Experimental Studies Of Therapeutic Effect Of Endostatin Gene Transferred Into Endometriosis Rat Model

The incidence of Endometriosis in child-bearing age women is 10 percent to 15 percent,even attain 50 percent in infertile women.The tendency is adscendent year by year.Traditional drug and operation have major secondary actions and higher recurrence rate.So it's important to seek new methods of treatment.In this article we established endometriosis model in Lewis rats successfully,injected the lipofectamine-endo-pBud compounds to the ectopic focus of the models and then observed the curative effect by analysising the changes of endostatin,vascular endothelial growth factor,matrix metalloproteinases -2,microvessel density and volums of ectopic focus after transfering endostatin gene into endometriosis rat model.We are going to explain the contents from three parts:the establishment of endometriosis model in lewis rats,endostatin gene transferred into endometriosis rat Model, observation of the curative effect after transfering endostatin gene into endometriosis rat model.Chapter one The Establishment of Endometriosis Model in Lewis RatsObjective:To establish endometriosis model in Lewis rats by deploying autotransplantation,to make a fundament for the next step.Methods:Using vaginal smear to observe estrous cycle of the Eighty-five 14-week-old female unpregnancy Lewis rats,choosing estruation to establish endometriosis model by deploying autotransplantation: Evaluated the achievement ratio by unaided eye 4 weeks after the operation.Measured length and width to calculate the volume of ectopic focus by sliding caliper.Choosing the successful ones for the next step.To confirm the models by histopathologic examination and to observe vegetal condition of ectopic focus in the posterior experiment.Results:1.Estrous cycle of Lewis rats is four to five days.All of the Eighty-five rats have continual Estrous cycle.There are copious nucleo-endothelial cells in preoestrus,profuse uclea Keratinocytes in estruation,multiple pattern nuclear leucocyte in metaoestrus and leucocyte together with a few endothelial cells in anestrum.2.In the experimentation 64 rats among all of the 85 ones formed ectopic focus successfully.The achievement ratio is 75.29%for using autotransplantation to construct endometriosis rat model.The ectopic focus of eleven rats was auantic,eight rats formed inflammatory necrotic cyst and two died.3.Volume of ectopic focus in two control groups was increased six weeks after operation than four week after operation.Conclusion:The model of auto-transplanted surgically Lewis rats is feasibility and repeatability,it is a suitable endometriosis animal model.Chapter two Endostatin Gene Transferred into Endometriosis Rat ModelObjective:To judge the result of ES gene transfection and investigate the probability of the way using in treatment of endometriosis from injecting the lipofectamine-endo-pBud compounds to ectopic focus of endometriosis rat model by Western-blot and real-time FQ-PCR.Methods:Choosing the sixty-four successful rat models as empirical study objects.Divided them randomly into three groups:endostatin transgenic group(Ⅰgroup),carrying agent control group(Ⅱgroup)and negative control group(Ⅲgroup).Injected 2μl lipofectamine-endo-pBud compounds using microinjector to ectopic focus ofⅠgroup,2μl lipofectamine-pBud compounds toⅡgroup and 2μl PBS toⅢgroup by the same way.Divided each group into 2 subgroups randomly two weeks later.Taked the ectopic focus tissues to measure the opposing expression of endostatin gene by real-time fluorescent quantitative PCR,measure the opposing expression of endostatin protein and expression of ES-HA fusion protein by Western-blot.Results:1.The opposing expressions of endostatin gene were detected by real-time fluorescent quantitative PCR,found the difference between endostatin transgenic group and in carrying agent control group and negative control group has statistical significance(P＜0.05),but the two control groups did not reach statistical significance(P＞0.05)two weeks after injecting lipofectinmine-endo-pBud compounds to the EF directly.2.ES-HA fusion protein can be detected in the ectopic focus in endostatin transgenic group.The opposing expression of endostatin protein is much higher in the ectopic focus of endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01). but the two control groups did not reach statistical significance(P＞0.05) two weeks after injecting lipofectinmine-endo-pBud compounds to the EF directly.Conclusion: ES gene and protein can expressed in endometriosis rats successfully by injecting lipofectinmine-endo-pBud compounds to the ectopic focus directly.Chapter three Observation of The Curative Effect after Transfering Endostatin Gene into Endometriosis Rat ModelObjective:To observe the curative effect by analysising the changes of endostatin,vascular endothelial growth factor,matrix metalloproteinases -2,microvessel density,volum of ectopic focus after transfering endostatin gene into endometriosis rat model.Methods:Drawed blood from vena caudalis of the 64 rat models in groupⅠ,ⅡandⅢto detect the level of endostatin and vascular endothelial growth factor in serum by ELISA.Detected the endostatin,MMP-2 and MVD in ectopic focus in groupⅠb,Ⅱb andⅢb by immunohistochemical assay,and measured the volume of the ectopia focus by vernier cursor.Results:1.The level of ES and VEGF in serum of endometriosis rat models were detected by enzyme-linked immunosorbentassay before and two weeks after transferring.Group comparison:the level of ES is much higher in endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01),but the two control groups did not reach statistical significance(P＞0.05);The level of VEGF is much lower in endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01),but the two control groups did not reach statistical significance(P＞0.05).Intra-group comparisio:in endostatin transgenic group the level of ES is increased(P＜0.01),and the level of VEGF is decreased(P＜0.01),but in the two control groups did not reach statistical significance(P＞0.05).There was an negative correlation between ES and VEGF in serum(r=-0.805).2.The expression of the ES,MMP-2 and MVD in ectopia focus were detected by irnmunohistochemical assay two weeks after transferring. The expression of ES is much higher in endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01),but the two control groups did not reach statistical significance(P＞0.05);The expression of MMP-2 is much lower in endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01),but the two control groups did not reach statistical significance(P＞0.05).The MVD is much lower in endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01),but the two control groups did not reach statistical significance(P＞0.05).And there was an negative correlation between the expression of ES and MMP-2 in ectopia focus(r=-0.700).3.The volume of the ectopia focus were detected by vernier cursor. Group comparison:the volume of the ectopia focus in three group before transferring did not reach statistical significance(P＞0.05).Intra-group comparisio:the volume of the ectopia focus is smaller in endostatin transgenic group than in carrying agent control group and negative control group(P＜0.01)two weeks after transferring,but the two control groups did not reach statistical significance(P＞0.05).The difference among three groups before and after transferring:volume of endostatin transgenic group is smaller than before(P＜0.01),and the volume of two control groups are bigger than before(P＜0.01).Conclusion:Injecting the lipofectamine-endo-pBud into ectopic focus has definite curative effect in endometriosis rat model.