| Epigenetics can be defined as alterations in DNA function without alterations in DNA sequence,it is a subjects of inheritance phenomenon described by DNA methylation spectra,genomic imprinting,histone modification,X chromosome inactivation and non-coding RNA etal.Of the total,DNA methylation is main form of epigenetics information and has widespread biological functions.These functions refer to germinal cell and embryo genetic recombination,development of tumor,inhibition of endogenous retrotransponsons,stabilization of X chromosome inactivation and centromeric function,T cell autoreactivity and autoimmunity,metabolic syndromes etal.There are two kinds of DNA methylation,de novo methylation catalyzed by DNMT3A,3B and maintainance methylation directed by DNMT1.DNA methylation reprogramming guided by DNMTs play an important role in gametogenesis,emboyro and out laver of nutritive tissue.Deletion of anv of DNMTs can cause serious influences on embryonic development and gestational proceeding,even embryonic lethality.Nowadays,more and more importance has been accorded on reproduction healthy,abortion in first trimester is a disease which causes intense anguish and injury to patients and their family.Spontaneous abortion is approximately 10-15 percent of total pregnancy,80 percent of which happened in first trimester.Abortion has many etiological factor,it contains genetic causes,autoimmune causes,anatomic causes,infectious causes,environmental causes,endocrine factors,hematologic disorders etal.Nevertheless,further exploration is needed on the pathomechanism, prevention,intervention,early treatment of abortion in first trimester.Thus,to sum up the points,to study and research the function of epigenetics in spontaneous abortion,to survey this desease in an new visual angle can conduce to find new theory to nosogenesis,provide new method for prevention,intervention,early treatment of abortion.Expecting news about development of reproduction area!Part One:Expression of DNMTs in villus and decidua of human spontaneous abortionObjective To determine the histological expression and localization of DNMT 1,3A,3B in villus and decidua of human spontaneous abortion and compares the expression difference of DNMTs under physiological and pathological conditions. Methods1,Immunohistochemistry assay:6 cases of Villus and decidua tissues were collected from spontaneous abortion and selective termination with normal pregnancy.After PFA fixation and paraffin embedding,microtome sections were analyzed by Power Vision immunohistochemistry.2,Western blot:16 cases of Villus and decidua tissues were collected from spontaneous abortion and selective termination with normal pregnancy.Western blot were used to determine the expression level of DNMTs.Results 1,Immunohistochemistry assay:(1)DNMT1 was expressed in both human villus and decidua.The expression of DNMT1 was localized in trophoblastic cell nucleus of villus.The localization of DNMT1 in decidua is mainly in nucleus of glandular epithelium.(2)DNMT 3A was also expressed in villus and decidua, localization was the same as DNMT1's.(3)There were no expression signals of DNMT 3B in villus and decidua,reason might be that the quantity of DNMT3B is very small or the first antibody was not suitable for immunohistochemistry.2,Western blot:(1)DNMT1 protein expression in villus of spontaneous abortion was lower than normal(P＜0.05)and there were no differences(P＞0.05) of DNMT1 expression level in decidua between two groups.(2)There were no significantly differences of DNMT3A protein level in villus and decidual between two groups(P＞0.05).(3)The strap of DNMT 3B in villus were too small to be visible to the naked eye,and there were no significantly differences of DNMT3B protein level in decidual between two groups(P＞0.05).ConclusionsVillus,but not decidual,of spontaneous abortion has methylation maintenance defect,de novo methylaion not changed yet.Part Two:Functions of DNMT1 in embryo implantationObjective To establish simple and effective embryo implantation model,provide perfect platform for the embryo implantation mechanism research.Detection the embryonic receptivity of endometrium model by cell expression of LIF.To deep study the mechanisrn of DNMTI in the occurrcnce and developmcnt of spontaneous abortion.Specific inhibitor of DNMT1 were used to determine its relationship to embryo implantation.Methods1,Healthy and unpregnancy ICR female mice were mated with males,then blastula were collected 96h later.Single layer of ishikawa cells,a well-differentiated human endometrial adenocarcinoma cell line were used as endometrium model.The implantation of embryos were observed and implantation rate of 24h,48h,72h were counted.2,Immunocytochemistry:ishikawa Cells which were adherent to Coverslips underwent PFA fixation and Triton X-100 permeation,embryonic acceptance of endometrium model was analyzed by PowerVision immunocytochemistry.3,Healthy and unpregnancy ICR female mice were mated with males,then blastula were collected 96h later.Embryos are devided into two groups in a stochastic process.One group was given DNMT1 specific inhibitor,another group was used as control.After 24h treatment,embryos were transfered above a single layer of ishikawa cells which were used as endometrium model.The implantation of embryos were observed and implantation rate of 48h and 72h were counted.Chemiluminescence Immunoassay was used to detect the quantity ofβ-CG in the nutritive medium.Results1,The experiment of embryo implantation model were repeated three times, implantation rate of 24h was 7%,implantation rate of 48h was 49%,implantation rate of 72h was 61%.2,LIF was expressed in ishikawa cell according to immunocytochemistry. 3,Functions of DNMT1 in embryo implantationThere was no statistic difference in secretory volume ofβ-CG between treatment group and control group(P=0.80).48h embryo implantation rate:treatment group 25%,control group 42%,there was statistical differences between two groups(P=0.033).72h embryo implantation rate:treatment group 61%,control group 77%,there was statistical differences. between two groups(P=0.029).Conclusions A stable and effective embryo implantation model of mouse in vitro was successfully developed.Endometrium model has some degree of embryonic acceptance.Inhibition of genomic methylation level in embryo could reduce the embryonic implantation rate. |
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