| Chapter one The Study SDF-1/CXCR4 mediated BMSCs on EndometrialRegeneration and Fibrosis Imbalances in the Pathogenesis ofIntrauterine AdhesionObjectives:To detect ESR1,stem cells,fibrosis and receptivity markers in human endometrial tissues,we study the relationship between BMSCs endometrial repairment and endometrial fibrosis in the process of Intrauterine adhesion formation,and explore the role of BMSCs in endometrial regeneration.Methods:1.The 56 cases suffered intrauterine adhesion were recruited in our center of hysteroscopy during Sep.2015 to Feb.2016.40 cases had adhesion with mild-to-moderate degree(9 to 18 scores),16 cases had adhesion with severe adhesion(?19 scores).The including criteria are:(1)the score?9 scores;(2)ages from 18-42 scores;(3)progesterone hormone>5 ng/ml.There are 20 cases suffered uterine septum for the same periodas the contrast.We collect the general information,and measure endometrial thickness by ultrasound.2.We obtain the endometrial tissues before operation.To detect the expression of ESR1,SDF-1,CXCR4,TGF-?1,MMP-9,VEGF and ?v?3by q RT-PCR,WB and IHC.Results:1.The endometrial thickness is different in every group in the luteum phase.The thickness with intrauterine adhesion is thinner than in the control group,with the increasing adhesion degree aggravation and thinner of thickness.2.ESR1 expression is different in every group.The expression of ESR1 in IUA groups is higher than in control group,with the increasing adhesion degree aggravation and higher of expression.3.The expression of SDF-1 is no obvious change in every group.The expression of CXCR4 is no obvious change in most groups,but the expression of CXCR4 in serious group is lower than in control group by IHC.4.The expression of TGF-?1 and MMP-9 are different in every group respectively.The expression in IUA groups is higher than in control group,with the increasing adhesion degree aggravation and higher of expression.5.The expression of VEGF is no obvious change in every group.The expression of ?v?3 is no obvious change in most groups,but the expression of ?v?3 in serious group is lower than in control group by IHC.Conclusions:After endometrium injured,endometrial repairment and fibrosis areimbalance in forming intrauterine adhesions.The extracellular matrix deposition and fibrosis,the expression of ESR1 increases,endometrium fibrosis reaction intensity,the inhibition of BMSCs repairing endometrium,the receptivity reduced possibly.Chapter two The Effect of Estrogen on SDF-1/CXCR4 mediated BMSCs in Endometrial Regeneration and Fibrosis in Teatment of IntrauterineAdhesionObjectives:Treatment with different dosage of estrogen after operation,we study the effect on the hypothalamic-pituitary-ovarian axis with different dosage of estrogen.Detection the expression of ESR1,stem cells,fibrosis and receptive markers,we study the effect of recovery with different dosage of estrogen.Methods:1.The 56 cases suffered intrauterine adhesion were recruited in our center of hysteroscopy during Sep.2015 to Feb.2016.40 cases had adhesion with mild-to-moderate degree(9 to 18 scores),16 cases had adhesion with severe adhesion(?19 scores).Ages from 18-42.2.One surgeon designated did these operations.A metal device packaged by anti-adhesion membrane was inserted into the uterine cavity.Three months later,the device was taken out of the cavity.3.The patients treated with different dosage after operation.EV 4mg(23 cases),EV 6mg(17 cases),EV 9mg(16 cases).In mild-moderatedegree,there are 28 cases,EV 4mg(19 cases),EV 9mg(9 cases).In severe degree,there are 11 cases,EV 4mg(4 cases),EV 9mg(7 cases).The patients accepted hysteroscopy exam after the first month and the third month postoperation.4.The serum level of FSH,E2,P were detected,to evaluate ovarian function and ovulation.5.The endometrial tissues were obtained before operation,after the first month and the third month postoperation.To detect the expression of ESR1,SDF-1,CXCR4,TGF-?1,MMP-9,VEGF,and ?v?3 by q RT-PCR,WB and IHC.6.Following the patients general information and pregnancy,after taking out of the