人类白细胞抗原-G基因甲基化和不明原因性自然流产的相关性研究
|
摘要
自然流产是常见的病理妊娠之一,发病率高,病因复杂。据统计,其发病率高达10%～15%,包括临床前流产在内的自然流产发生率甚至高达70%。在对自然流产病因的研究中,感染、染色体异常、生殖器畸形及内分泌疾病等已研究得比较清楚,但仍有高达60～70%的患者病因不明。近几年研究表明,不明原因的自然流产主要与免疫因素有关。目前对自身免疫性自然流产如抗磷脂综合征等研究较多,同种免疫性自然流产如封闭抗体缺乏也做了一些研究,治疗已经取得一定疗效,但仍有部分患者治疗效果不理想,成为临床上难治性不育的主要原因,这给患者带来沉重的心理负担,造成家庭的不稳定,并成为一个重要的社会问题,因此需要对自然流产的病因及治疗进行更深入的研究。
近年免疫遗传学表明免疫与遗传有关,基因表达量的异常可以引起免疫功能的紊乱,导致免疫性疾病的发生,这为探讨免疫性疾病的发病机制开辟了新途径。那么,不明原因性自然流产患者是否存在免疫遗传的异常呢?
最新国内外研究表明,人类白细胞抗原-G(human leukocyteantigen-G,HLA-G)的下降能引起机体免疫失调,导致胚胎受到免疫攻击而流产。自然流产患者体内HLA-G抗原表达异常是否与基因异常有关,是否影响蜕膜自然杀伤(natural killer,NK)细胞的功能,目前尚不清楚。因此,本课题研究不明原因性自然流产患者绒毛外滋养细胞HLA-G基因表达水平的变化,HLA-G基因甲基化程度在体外对绒毛外滋养细胞的HLA-G基因表达水平和蜕膜NK细胞功能的影响,探讨HLA—G基因甲基化与不明原因性自然流产的关系,旨在从基因水平调控免疫应答反应,为最终解决自然流产这一常见病理妊娠提供理论依据。同时也为阐明自然流产的免疫遗传学发病机制开辟新途径,为临床上有效治疗不明原因性自然流产寻找新方案。
论文分为四部分。第一部分用不明原因性自然流产患者和正常妇女的早孕绒毛组织进行原代绒毛外滋养细胞培养;第二部分探讨不明原因性自然流产患者和正常早孕妇女的绒毛外滋养细胞上HLA-G基因在mRNA、蛋白水平的表达及甲基化程度是否存在差异;第三部分探讨不明原因性自然流产患者和正常妇女的子宫蜕膜NK细胞的亚型和功能的差异,以及HLA-G抗原浓度对NK细胞功能的影响;第四部分探讨去甲基化药物对于HLA-G基因在甲基化程度、mRNA、蛋白水平表达的影响。
第一章人早孕期绒毛外滋养细胞的原代细胞培养及鉴定
目的:
培养出原代绒毛外滋养细胞,为本研究提供人绒毛外滋养细胞。
方法:
获取不明原因性自然流产患者(研究组)和正常早孕妇女(对照组)的绒毛组织,各20例。用原代培养方法培养出绒毛外滋养细胞,进一步纯化,并用免疫细胞化学染色(角蛋白CK17、波形蛋白、HLA-G抗原)鉴定绒毛外滋养细胞。
结果:
研究组和对照组均成功培养出原代绒毛外滋养细胞,台盼兰染色显示大于90%绒毛外滋养细胞存活,用波形蛋白、角蛋白、HLA-G抗原进行免疫细胞化学染色,显示所培养的细胞为绒毛外滋养细胞。
第二章不明原因性自然流产患者绒毛外滋养细胞上HLA-G基因的表达水平
目的:
研究不明原因性自然流产患者HLA-G基因mRNA、蛋白水平的表达及是否存在甲基化,,探讨HLA—G基因甲基化对HLA-G基因表达水平的影响。
方法:
采用Western Blot比较两组绒毛外滋养细胞HLA-G基因在蛋白质水平的表达情况;采用免疫细胞化学染色比较两组绒毛外滋养细胞HLA-G抗原的表达情况;采用RT-PCR比较两组绒毛外滋养细胞HLA-G基因在转录水平的表达情况;采用甲基化特异性PCR比较两组绒毛外滋养细胞HLA-G基因甲基化程度。
结果:
1、经Gelpro analyzer分析软件分析图像,研究组绒毛外滋养细胞上HLA-G蛋白/GAPDH比值为0.669±0.016,对照组为1.276±0.030。两组比值经过两独立样本秩和检验比较,研究组绒毛外滋养细胞HLA-G蛋白表达低于对照组(P＜0.01),差异有显著性。
2、研究组绒毛外滋养细胞HLA-G染色成黄色,对照组绒毛外滋养细胞染色呈棕黄或棕褐色。综合染色强度和阳性细胞所占比例进行平均半定量处理,两组绒毛外滋养细胞的HLA-G抗原表达强度经x~2检验,研究组绒毛外滋养细胞的HLA-G抗原表达强度低于对照组(P＜0.01),差异具有显著性。
3、经Gelpro analyzer分析软件分析图像,研究组绒毛外滋养细胞上HLA-G mRNA/β-actin比值为0.151±0.012,对照组为1.826±0.054。两组比值经过两独立样本秩和检验比较,研究组绒毛外滋养细胞上HLA-G mRNA水平低于对照组(P＜0.01),差异有显著性。
4、研究组中共有11例绒毛外滋养细胞上HLA-G基因存在甲基化状态,而9例HLA-G基因呈去甲基化状态;对照组绒毛外滋养细胞上HLA-G基因呈去甲基化状态。
结论:
1、不明原因性自然流产患者绒毛外滋养细胞上HLA-G基因表达水平下降;
