each side uterine horn were treated with 0.3 ml auto-cross-linked hyaluronic acid gel injected into uterine cavity isolation.(3)ASCs group: each side uterine horn were given1x106 GFP-ASCs along the longitudinal axis of intramural injection;(4)SIS group: SIS combined with mould were implanted into uterine cavity.(5)ASCs+SIS group: 1x106 GFP-ASCs composite SIS were transplanted into the bilateral uterine horns.Uterine specimens were collected at 7 d,14 d and 21 d after treatment.3 Uterine specimens collected for the following tests:1)The number of endometrial glands and the endometrial thickness were observed with HE staining.The fibrosis area percent were calculated by IPP6.0 through Masson staining.2)Receptivity factors,such as leukocyte inhibitory factor(LIF),homeboxe-A10(HOXA10),and gene pathogenesis related factors,such as TGF-beta1,Smad3 were detected by qRT-PCR after extraction mRNA from uterine tissue.3)The expression of TGF-beta1 and Smad3 protein in the uterus tissue was detected by Western blot.4)The expression and location of epithelial keratin(AE1/AE3)and estrogen receptor-?(ER-?)was detected by immunohistochemical staining.5)The localization of GFP-ASCs in frozen sections of uterus was observed by fluorescence microscope.4 Three rats were involved in each group: the right side horns in rat model were treated and the other ones were left for control.60 days after treatment,the rats were mated with male rats.Evaluated the function of horns by the pregnancy outcomes.Results:1 Changes of pathological morphology indexes:1)The endometrial morphology had different degrees of recovery in four treatment group,especially in Gel group and ASCs+SIS group.Masson staining showed that the formation of adhesions in uterine cavity and moreblue collagen fiber deposition,more messy,irregular arrangement in model group.In Gel group,SIS group and ASCs+SIS group,the fibers were lighter in color and became shallower with the prolongation of treatment time.The fiber deposition of ASCs group was serious,and the arrangement was a little messy.2)The number of endometrial glands: at 7 d,there was no significant difference among all 5 groups(P>0.05).At 14 d,the number of glands in Gel group and ASCs+SIS group were significantly higher than that in model group(3.75 vs 2.75,P=0.019 3.94 vs 2.75,P=0.009);the number in ASCs+SIS group was higher than those in ASC group and SIS group(3.94 vs 2.93,P=0.019;3.94 vs 2.44,P=0.001).At 21 d,the number of glands Gel group and ASCs+SIS group were significantly higher than that in model group(5.56 vs2.62,P=0.000 4.21;vs 2.62,P=0.033);the number of gland in SIS group was similar to that in ASCs+SIS group(4.21 vs 3.94,P=0.001).3)The thickness of endometrium: at 7 d,the thickness were higher ASCs group and ASCs+SIS group than that in model group(636.06 ?m vs 419.88?m,P=0.000;457.47 ?m vs 419.88 ?m,P=0.018),and higher than that of Gel group(636.06 ?m vs 396.62 ?m,P=0.000;457.47 ?m vs 396.62 ?m;P=0.006).At 14 d,the thickness in ASCs group and ASCs+SIS group were higher than that in model group(528.98 ?m vs 315.92 ?m,P=0.008;498.33 ?m vs 315.92?m,P=0.022).At 21 d,endometrial thickness in four treatment groups were thicker than that in model group(561.98 ?m vs 319.02 ?m,P=0.009;776.22?m vs 319.02 ?m,P=0.000;590.11 ?m vs 319.02 ?m,P=0.004;538.03 ?m vs319.02 ?m,P=0.018).4)The fibrosis area percent: at 7 d,the fibrosis area percent was lowest in SIS group(14.50%).At 14 d,the percent in Gel group,SIS group and ASCs+SIS group were lower than that of model group(16.33% vs 30.44%P=0.000;29.40% vs 30.44%,P=0.002;16.18% vs 30.44%,P=0.000).At 21 d,the degree of fibrosis was lower in SIS group and ASCs+SIS group than that of model group(12% vs 20.08%,P=0.000;11.28% vs 20.08%,P=0.002).2 The expression level of TGF-beta1,Smad3,Hoxa10 and LIF mRNA1)TGF-beta1 mRNA: at 7 d,the level of TGF-beta1 was lower in Gel group and SIS group than that in model group(0.55-fold vs 1-fold,P=0.039;0.42-fold vs 1-fold,P=0.010).At 14 d,the level of TGF-beta1 in Gel group