| Objective: Intrauterine adhesions (IUA) refers to the whole or part ofintrauterine adhesions, caused by irregular uterine cavity operation, theinduced abortion technique, secondary infection, and so on. The incidence ofIUA is20%-30%. Patients often have the symptoms of menstrual reduce,amenorrhea and infertility. This adverse outcome is mainly manifested in theinfertility crowd gradually increasing. Even if the surgery, the rate ofadhesions forming is50%again. lAlong with the development of assistedreproductive technology, many relevant problems are solved, but the mostfailure cases of assisted pregnancy are caused by intrauterine adhesions orfibrosis.Now the mechanism of intrauterine fibrosis is unclear, there is still a lackof effective means of diagnosis and prevention both at home and abroad.Therefore relevant basic and clinical research is badly need to clarify themechanism of endometrial fibrosis, so as to develop new prevention andtreatment technology.The present study aims to take the human endometrial epithelium cellsand stromal cells as seed cells, Ⅰtype liquid collagen of rat tail and Matrigelglue as biological scaffolds, to investigate the reconstruction of human threedimensional endometrium structure in vitro, and to analyses the morphologyand immunohistochemical features. Thus the beneficial experiment model andthe experiment basis of reconstructing a complete human three dimensionalendometrium structure in vitro can be provided for endometrial study. Fromthe cell, gene and protein level, to investigate the effect Ang-(l-7) and AngⅡon endometrium and the number and activity of endometrial stromal cells andendometrial epithelium cells; the new ideas and theoretical basis can be provided for the prevention of endometrial adhesion or fibrosis.Methods:1The isolation, purification, culture and characterization of humanendometrial epithelium cells (EECs) and endometrial stromal cells (ESCs)EECs and ESCs from human endometrium at the proliferative phasewere isolated by using enzymolysis and differential centrifugation method.The amount of living cells and the cell vitality were observed by using4%trypan blue staining; Morphology and structure of endometrial cells wereobserved with a light microscope, inverted phase contrast microscope,Hematoxylin and eosin (H&E) staining; Endomentrial cells were verified byimmunocytochemical staining. The different concentration combination ofestrogen hormone and progestational hormone to promote the proliferation ofendometrium cells were examined by MTT method.2Restruction, culture and characterization of human three dimensionalendometrium structure in vitroⅠ type liquid collagen of rat tail was prepared, Ⅰtype liquid collagen ofrat tail and Matrigel as biological scaffolds, the human endometrial epitheliumcells at Original generation and stromal cells at the proliferative phase as seedcells, endometrial stromal cells were vaccined in collagen to form the stromalcells-collagen compound, Waiting for the solidification, Matrigel was coveredon the compound, endometrial epithelium cells were vaccined on the Matrigel,the Tissue-engineered endometrium was reconstructed according to the naturalendometrial structure. Hematoxylin and eosin staining andimmunohistochemical staining were used to analyses Morphology andstructure of engineered endometrium.3The effect of AngⅡon the proliferation of EECsThe EECs were cultured in vitro, the effect of different concentrationAngⅡ(10-6mol/L,10-7mol/L,10-8mol/L,10-9mol/L) on the proliferation ofEECs was observed by MTT mothed for24h,48h and72h.4The effect of Ang (1-7) and AngⅡon the proliferation of EECsThe EECs were cultured in vitro, the effect of different concentration Ang-(1-7)(10-5mol/L,10-6mol/L,10-7mol/L,10-8mol/L) and AngⅡ(10-6mol/L)on the proliferation of EECs was observed by MTT mothed for24h,48h and72h.5The effect of Ang(1-7) and AngⅡon the activity of EECsThe EECs were cultured, then the cells were divided into control group,AngⅡ(10-6mol/L) group, Ang-(1-7)(10-5mol/L) group and AngⅡ(10-6mol/L)+Ang-(1-7)(10-5mol/L) group. The protein expression level of α-SMA andE-cadherin was detected by the method of immunocytochemistry and Westernblot when the cells were cultured for72h; The content of Fibronectin (FN)and collagen type I (Col I) in the cultured supernatants was measured byenzyme-linked immunosorbent assay (ELISA); The mRNA expression levelof α-SMA, E-cadherin, FN and Col I was detected by real-time poly merasechain reaction (real-time PCR).6The effect of AngⅡon the proliferation