2、HLA-G基因表达水平下降部分与基因甲基化有关,还有一部分可能与其他基因调控有关。
第三章不明原因性自然流产患者子宫蜕膜NK细胞亚型及功能变化的研究
目的:
研究不明原因性自然流产患者的子宫蜕膜NK细胞是否存在亚型及功能的变化。
方法:
用流式细胞仪检测研究组和对照组的子宫蜕膜NK细胞D56~+CD16~+亚型和CD56~+CD16~-亚型;并将两组的子宫蜕膜自然杀伤细胞与绒毛外滋养细胞共同培养,采用四甲基偶氮唑蓝(MTT)比色法检测两组子宫蜕膜自然杀伤细胞对绒毛外滋养细胞的影响。
结果:
1、研究组蜕膜CD56~+CD16~+NK细胞比例为20.327±2.495%,对照组为10.560±1.050%,两者经x~2检验,研究组CD56~+CD16~+亚型比例显著高于于对照组,差异有显著性(P＜0.05);CD56~+CD16~-NK细胞比例为70.538±4.637%,对照组为76.349±3.871%,两者经x~2检验,研究组CD56~+CD16~-亚型比例低于于对照组,但差异无统计学意义(P＞0.05)。
2、研究组蜕膜NK细胞杀伤活性为18.755±2.378%,有17例存在杀伤活性异常增高,杀伤活性异常增高率为85%;对照组蜕膜NK细胞杀伤活性为8.195±1.582%,有4例存在杀伤活性异常增高,杀伤活性异常增高率为20%。两组经x~2检验,研究组蜕膜NK细胞杀伤活性显著高于对照组(P＜0.05),研究组蜕膜NK细胞活性异常增高率显著高于对照组(P＜0.01)。
3、0umol/L、10 umol/L和100 umol/L HLA-G抗原下自然流产患者蜕膜NK细胞的杀伤活性分别为18.755±2.378%、12.065±1.983%和8.209755±1.045%,三组经多样本x~2检验,组间蜕膜NK细胞杀伤活性有显著性差异(P＜0.05)。
结论:
1、自然流产患者的CD56~+CD16~+亚型NK细胞比例增高,CD56~+CD16~-亚型NK细胞比例减少,同时杀伤活性异常增高,导致免疫功能紊乱;
2、免疫功能紊乱可能与HLA-G基因水平下调、基因甲基化有关。
3、免疫遗传异常是不明原因性自然流产的重要因素。
第四章5'—氮杂2'—脱氧胞苷对不明原因性自然流产患者绒毛外滋养细胞HLA-G基因甲基化状态及表达水平的影响
目的:
研究去甲基化药物对HLA-G基因甲基化程度及表达水平的影响。
方法:20例不明原因性自然流产患者随机分为处理组和未处理组,每组各10例,处理组经甲基化转移酶抑制剂5'—氮杂2'—脱氧胞苷处理原代绒毛外滋养细胞。采用Western Blot、免疫细胞化学染色、RT-PCR、甲基化特异性PCR比较两组绒毛外滋养细胞HLA-G基因在mRNA、蛋白质水平的表达情况及甲基化程度。
结果:
1、经Gelpro analyzer分析软件分析图像,处理组绒毛外滋养细胞上HLA-G蛋白/GAPDH比值为1.245±0.047,未处理组为0.652±0.017。两组比值经过两独立样本秩和检验比较,处理组绒毛外滋养细胞HLA-G蛋白表达高于对照组(P＜0.01),差异有显著性。
2、处理组绒毛外滋养细胞HLA-G染色呈棕黄或棕褐色,未处理组绒毛外滋养细胞染色成黄色。综合染色强度和阳性细胞所占比例进行平均半定量处理,两组绒毛外滋养细胞的HLA-G抗原表达强度经x~2检验,研究组绒毛外滋养细胞的HLA-G抗原表达强度高于对照组(P＜0.01),差异具有显著性。
3、经Gelpro analyzer分析软件分析图像,研究组绒毛外滋养细胞上HLA-G mRNA/β-actin比值为2.018±0.083,对照组为0.147±0.011。两组比值经过两独立样本秩和检验比较,研究组绒毛外滋养细胞上HLA-G mRNA水平高于对照组(P＜0.01),差异有显著性。
4、处理组绒毛外滋养细胞上HLA-G基因呈去甲基化状态;部分未处理组绒毛外滋养细胞上HLA-G基因存在甲基化状态,而部分未处理组绒毛外滋养细胞上HLA-G基因呈去甲基化状态。
结论:
经过去甲基化药物处理后,自然流产患者HLA-G基因呈去甲基化状态,基因表达水平上调,去甲基化治疗可望作为自然流产新的治疗方法。
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 Identification
Objective:
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 Cells
Objiective:
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 Abortion
Objective:
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 cells
Objective:
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.
