Correlation Between Methylation Of Human Leukocyte Antigen-G And Unexplained Spontaneous Abortion

Spontaneous abortion is a familiar pathologic pregnancy,its' incidence is about 10～15%.And there was reported incidence of spontaneous abortion even is 70%including subclinic abortion.Pathogeny of spontaneous abortion is very complex,some were well known to us,just like infection,chromosome abnormity,malformation of genitalia and incretion disease,and so on.Recently,many research indicated unplained spontaneous abortion was related with immunity.At present,there are many research about autoimmunity abortion just like Antiphospholipid syndrome which had good curative effect;there had some study about alloimmune abortion,but its' curative effect is not satisfied.Maybe there still are some unknown pathogeny result in bad curative effect,in turn there are many yeld patients nowadays.This is a serious social problem so that we must do our hard to investigate the pathogeny of unexplained spontaneous abortion,which can guide therapy in turn.Heredity-immunology consider that there are some correlation between heredity and immunology,abnormity of gene expression canmake immune disfunction,in turn lead to immune diseases.This is a new method to investigate pathogent of immune diseases.Now researchers shown decrease of human leukocyte antigen-G(HLA-G)make disfunction of economy immunity.Economy immunity attack embryo,so that abortion occur as a result.But whether decrease of HLA-G in spontaneous abortion relate to abnormity of gene expression or not,this still not clear.This paper research in unexplained spontaneous abortion patients HLA-G gene expression in extravillous trophoblast(EVT),gnen methlation influence HLA-G gene expression,and natural killer(NK) cells' function,in order to discuss the ralationship between HLA-G gnen methlation and unexplained spontaneous abortion.It's a new method to elucidate pathogenic mechanisnm of spontaneous abortion in immunity,and maybe can find out effective therapy in clinic.This paper is dividee into four parts.In the first part,we cultivate original extravillous trophoblast cells including unexplained spontaneous abortion patients and normal pregnant women,so that there wre enough extravillous trophobalst cells to investigate.In the second part,cultivated extravillous trophoblast cells extract DNA,RNA and protein,learn about whether HLA-G gene expression in DNA,RNA and protein level wre different between unexplained spontaneous abortion patients and normal pregnant women.In the third part,we used drug to investigate gnen hypermethylation's influence to HLA-G gene expression in DNA,RNA and protein level.In the fourth part,we compare NK cells' subtypes and function in unexplained spontaneous abortion patients and normal pregnant women. Chapter 1 Human Extravillous Trophoblast Cells' Orginal Cultivation and IdentificationObjective:In order to investgate HLA-G gnen expression in human extravillous trophoblast cells,we did original cultivation of extravillous trophoblast cells of unexplained spontaneous abortion patients and normal pregnant women.Methods:We obtaines 20 research groups and 20 control groups' villus,and isolated extravillous trophoblast cells,cultivated original human extravillous trophoblast cells.Then we used immune cytochemistry identified extravillous trophoblast cells.Results:Two groups both cultivated original extravillous trophoblast cells,above 90%extravillous trophoblast cells were alive,used keratin and CK17 shown cultivated cells were extravillous trophoblast cells. Chapter 2 Unexplained Spontaneous Abortion's patients HLA-G Gene Expression in Extravillous Trophoblast CellsObjiective:In order to learn about whether methylation of HLA-G gene infulence HLA-G gene expression,we investigated methylation of HLA-G gene and it's expression in extravillous trophoblast cells of unexplained spontaneous abortion patients and normal pregnant women.Methods:DNA,Total RNA and protein were extracted from two groups' cultivated extravillous trophoblast cells.HLA-G gene expression at protein level were detected by Western Blot and immune cytochemistry;HLA-G gene expression at RNA level were detected by RT-PCR;HLA-G gene expression at DNA level were detected by MSP.Results:1,Research group's HLA-G gene expression at protein level in extravillous trophoblast cells were lower than