IUD.Results:1.General conditions1.1 Hysteroscopy exam: There are two patients happened the second adhesion at the first month postoperation,they are all serious degree.There is no new adhesion happened in later month.The cavity become normal,and endometrium nearly covered whole area of cavity.1.2 The effect of Estrogen therapy on ovarian functionIn EV 4mg group,the ovulation inhibition rate is 8.69%(2/23);In EV 6mg group,the ovulation inhibition rate is 58.82%(10/17);In EV9 mg group,the ovulation inhibition rate is 93.75%(15/16).Estrogen therapy Finished,In EV 4mg group,the ovulation inhibition rate is 4.34(1/23);In EV 6mg group,the ovulation inhibition rate is 17.64%(3/17);In EV 9mg group,the ovulation inhibition rate is56.25%(9/16).IUD extracted after 3 months postoperation,the amount of menstruation become normal in 53 patients,the effective rate is 94.6%(53/56),and there is no change in 3 patients.13 patients extracted IUD have pregnancy in 3 months,pregnancy rate is 23.21%(13/56),the recent recovery rate is high.2.The expression of ESR1 is decreased postoperation.3.The expression of SDF-1 goes through the process of falling after rising first,the expression level preoperation is similar to the level of 3months postoperation.The expression of CXCR4 is no obvious change before and after treatment.4.The expression of TGF-?1 and MMP-9 are decreased postoperation,but the expression of TGF-?1 in EV 9mg group is no obvious change.The expression of MMP-9 in severe group and EV 9mg group is no obvious change.5.The expression of VEGF is no obvious change before and after treatment.The expression of ?v?3 is risen postoperation in EV 4mg group and mild-moderate group,but the expression in EV 9mg group and serious group are no obvious change.Conclusions:the recovery of uterine shape,inhibition endometrial fibrosis,increase the area of endometrium,acceleration endometrium regeneration,improvement amount of menstruation and pregnancy rate,when the patients accepted TCRA and insertion IUA and Estrogen therapy.The effect of BMSCs regeneration is little in the following 3 months.Different Estrogen dosage has different effect on ovarian function,Estrogen with large dosage has obvious inhibition.The effect of Severe IUA is poor.Endometrium fibrosis maybe inhibited and endometrium1.3 menstruation and pregnancyregeneration maybe improved by Estrogen with moderate dosage therapy compared with the large one,it seems to be advantageous.Chapter three Establishment and Identification of the SD Rat Model ofIntrauterine AdhesionObjective:We set up the SD rat model of intrauterine adhesion to verificate the consistency between the rat sample and human sample.In order to provide experimental basis for further study of stem cells transplantation therapy.Methods:1.Ten SD rats,weighted about 200-250 g,were divided into two groups in self control study: model group(perfusing alcohol into the left uterine cavities)and control group(perfusing saline into the right uterine cavities).2.Identify whether the model was established successfully by observing the endometrium morphological change with Hematein Eosin(HE)staining,measuring the endometrium thickness,detecting fibrosis severity with Masson staining,examinating the expression of ESR1,TGF-?1 with WB and q PCR which were the markers of endometrial cells.Results:1.Compared with control group,the endometrium lining was thinnerand flat in left cavity.The glands were sparse and the endometrial interstitial edema.2.The rate of fibrosis area increased obviously compared with control group.3.The expression of ESR1 was significantly higher than that of control group.4.The expression of TGF-?1 was significantly higher than that of control group.Conclusions:Successful rat model of intrauterine adhesion could be established by perfusing alcohol into uterine cavities,it corresponds with clinical human sample,and the success rate was 100%.The expressions of ESR1,TGF-?1 were significantly higher than those of control group.The change corresponds with the change of clinical human sample. |
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