近年免疫遗传学表明免疫与遗传有关,基因表达量的异常可以引起免疫功能的紊乱,导致免疫性疾病的发生,这为探讨免疫性疾病的发病机制开辟了新途径。那么,不明原因性自然流产患者是否存在免疫遗传的异常呢?
最新国内外研究表明,人类白细胞抗原-G(human leukocyteantigen-G,HLA-G)的下降能引起机体免疫失调,导致胚胎受到免疫攻击而流产。自然流产患者体内HLA-G抗原表达异常是否与基因异常有关,是否影响蜕膜自然杀伤(natural killer,NK)细胞的功能,目前尚不清楚。因此,本课题研究不明原因性自然流产患者绒毛外滋养细胞HLA-G基因表达水平的变化,HLA-G基因甲基化程度在体外对绒毛外滋养细胞的HLA-G基因表达水平和蜕膜NK细胞功能的影响,探讨HLA—G基因甲基化与不明原因性自然流产的关系,旨在从基因水平调控免疫应答反应,为最终解决自然流产这一常见病理妊娠提供理论依据。同时也为阐明自然流产的免疫遗传学发病机制开辟新途径,为临床上有效治疗不明原因性自然流产寻找新方案。
论文分为四部分。第一部分用不明原因性自然流产患者和正常妇女的早孕绒毛组织进行原代绒毛外滋养细胞培养;第二部分探讨不明原因性自然流产患者和正常早孕妇女的绒毛外滋养细胞上HLA-G基因在mRNA、蛋白水平的表达及甲基化程度是否存在差异;第三部分探讨不明原因性自然流产患者和正常妇女的子宫蜕膜NK细胞的亚型和功能的差异,以及HLA-G抗原浓度对NK细胞功能的影响;第四部分探讨去甲基化药物对于HLA-G基因在甲基化程度、mRNA、蛋白水平表达的影响。
第一章人早孕期绒毛外滋养细胞的原代细胞培养及鉴定
目的:
培养出原代绒毛外滋养细胞,为本研究提供人绒毛外滋养细胞。
方法:
获取不明原因性自然流产患者(研究组)和正常早孕妇女(对照组)的绒毛组织,各20例。用原代培养方法培养出绒毛外滋养细胞,进一步纯化,并用免疫细胞化学染色(角蛋白CK17、波形蛋白、HLA-G抗原)鉴定绒毛外滋养细胞。
结果:
研究组和对照组均成功培养出原代绒毛外滋养细胞,台盼兰染色显示大于90%绒毛外滋养细胞存活,用波形蛋白、角蛋白、HLA-G抗原进行免疫细胞化学染色,显示所培养的细胞为绒毛外滋养细胞。
第二章不明原因性自然流产患者绒毛外滋养细胞上HLA-G基因的表达水平
目的:
研究不明原因性自然流产患者HLA-G基因mRNA、蛋白水平的表达及是否存在甲基化,,探讨HLA—G基因甲基化对HLA-G基因表达水平的影响。
方法:
采用Western Blot比较两组绒毛外滋养细胞HLA-G基因在蛋白质水平的表达情况;采用免疫细胞化学染色比较两组绒毛外滋养细胞HLA-G抗原的表达情况;采用RT-PCR比较两组绒毛外滋养细胞HLA-G基因在转录水平的表达情况;采用甲基化特异性PCR比较两组绒毛外滋养细胞HLA-G基因甲基化程度。
结果:
1、经Gelpro analyzer分析软件分析图像,研究组绒毛外滋养细胞上HLA-G蛋白/GAPDH比值为0.669±0.016,对照组为1.276±0.030。两组比值经过两独立样本秩和检验比较,研究组绒毛外滋养细胞HLA-G蛋白表达低于对照组(P＜0.01),差异有显著性。
2、研究组绒毛外滋养细胞HLA-G染色成黄色,对照组绒毛外滋养细胞染色呈棕黄或棕褐色。综合染色强度和阳性细胞所占比例进行平均半定量处理,两组绒毛外滋养细胞的HLA-G抗原表达强度经x~2检验,研究组绒毛外滋养细胞的HLA-G抗原表达强度低于对照组(P＜0.01),差异具有显著性。
3、经Gelpro analyzer分析软件分析图像,研究组绒毛外滋养细胞上HLA-G mRNA/β-actin比值为0.151±0.012,对照组为1.826±0.054。两组比值经过两独立样本秩和检验比较,研究组绒毛外滋养细胞上HLA-G mRNA水平低于对照组(P＜0.01),差异有显著性。
4、研究组中共有11例绒毛外滋养细胞上HLA-G基因存在甲基化状态,而9例HLA-G基因呈去甲基化状态;对照组绒毛外滋养细胞上HLA-G基因呈去甲基化状态。
结论:
1、不明原因性自然流产患者绒毛外滋养细胞上HLA-G基因表达水平下降;
2、HLA-G基因表达水平下降部分与基因甲基化有关,还有一部分可能与其他基因调控有关。
第三章不明原因性自然流产患者子宫蜕膜NK细胞亚型及功能变化的研究
目的:
研究不明原因性自然流产患者的子宫蜕膜NK细胞是否存在亚型及功能的变化。
方法:
用流式细胞仪检测研究组和对照组的子宫蜕膜NK细胞D56~+CD16~+亚型和CD56~+CD16~-亚型;并将两组的子宫蜕膜自然杀伤细胞与绒毛外滋养细胞共同培养,采用四甲基偶氮唑蓝(MTT)比色法检测两组子宫蜕膜自然杀伤细胞对绒毛外滋养细胞的影响。
结果:
1、研究组蜕膜CD56~+CD16~+NK细胞比例为20.327±2.495%,对照组为10.560±1.050%,两者经x~2检验,研究组CD56~+CD16~+亚型比例显著高于于对照组,差异有显著性(P＜0.05);CD56~+CD16~-NK细胞比例为70.538±4.637%,对照组为76.349±3.871%,两者经x~2检验,研究组CD56~+CD16~-亚型比例低于于对照组,但差异无统计学意义(P＞0.05)。
2、研究组蜕膜NK细胞杀伤活性为18.755±2.378%,有17例存在杀伤活性异常增高,杀伤活性异常增高率为85%;对照组蜕膜NK细胞杀伤活性为8.195±1.582%,有4例存在杀伤活性异常增高,杀伤活性异常增高率为20%。两组经x~2检验,研究组蜕膜NK细胞杀伤活性显著高于对照组(P＜0.05),研究组蜕膜NK细胞活性异常增高率显著高于对照组(P＜0.01)。
3、0umol/L、10 umol/L和100 umol/L HLA-G抗原下自然流产患者蜕膜NK细胞的杀伤活性分别为18.755±2.378%、12.065±1.983%和8.209755±1.045%,三组经多样本x~2检验,组间蜕膜NK细胞杀伤活性有显著性差异(P＜0.05)。
结论:
1、自然流产患者的CD56~+CD16~+亚型NK细胞比例增高,CD56~+CD16~-亚型NK细胞比例减少,同时杀伤活性异常增高,导致免疫功能紊乱;
2、免疫功能紊乱可能与HLA-G基因水平下调、基因甲基化有关。
3、免疫遗传异常是不明原因性自然流产的重要因素。
第四章5'—氮杂2'—脱氧胞苷对不明原因性自然流产患者绒毛外滋养细胞HLA-G基因甲基化状态及表达水平的影响
目的:
研究去甲基化药物对HLA-G基因甲基化程度及表达水平的影响。
方法:20例不明原因性自然流产患者随机分为处理组和未处理组,每组各10例,处理组经甲基化转移酶抑制剂5'—氮杂2'—脱氧胞苷处理原代绒毛外滋养细胞。采用Western Blot、免疫细胞化学染色、RT-PCR、甲基化特异性PCR比较两组绒毛外滋养细胞HLA-G基因在mRNA、蛋白质水平的表达情况及甲基化程度。
结果:
1、经Gelpro analyzer分析软件分析图像,处理组绒毛外滋养细胞上HLA-G蛋白/GAPDH比值为1.245±0.047,未处理组为0.652±0.017。两组比值经过两独立样本秩和检验比较,处理组绒毛外滋养细胞HLA-G蛋白表达高于对照组(P＜0.01),差异有显著性。
2、处理组绒毛外滋养细胞HLA-G染色呈棕黄或棕褐色,未处理组绒毛外滋养细胞染色成黄色。综合染色强度和阳性细胞所占比例进行平均半定量处理,两组绒毛外滋养细胞的HLA-G抗原表达强度经x~2检验,研究组绒毛外滋养细胞的HLA-G抗原表达强度高于对照组(P＜0.01),差异具有显著性。
3、经Gelpro analyzer分析软件分析图像,研究组绒毛外滋养细胞上HLA-G mRNA/β-actin比值为2.018±0.083,对照组为0.147±0.011。两组比值经过两独立样本秩和检验比较,研究组绒毛外滋养细胞上HLA-G mRNA水平高于对照组(P＜0.01),差异有显著性。
4、处理组绒毛外滋养细胞上HLA-G基因呈去甲基化状态;部分未处理组绒毛外滋养细胞上HLA-G基因存在甲基化状态,而部分未处理组绒毛外滋养细胞上HLA-G基因呈去甲基化状态。
结论:
经过去甲基化药物处理后,自然流产患者HLA-G基因呈去甲基化状态,基因表达水平上调,去甲基化治疗可望作为自然流产新的治疗方法。
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 Identification
Objective:
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 Cells
Objiective:
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 Abortion
Objective:
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 cells
Objective:
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.