of ESCsThe human ESCs were cultured in vitro, the effect of differentconcentration AngⅡ(10-6mol/L,10-7mol/L,10-8mol/L,10-9mol/L) on theproliferation of ESCs was observed by MTT mothed for24h,48h and72h.7The effect of Ang(1-7) and AngⅡon the proliferation of ESCsThe ESCs were cultured in vitro, the effect of different concentrationAng(1-7)(10-5mol/L,10-6mol/L,10-7mol/L,10-8mol/L) and AngⅡ(10-6mol/L)on the proliferation of ESCs was observed by MTT mothed for24h,48h and72h.8The effect of Ang (1-7) and AngⅡon the activity of ESCsThe ESCs were cultured, then the cells were divided into control group,AngⅡ(10-6mol/L)group, Ang-(1-7)(10-5mol/L) group and AngⅡ(10-6mol/L)+Ang-(1-7)(10-5mol/L)group. The protein expression level of α-SMA,transforming growth factor β1(TGF-β1) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry and Westernblot when the cells were cultured for72h; The content of TGF-β1, IGF-I, ColI and FN in the cultured supernatants was measured by ELISA; The mRNAexpression level of α-SMA, TGF-β1, IGF-I, Col I and FN was detected by real-time PCR.9The effect of Ang-(1-7) and AngⅡon the activity of the Tissue-engineeredendometriumThe human Tissue-engineered endometrial were cultured, then humanTissue-engineered endometrial were divided into control group, AngⅡ(10-6mol/L)group, Ang-(1-7)(10-5mol/L) group and AngⅡ(10-6mol/L)+Ang-(1-7)(10-5mol/L); The protein expression level of α-SMA, TGF-β1and IGF-1endometrial was test by Western blot; The mRNA expression level of α-SMA,TGF-β1and IGF-I was detected by real-time PCR.Results:1The isolation, purification, culture and characterization of human EECs andESCsThe EECs were observed under an inverted phase-contrast microscope: Amass of EECs which were just isolated were clouds shaped, the purity ofEECs can reach about90%by the using differential centrifugation method,Trypan blue staining showed that the viability of EECs was92%～96%, thecells attached growth after cultured for24h. Most of EECs formed tightlypacked whorls and could grow to confluence after3day culture, a single EECshowed the round or elliptical, the EEC had a single nuclear which was large,round and located in the center of the cell, its nucleoli was observed obviously.EECs could not pass generation with DMEM/F12and10%fetal calf serumculture.H&E staining showed: a single EEC showed the round or elliptical underlight microscope after3days culture, the EEC had a single nuclear which waslarge, round and located in the center of the cell, its nucleoli was observedobviously; Cytoplasmic relatively more, granular and eosinophilic.The result of immunocytochemical staining showed: the EEC waspositive by stained cytokeratin antibody, the cytoplasm was stained claybank,the nucleus is negative, while the EEC was negative by stained vimentinantibody.The ESCs were observed under an inverted phase-contrast microscope: The ESCs which were just isolated mostly remained round and singlescattered, the purity of ESCs can reach about92%by the using differentialcentrifugation method, Trypan blue staining showed that the viability of ESCwas95%～98%. When they were cultured in the culturing bottle, the cellsattached the bottle after cultured for2h and grew after cultured for12h, thenthey grew as spindle and polygonal-shaped cells with long cytoplasmicprocesses. They usually reached confluence after3～4days culture, ESCscould pass3～4generation with DMEM/F12and10%fetal calf serum culturein vitro, the ESC had a single nuclear which was large, round and located inthe center of the cell, its nucleoli was observed obviously.H&E staining showed: a single ESC showed the spindle, polygon or starunder light microscope after3days culture, the ESC had a single nuclearwhich showed large, round or oval and located in the center of the cell, itsnucleoli was observed obviously and chromatin loose; Cytoplasmic relativelymore, granular and eosinophilic.The result of immunocytochemical staining showed: the ESCs waspositive by stained vimentin antibody, the cytoplasm was stained claybank,the nucleus is negative, while the ESCs was negative by stained cytokeratinantibody.The effect of E2and P4on the proliferation of human endometrial cellsby MTT method: The result showed that the proliferation of EECs wasinsignificantly by the treatment of E2or P4alone (P>0.05), yet a combinationof100nmol/L E2and10nmol/L P4could promote its proliferation greatly(P<0.05). In the case of ESCs, the treatment of100nmol/L P4alone couldpromote the proliferation significantly (P<0.05). The study found that theEECs can be passed