and ASCs+SIS group was lower than that of model group(0.49-fold vs 1-fold P=0.001;0.48-fold vs 1-fold,P=0.001);the difference among all groups was no significant at 21 d(P>0.05).2)Smad3 mRNA: at 14 d,the expression of Smad3 in ASCs+SIS group was lower than that in model group(0.73-fold vs 1-fold,P=0.033).At 7 d and21 d,there were no significant differences among groups(P>0.05).3)Hoxa10 mRNA: at 7 d,there were no significant differences among groups(P>0.05).At 14 d,the expression of Hoxa10 in Gel group,SIS group and ASCs+SIS group were significantly higher than that in model group(4.17-fold vs 1-fold,P=0.006;4.84-fold vs 1-fold,P=0.020;4.02-fold vs;1-fold,P=0.026).At 21 d,the expression of Hoxa10 in Gel group and ASCs+SIS group were higher than that in model group(4.47-fold vs 1-fold,P=0.031;5.61-fold vs 1-fold,P=0.005).Hoxa10 mRNA expression in ASCs+SIS group was higher than that in group ASCs(5.61-fold vs 1.64-fold,P=0.015).4)LIF mRNA: at 7 d,there were no significant differences among all groups.At 14 d,the expression level of LIF mRNA was significantly higher in ASCs group and ASCs+SIS group than that in model group(1.76-fold vs1-fold,P=0.002;2.46-fold vs 1-fold,P=0.000).At 21 d,the level in ASCs+SIS group was significantly higher than that in model group(2.21-fold vs,1-fold,P=0.027).3 Western blot1)The expression level of TGF-beta1 protein: at 7 d after treatment,the expression of Gel group,SIS group and ASCs+SIS group was lower than that of model group and ASCs group.At 14 d,the expression of the four treatment groups were slightly less.At 21 d,TGF-beta1 expression of five groups were similar.2)The expression level of Smad3 protein: at 7 d after treatment,theSmad3 expression in SIS group and ASCs+SIS group were lower than those in model group,Gel group and ASCs group.At 14 d,the expression in Gel group and SIS group was decreased,lower than that in model group.At 21 d,Smad3 expression in four treatment groups and model group were similar.4 Immunohistochemistry1)The expression of AE1/AE3 protein: at 7 d,the expression of AE1/AE3 in Gel group and SIS group were significantly higher than that in model group(P=0.011;P=0.000).At 14 d,the expression in Gel group and SIS group was significantly higher than that in model group(P=0.001;P=0.049).At 21 d,the expression of AE1/AE3 was still significantly higher in Gel group,SIS group and ASCs+SIS group than the model group(P=0.002;P=0.000;P=0.008).2)The expression of ER-? protein: at 7 d,the expression of ER-? in Gel group and SIS group were significantly higher than that in model group(P=0.004;P=0.000).At 14 d,the expression in Gel group was significantly higher than that in model group(P=0.004).At 21 d,the expression of AE1/AE3 in Gel group,SIS group and ASCs+SIS group were significantly higher than that in the model group(P=0.001;P=0.001;P=0.000).5 The GFP-ASCs were observed in endometrial stroma in frozen section in ASCs group and ASCs+SIS group after transplantation under immunofluorescence microscopy.The presence of GFP-ASCs were decreased with the extension of time for observation in ASCs+SIS group.Non GFP-ASCs were found in glandular epithelial position.6 Pregnancy outcomesThe fetus number in left horns were 0~3.Embryos in Gel treatment horns increased to 6~9.Embryos of ASCs treatment horns were 2~5.Embryos in SIS treatment horns were 4~6.Embryos in ASCs+SIS treatment horns increased to 7~9,which was similar to the Gel treatment group.Conclusions:1 Auto-cross-linked hyaluronic acid gel can effectively prevent the IUAs adhesion formation,help to improve the endometrial morphology,andimprove the endometrial receptivity for better pregnancy outcomes.2 ASCs in situ muscle layer injection can not significantly improve the endometrial regeneration.3 SIS with or without ASCs transplantation could significantly improve the endometrial pathological index,reduce fibrosis,promote endometrial growth,improve endometrial receptivity,improve pregnancy.The effect of SIS with ASCs is better. |
PDF Full Text Request