引文
[1] Ishitani A,Sageshima N,Lee N,et al.Ptotein expression and peptide binding suggest unique and interacting functional roles for HLA-E,F and G in maternal-placental immune recognition[J] J Immunol.2003,171:1376-1384;
[2] Rabot M,Tabiasco J,Polgar B,et al. HLA class I/NK cell receptor interaction in early human decidua basalis: possible functional consequences[J].Chem Immunol Allergy.2005,89:72-83.
[3] Emmer Pm,Steegers EA,Kerstens HM,et al.Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies[J]. Hum Reprod.2002,17(4): 1072-80.
[4] Hviid TV,Hylenius S,Rorbye C,et al.HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels[J]. Immunogenetics.2003,55(2):63-79.
[5] Varla-Leftherioti M.The significance of the women's repertoire of natural killer cell receptors in the maintenance of pregnancy[J].Chem Immunol Allergy. 2005, 89:84-95.
[6] Philippe M,Gael M,Philippe R,et al.HLA-G gene repression is reversed by demethylation[J]. Proc Natl Acad Sci.2003,100(3):1191-1196.
[7] Guillaudeux T, Rodriguez AM, Girr M, et al. Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta[J].J Immunol. 1995,154(7):3283-3299.
[8] Onno, M,Amiot L,Bertho N,et al.CpG methylation patterns in the 5' part of the nonclassical HLA-G gene in peripheral blood CD34+ cells and CD2+ lymphocytes[J]. Munksgaard International Publishers. 1997,49(4):356-364.
[9] Munz C,Holmes N,King A,et al.Human histocompatibility leukocyte antigen(HLA)-G molecules inhibit NKAT3 expressing natural killer cells[J].J Exp Med,1997,185(3):385-392.
[10]彭冰,刘淑芸,邢爱耕等。人类白细胞抗原G、E在人胎盘组织中的表达及意义[J]。现代妇产科进展,2006,15(7):525-530;
[11]Joanna LJ,Peter RS,Lawrence WC.Cytotrophoblast differentiation in the first trimester of pregnancy:evidence for separate progenitors of extravillous trophoblasts and syncytiotrophoblast[J].Reproduction.2005,130:95-103;
[12]Shiroishi M,Tsumoto K,Amano K et al.Human inhibitory receptors Ig-like transcript 2(ILT2)and ILT4 compete with CD8 for MHC class Ⅰ binding and bind preferentially to HLA-G[J].Proc.Natl.Acad.Sci.2003,100:8856-8861;
[13]Morales PJ,Pace JL,Platt JS,et al.Placenta cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype[J].J.Immunol.2003,171:6215-6224;
[14]Contini P,Ghio M,Poggi A,et al.Soluble HLA-A,B,G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8ligation[J].Eur.J.Immunol.2003,33:125-134;
[15]Kanai T,Fujii T,Unno N,et al.Hurnan leukocyte antigen-G expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood[J].Am J Reprod Immunol.2001,45(2):94-99;
[16]Patel RN,Quack KC,Hill JA,et al.Expression of membraneboundHLA-G at the maternal-fetal interface is not associated with pregnancy maintenance among patients with idiopathic recurrent pregnancy loss[J].Mol Hum Renrod.2003.9.551-557.
[17]王炎秋,陈士岭,邢福祺。HLA-G蛋白在滋养层细胞的表达及意义[J]。第一军医大学学报。2005,25(12):1488-1490;
[18]King A,Hiby SE,Gardner L,et al.Recognition of trophoblast HLA class Ⅰmolecules by decidual NK cell receptors—a review[J].Placenta.2000,21:S81-85;
[19]Chrysoula,Dosiou,Linda C.Natural killer cells in pregnancy and recurrent pregnancy loss:Endocrine and immunologic perspectives[J].Endocr Rev.2005,26(1):44-62;
[20]Kuhbav GO,Critchley HO,Bowen JM,et al.Prolactin induces ERK,phosphorylation in epithelial and CD56+ natural killer cells of the human endometrium[J].J Clin Endocrinlo Metab.2002,87:2329-2335;
[21]Borrego F,Kabat J,Kim DK,etal.Structure and function of major histocompatibility complex(MHC)class I specific receptors expressed on human natural killer(NK)cells[J].Mol Immunol,2002,38(9):637-660.