to5～6generation when the EECs and a small amount ofESCs were mixed cultured with100nmol/L E2and10nmol/L P4.2Culture and characterization of human three dimensional endometriumstructure in vitroThe three-dimensional tissue-engineered endometrium constructs wascultured with DMEM/F12and10%fetal calf serum. After2or3days, the collagen began to contract. The constructs contracted gradually during thedays of culture. Four glass columns standing in the molds resisted thecontraction and prevented the constructs from thickening. In the meantime,they provided a static stretch.The reconstructed endometria were cultured for14days in vitro.Hematoxylin and eosin staining showed that the EECs and ESCs were in goodcondition, the EECs formed a monolayer on top of the reconstructedendometrium. Many cells in the epithelial layer showed columnar morphologysimilar to luminal epithelial cells in vitro.Immunohistochemical staining showed that the cells in the epitheliallayer were positive by stained cytokeratin antibody, the cytoplasm was stainedclaybank, while the cells in the stromal layer were negative; the cells in thestromal layer were positive by stained vimentin antibody, the cytoplasm wasstained claybank, while the cells in the epithelial layer were negative.3The proliferation effect of AngⅡon the of EECsThe EECs were cultured with different concentration AngⅡfor24h,48hand72h, the number changes of EECs was observed by the detecting purplecrystal of each culture hole absorbance value (A) in550nm, the result showedthat AngⅡcould promote EECs proliferation in the way of dose dependenceand time dependence, that is to say, the longer of incubation time, proliferationeffect of EECs more apparent; the higher concentration of Ang Ⅱ,proliferation effect of EECs more apparent; Compared with the control group,the proliferation effect of EECs in different concentration groups have notstatistical significance for24h(P>0.05), while the proliferation effect of EECsin different concentration groups have statistical significance for48h and72h(P<0.05).4The effect of AngⅡ and Ang-(1-7) on the proliferation of EECsAfter the cells had been cultured for24h,48h and72h, the change innumber of the EECs were observed by the detecting purple crystal in eachculture well at an absorbance value (A) at550nm, the result showed thatcompared with the control group, the number of EECs in the Ang-(1-7) group has no significant change (P>0.05), while AngⅡsignificantly promoted EECsproliferation in a time dependent manner(P<0.05), compared with the AngⅡgroup, the number of EECs was significantly decreased in AngⅡ+Ang-(1-7)group.5The effect of AngⅡand Ang-(1-7) on the activity of EECsThe results of the immunocytochemistry staining and western blotsshowed that compared with the control group, the expression of a-SMA andE-cadherin proteins in the Ang-(1-7) group was not significantly changed, butthe expression level of a-SMA proteins was significantly increased while theexpression level of E-cadherin proteins was significantly decreased in the AngⅡgroup; Compared with the AngⅡgroup, the expression level of a-SMAproteins was significantly decreased while the expression level of E-cadherinproteins was significantly increased in the AngⅡ+Ang-(1-7) group.The result of ELISA showed that compared with the control group, theCol I and FN protein in the cultured supernatants in the Ang-(1-7) group hasno significant change, and increased significantly in AngⅡgroup. Comparedwith the AngⅡgroup, the amount of these proteins in the AngⅡ+Ang-(1-7)group was decreased significantly.The real time PCR showed that compared with the control group, themRNA levels of α-SMA, E-cadherin, Col I and FN in the Ang-(1-7) groupshowed no significant change, and the mRNA levels of α-SMA, Col I and FNwas significantly increased while the mRNA levels of E-cadherin wassignificantly decreased in the AngⅡgroup. Compared with the AngⅡgroup,the mRNA levels of α-SMA, Col I and FN was significantly decreased whilethe mRNA levels of E-cadherin was significantly increased in the AngⅡ+Ang-(1-7) group.6The proliferation effect of AngⅡon the of ESCsThe ESCs were cultured with different concentration AngⅡfor24h,48hand72h. the number changes of ESCs was observed by the detecting purplecrystal of each culture hole absorbance value (A) in550nm, the result showedthat AngⅡcould promote ESCs proliferation in the way of dose dependence and time dependence, that is to say, the longer of incubation time, proliferationeffect of ESCs more apparent; the higher concentration