control group,there was significant difference between the two groups(P＜0.01).Research group's HLA-G gene expression at antigen level in extravillous trophoblast cells were lower than control group,there was significant difference between the two groups(P＜0.01).2,Research group's HLA-G gene expression at mRNA level in extravillous trophoblast cells were lower than control group,there was significant difference between the two groups(P＜0.01).3,11 of research group's HLA-G gene expression at DNA level in extravillous trophoblast cells were methylation,9 were hypermethlation,whereas,control group were all hypermethylation.Conclusion:1,HLA-G gene expression was decreased in unexplained spontaneous abortion.2,Methylation of HLA-G gene is one of mechanism leading to decrease of HLA-G gene expression in unexplained spontaneous abortion;but decrease of HLA-G gene expression in some patients of unexplained spontaneous abortion was not correlated with gene methylation,maybe other gene regulation method involved in that. Chapter 3 Subtypes and Function of Decidual NK cells in Unexplained Spontaneous AbortionObjective:In order to learn about whether decidual NK cells heve chang in subtype and function in unexplained spontaneous abortion.Methods:Subtype of decidual NK cells were detected by Flow cytometry(CD56 and CD16),andput decidual Nkcells and extravillous trophoblast cells into one bottle,then co-cultivated 48 hours,function of decidual NK cells were detected by MTT assay.Results:1,The percen of research group's CD56~+CD16~+ NK cells was 20.327±2.495%,whereas The percen of control group was 10.560±1.050%,The percen of research group was significant higher than control group(P＜0.05).The percen of research group's CD56~+CD16~- NK cells was 70.538±4.637%,whereas The percen of control group was 76.349±3.871%,there was not significant difference between two group(P＞0.05).2,Research group's activity was 18.755±2.378%,control group's activity was 8.195±1.582%,The activity of research group was significant higher than control group(P＜0.05).The precent of abnormal increase about research group's activity was 85%,control group's activity was 17%,The The precent of abnormal increase about research group's activity was significant higher than control group(P＜0.01).3,NK cells activity was 18.755±2.378%,12.065±1.983%, 8.209755±1.045%at 0 umol/L,10 umol/L和100 umol/L HLA-G.The activity of three groups were significant different(P＜0.05).Conclusion:1,The percent of CD56~+CD16~+ NK cells were increased,and CD56~+CD16~- NK cells,and abnormal increase of NK cells' activity,which reslt in immune disfunction.2,HLA-G can inhibit NK cells' function,Abnormal increase of NK cells' activity maybe is connected with methylation of HLA-G gene.3,Heredity-immunology maybe is a important factor in unexplained spontaneous abortion Chapter 4 Effect of 5Aza-dc to Unexplained Spontaneous Abortion HLA-G Gene Methylation and Expression in Extravillous Trophoblast cellsObjective:In order to learn about effect of 5Aza-dc to HLA-G gene methylation and expression.Methods:20 unexplained spontaneous abortion patients were divide into deal group and undeal group,every group is 10 ptients by control.deal group's extravillous trophoblast cells were deal with 5Aza-dc.HLA-G gene expression at protein level were detected by Western Blot and immune cytochemistry;HLA-G gene expression at RNA level were detected by RT-PCR;HLA-G gene expression at DNA level were detected by MSP.Results:1,Deal group's HLA-G gene expression at protein level in extravillous trophoblast cells were higher than undeal group,there was significant difference between the two groups(P＜0.01).Deal group's HLA-G gene expression at antigen level in extravillous trophoblast cells were lower than undeal group,there was significant difference between the two groups(P＜0.01).2,Deal group's HLA-G gene expression at mRNA level in extravillous trophoblast cells were lower than undeal group,there was significant difference between the two groups(P＜0.01).3,Some undeal group's HLA-G gene expression at DNA level in extravillous trophoblast cells were methylation,some were methlation partly,whereas,deal group were all hypermethylation.Conclusion:After using 5Aza-dc,methylation of HLA-G gene became normal level and HLA-G gene expression was increased,which maybe become a new curative project to spontaneous abortion.