[22]Dosiou Chrysoula,Giudice LC.Natural killer cells in pregnancy and recurrent pregnancy loss:endocrine and immunologic perspectives[J].Endocr Rev.2005,26(1):44-62;
[23]Santi DV,Garrett CE,Barr PJ.On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs[J].Cell.1983,33:9;
[24]Wold AS,Arici A.Natural killer cells and reproduction failure[J].Curr Op in Obstet Gynecol,2005,17(3):237-341;
[25]Matsubayashi H,Shida M,Kondo A,et al.Preconception peripheral natural killer cell activity as a predictor of pregnancy outcome in patients with unexplained infertility[J].Am J Reprod Immunol,2005,53(3):126-131;
[26]李昭荣、韩香、吕海侠等。蜕膜自然杀伤细胞与不明原因性反复流产的关系[J]。生殖医学杂志,2005,14(2):68-71;
[27]HitomiSasaki,Xiao-ChunXu,T.Mohanakumar.HLA-E and HLA-G Expression on Porcine Endothelial Cells Inhibit Xenoreactive Human NK Cells Through CD94/NKG2-Dependent and -Independent Pathways[J].The Journal of Immunology,1999,163:6301-6305
[28]PietroForte,LaszloPazmany,UlrikeB,et al.HLA-G Inhibits Rolling Adhesion of Activated Human NK Cells on Porcine Endothelial Cells[J].The Journal of Immunology,2001,167:6002-6008.
[29]Barakonyi A,Kovacs KT,Miko E,et al.Recognition of nonclassical HLA class Ⅰantigens by gamma delta T cells during pregnancy[J].J Immunol.2002,15;168(6):2683-8.
[30]Sageshima N,Ishitani A,Omura M,et al.Necrotic feature of the trophoblasts lacking HLA-G expression in normal and pre-eclamptic placentas[J].Am J Reprod Immunol.2003,49(3):174-82.
[31]Christman JK.5-azacytidine and 5-aza-2-deoxycyfdine as inhibitors of DNA methylation:mechanistic studies and their implications for cancer therapy[J].ONcogene,2002,21:5483-5495.
[1] Emmer Pm,Steegers EA,Kerstens HM,et al.Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies[J]. Hum Reprod.2002,17(4): 1072-80.
[2] Hviid TV, Larsen LG, Hogh AM and Bzorek M.HLA-G expression inplacenta in relation to HLA-G genotype and polymorphisms[J]. Am JReprod Immunol. 2004,52:212-217.
[3] Ingman WV,Robertson SA.Defining the actions of transforming growth factor beta in reproduction[J].Bioessays.2002,24:904-914.
[4] LeMaoult J, Le Discorde M, Rouas-Freiss N,et al.Biology and functions of human leukocyte antigen-G in health and sickness[J] .Tissue Antigens.2003, 62:273-284.
[5] Joanna LJ,Peter RS,Lawrence WC.The isolation and characterization of a population of extravillous cytotrophoblast progenitors from first trimester human placenta[J] .Human Reproduction,2007,22(8):2111-2119.
[6] Creput C, Le Friec G, Bahri R,et al.Detection of HLA-G in serum and graft biopsy associated with fewer acute rejections following combined liverkidney transplantation: possible implications for monitoring patients[J].Hum Immunol [J]. 2003,64:1033-1038.
[7] Ishitani A, Sageshima N, Lee N, et al.Protein and peptide binding suggest unique and interacting functional roles for HLA-E, expression F, and G in maternalplacental immune recognition[J]. J Immunol.2003,171:1376-1384.
[8] Rebmann V, Busemann A, Lindemann M,et al.Detection of HLA-G5 secreting cells[J] .Hum Immunol.2003,64:1017-1024.
[9] Philippe LB,Valerie M.HLA-G and pregnancy [J] Journal of Reproduction and Fertility,1997,2:7-13.
[10]Rabot M,Tabiasco J,Polgar B,et al.HLA class I/NK cell receptor interaction in early human decidua basalis:possible functional consequences.Chem Immunol Allergy.2005;89:72-83.
[11]王炎秋,陈士岭,邢福祺。HLA-G蛋白在滋养层细胞的表达及意义[J]。第一军医大学学报,2005,25(12):1488-1490。
[12]Chrysoula,Dosiou,Linda C.Natural killer cells in pregnancy and recurrent pregnancy loss:Endocrine and immunologic perspectives[J].Endocr Rev.2005,26(1):44-62;
[13]Kuhbav GO,Critchley HO,Bowen JM,et al.Prolactin induces ERK,phosphorylation in epithelial and CD56+ natural killer cells of the human endometrium[J].J Clin Endocrinlo Metab.2002,87:2329-2335;
[14]Borrego F,Kabat J,Kim DK,etal.Structure and function of major histocompatibility complex(MHC)class Ⅰ specific receptors expressed on human natural killer(NK)cells[J].Mol Immunol,2002,38(9):637-660.
[15]Menier C,Riteau B,Carosella ED,et al.MICA triggering signal for NK cell tumor lysis is counteracted by HLA-Gl-mediated inhibitory signal[J].Int J Cancer.2002,100:63-70.
[16]Sala FG,Del Moral PM,Pizzato N,et al.The HLA-G~*0105N null allele induces cell surface expression of HLA-E molecule and promotes CD94/ NKG2Amediated recognition in JAR choriocarcinoma cell line[J].Immunogenetics,2002,4:238-242.
[17]Hunt JS,Petroff MG,McIntire RH,et al.HLA-G and immune tolerance in pregnancy[J].FASEB J.2005,19(7):681-93.
[18]Rieger L,Hofmeister V,Probe C,et al.Th_1 and Th_2 like cytokine production by first trimester decidual large granular lymphocytes is influenced by HLA-G and HLA-E[J].Mol Hum Reprod,2002,8(3):255-261.