of Ang Ⅱ,proliferation effect of ESCs more apparent; Compared with the control group,the proliferation effect of ESCs in the different concentration AngⅡgroupshave not statistical significance for24h (P>0.05), while the proliferationeffect of ESCs in the different concentration AngⅡgroups have statisticalsignificance for48h and72h (P<0.05).7The effect of AngⅡand Ang-(1-7) on the proliferation of ESCsAfter the cells had been cultured for24h,48h and72h, the change innumber of the ESCs were observed by the detecting purple crystal in eachculture well at an absorbance value (A) at550nm, the result showed thatcompared with the control group, the number of ESCs in the Ang-(1-7) grouphas no significant change (P>0.05), while AngⅡsignificantly promoted ESCsproliferation in a time dependent manner (P<0.05), compared with the AngⅡgroup, the number of EECs was significantly decreased in AngⅡ+Ang-(1-7)(10-5mol/L) group.8The effect of AngⅡand Ang-(1-7) on the activity of ESCsThe results of the immunocytochemistry staining and western blotsshowed that compared with the control group, the expression of α-SMA,TGF-β1and IGF-I proteins in the Ang-(1-7) group was not significantlychanged, but they were significantly increased in the AngⅡgroup; comparedwith the AngⅡgroup, the expression of these proteins in the AngⅡ+Ang-(1-7)group decreased significantly.The result of ELISA showed that compared with the control group, theTGF-β1, IGF-I, FN and Col I protein in the cultured supernatants in theAng-(1-7) group has no significant change, and increased significantly in AngⅡgroup. Compared with the AngⅡgroup, the amount of these proteins in theAngⅡ+Ang-(1-7) group was decreased significantly.The real time PCR showed that compared with the control group, themRNA levels of α-SMA, TGF-β1, IGF-I, FN and Col I in the Ang-(1-7) groupshowed no significant change, and were significantly increased in the AngⅡ group. Compared with the AngⅡgroup, these mRNA levels in the AngⅡ+Ang-(1-7) group decreased significantly.9The effect of AngⅡand Ang-(1-7) on the activity of human endometrialtissueAfter endometrial tissue were cultured for72h, the result of Western blotshowed: Compared with the control group, the proteins expression of α-SMA,TGF-β1and IGF-I in the Ang-(1-7) group was not significantly changed, butthey were significantly increased in the AngⅡgroup(P<0.05); compared withthe AngⅡgroup, the expression of these proteins in the AngⅡ+Ang-(1-7)group decreased significantly(P<0.05).After endometrial tissue were cultured for72h, the result of real timePCR showed: Compared with the control group, the mRNA expression ofα-SMA, TGF-β1and IGF-I in the Ang-(1-7) group was not significantlychanged, but they were significantly increased in the AngⅡgroup(P<0.05);compared with the AngⅡgroup, the expression of these mRNAs in the AngⅡ+Ang-(1-7) group decreased significantly(P<0.05).Conclusion:1The human endometrial epithelial cells (EECs) and endometrial stromalcells (ESCs) were isolated, purified, cultured and characterized successfully.2Human three dimensional endometrium structure was cultured andcharacterized successfully in vitro.3Ang Ⅱ could promote EECs proliferation in the way of dosedependence and time dependence.4AngⅡ c ould promote the activaty of EECs which can express musclefibroblasts specific protein α-SMA and change the secretory function andmake the synthesis of Col Ⅰ andFN increase, while the expression level ofE-cadherin decreased.5Ang-(1-7) could inhibit AngⅡ o n theproliferation of EECs; Ang-(1-7)could inhibit Ang Ⅱ on the activation of EECs by inhibiting the expressionlevel of muscle fibroblasts specific protein α-SMA, changing the secretoryfunction and making the synthesis of ColⅠ andFN decrease, while the expression level of E-cadherin increased.6AngⅡcould promote ESCs proliferation in the way of dose dependenceand time dependence.7AngⅡ could promote the activaty of ESCswhich can express musclefibroblasts specific protein α-SMA, change the secretory function and makethe synthesis of Col Ⅰ, FN, TGF-β1and IGF-I increased.8Ang-(1-7) could inhibit Ang Ⅱo n theproliferation of ESCs; Ang-(1-7)could inhibit AngⅡ on the activation of ESCs by inhibiting the expressionlevel of muscle fibroblasts specific protein α-SMA, changing the secretoryfunction and making the synthesis of ColⅠ, FN, TGF-β1and IGF-I decrease.9Ang-(1-7) could inhibit AngⅡ on the expression level ofα-SMA,TGF-β1and IGF-I of endometrial tissue so that Endometrial fibrosis beinhibited. |
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