[19]Varla-Leftherioti M.The significance of the women's repertoire of natural killer cell receptors in the maintenance of pregnancy[J].Chem Immunol Allergy. 2005,89:84-95.
[20] Shiroishi M,Tsumoto K,Amano K et al.Human inhibitory receptors Ig-like transcript 2(ILT2) and ILT4 compete with CD8 for MHC class I binding andbind preferentially to HLA-G.Proc. Natl. Acad.Sci. 2003,100:8856-8861;
[21] Morales PJ,Pace JL,Platt JS,et al.Placenta cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype.J. Immunol.2003,171:6215-6224;
[22]Contini P,Ghio M,Poggi A,et al.Soluble HLA-A,B,G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation.Eur. J. Immunol.2003,33:125-134;
[23]Kanai T,Fujii T,Unno N,et al.Human leukocyte antigen-G expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood.Am J Reprod Immunol.2001,45(2):94-99;
[24]Fournel S,Aguerre GM,Huc X,et al.Cutting edge:soluble HLA-G1 triggers CD95/CD95 ligand mediated apoptosis in activated CD8+ cells by interacting with CD8[J] J Immunol,2000,164(12):6100-6104.
[25] Forte P,Pazmany L,Matter UB,et al. HLA-Ginhibits rolling adhesion of activated human NK cells on porcine endothelial cells[J].J Immunol,2001,167(10):6002— 6008.
[26]Rukavina D,Vince GRoles of agtokines and immune cell at the interface—a workshop report[J].Placenta,2000,21:97-98.
[27] Pfeiffer KA, Rebmann V ,Ven K.Soluble histocompatibility antigen levels in early pregnancy after IVF[J]. Hum Immunol.2002,61:559-564.
[28] Emmer Pm,Steegers EA,Kerstens HM,et al.Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies[J].Hum Reprod.2002 ,17(4):1072-80.
[29]Hviid TV,Hylenius S,Rorbye C,et al.HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels[J]. Immunogenetics.2003,55(2):63-79.
[30] Hviid TV, Hylenius S, Lindhard A,et al.Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome[J]. Tissue Antigens.2004,64:66-69.
[31] Patel RN, Quack KC, Hill JA,et al.Expression of membraneboundHLA-G at the maternal-fetal interface is not associated with pregnancy maintenance among patients with idiopathic recurrent pregnancy loss[J]. Mol Hum Reprod. 2003, 9: 551-557.
[32] Hviid TV,Hylenius S,Rorbye C,et al. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels[J].Immunogenetics. 2003,55(2):63-79.
[33] Pfeiffer KA,Fimmers R,Engels G,et al. The HLA-G genotype is potentially associated with idiopathic recurrent spontaneous abortion[J].Mol Hum Reprod. 2001,7(4):373-378.
[34] Barakonyi A,Kovacs KT,Miko E,et al. Recognition of nonclassical HLA class I antigens by gamma delta T cells during pregnancy. J Immunol.2002 Mar 15;168(6):2683-8.
[35] Sageshima N,Ishitani A,Omura M,et al. Necrotic feature of the trophoblasts lacking HLA-G expression in normal and pre-eclamptic placentas.Am J Reprod Immunol. 2003 Mar;49(3): 174-82.
[36] Varla-Leftherioti M.The significance of the women's repertoire of natural killer cell receptors in the maintenance of pregnancy. Chem Immunol Allergy.2005;89:84-95.
[37] Yie SM, Li LH, Li YM,et al.HLA-G protein concentrations in maternal serum and placental tissue are decreased in preeclampsia[J]. Am J Obstet Gynecol. 2004,191:525-529.
[38]Hylenius S, Nybo Andersen AM, Melbye M,et al.Association between HLA-G genotype and risk of pre-eclampsia: a case-control study using family triads[J]. Mol Hum Reprod.2004,10:237-246
[39] Nagamatsu T, Fujii T, Yamashita T,et al.Hypoxia does not reduce HLA-G expression on extravillous cytotrophoblasts[J]. J Reprod Immunol. 2004, 63: 85-95.
[40] Hviid TV,Christiansen OB,Johansen JK,et al.Characterization of a new HLA-G allele encoding a nonconservative amino acid substitution in the alpha3 domain (exon4) and its relevance to certain complications in pregnancy[J].Immunogenetics,2001,53(1):48-53.
[2] Rabot M,Tabiasco J,Polgar B,et al. HLA class I/NK cell receptor interaction in early human decidua basalis: possible functional consequences[J].Chem Immunol Allergy.2005,89:72-83.
[3] Emmer Pm,Steegers EA,Kerstens HM,et al.Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies[J]. Hum Reprod.2002,17(4): 1072-80.
[4] Hviid TV,Hylenius S,Rorbye C,et al.HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels[J]. Immunogenetics.2003,55(2):63-79.
[5] Varla-Leftherioti M.The significance of the women's repertoire of natural killer cell receptors in the maintenance of pregnancy[J].Chem Immunol Allergy. 2005, 89:84-95.
[6] Philippe M,Gael M,Philippe R,et al.HLA-G gene repression is reversed by demethylation[J]. Proc Natl Acad Sci.2003,100(3):1191-1196.
[7] Guillaudeux T, Rodriguez AM, Girr M, et al. Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta[J].J Immunol. 1995,154(7):3283-3299.
[8] Onno, M,Amiot L,Bertho N,et al.CpG methylation patterns in the 5' part of the nonclassical HLA-G gene in peripheral blood CD34+ cells and CD2+ lymphocytes[J]. Munksgaard International Publishers. 1997,49(4):356-364.
[9] Munz C,Holmes N,King A,et al.Human histocompatibility leukocyte antigen(HLA)-G molecules inhibit NKAT3 expressing natural killer cells[J].J Exp Med,1997,185(3):385-392.
[10]彭冰,刘淑芸,邢爱耕等。人类白细胞抗原G、E在人胎盘组织中的表达及意义[J]。现代妇产科进展,2006,15(7):525-530;
[11]Joanna LJ,Peter RS,Lawrence WC.Cytotrophoblast differentiation in the first trimester of pregnancy:evidence for separate progenitors of extravillous trophoblasts and syncytiotrophoblast[J].Reproduction.2005,130:95-103;
[12]Shiroishi M,Tsumoto K,Amano K et al.Human inhibitory receptors Ig-like transcript 2(ILT2)and ILT4 compete with CD8 for MHC class Ⅰ binding and bind preferentially to HLA-G[J].Proc.Natl.Acad.Sci.2003,100:8856-8861;
[13]Morales PJ,Pace JL,Platt JS,et al.Placenta cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype[J].J.Immunol.2003,171:6215-6224;
[14]Contini P,Ghio M,Poggi A,et al.Soluble HLA-A,B,G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8ligation[J].Eur.J.Immunol.2003,33:125-134;
[15]Kanai T,Fujii T,Unno N,et al.Hurnan leukocyte antigen-G expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood[J].Am J Reprod Immunol.2001,45(2):94-99;
[16]Patel RN,Quack KC,Hill JA,et al.Expression of membraneboundHLA-G at the maternal-fetal interface is not associated with pregnancy maintenance among patients with idiopathic recurrent pregnancy loss[J].Mol Hum Renrod.2003.9.551-557.
[17]王炎秋,陈士岭,邢福祺。HLA-G蛋白在滋养层细胞的表达及意义[J]。第一军医大学学报。2005,25(12):1488-1490;
[18]King A,Hiby SE,Gardner L,et al.Recognition of trophoblast HLA class Ⅰmolecules by decidual NK cell receptors—a review[J].Placenta.2000,21:S81-85;
[19]Chrysoula,Dosiou,Linda C.Natural killer cells in pregnancy and recurrent pregnancy loss:Endocrine and immunologic perspectives[J].Endocr Rev.2005,26(1):44-62;
[20]Kuhbav GO,Critchley HO,Bowen JM,et al.Prolactin induces ERK,phosphorylation in epithelial and CD56+ natural killer cells of the human endometrium[J].J Clin Endocrinlo Metab.2002,87:2329-2335;
[21]Borrego F,Kabat J,Kim DK,etal.Structure and function of major histocompatibility complex(MHC)class I specific receptors expressed on human natural killer(NK)cells[J].Mol Immunol,2002,38(9):637-660.
[22]Dosiou Chrysoula,Giudice LC.Natural killer cells in pregnancy and recurrent pregnancy loss:endocrine and immunologic perspectives[J].Endocr Rev.2005,26(1):44-62;
[23]Santi DV,Garrett CE,Barr PJ.On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs[J].Cell.1983,33:9;
[24]Wold AS,Arici A.Natural killer cells and reproduction failure[J].Curr Op in Obstet Gynecol,2005,17(3):237-341;
[25]Matsubayashi H,Shida M,Kondo A,et al.Preconception peripheral natural killer cell activity as a predictor of pregnancy outcome in patients with unexplained infertility[J].Am J Reprod Immunol,2005,53(3):126-131;
[26]李昭荣、韩香、吕海侠等。蜕膜自然杀伤细胞与不明原因性反复流产的关系[J]。生殖医学杂志,2005,14(2):68-71;
[27]HitomiSasaki,Xiao-ChunXu,T.Mohanakumar.HLA-E and HLA-G Expression on Porcine Endothelial Cells Inhibit Xenoreactive Human NK Cells Through CD94/NKG2-Dependent and -Independent Pathways[J].The Journal of Immunology,1999,163:6301-6305
[28]PietroForte,LaszloPazmany,UlrikeB,et al.HLA-G Inhibits Rolling Adhesion of Activated Human NK Cells on Porcine Endothelial Cells[J].The Journal of Immunology,2001,167:6002-6008.
[29]Barakonyi A,Kovacs KT,Miko E,et al.Recognition of nonclassical HLA class Ⅰantigens by gamma delta T cells during pregnancy[J].J Immunol.2002,15;168(6):2683-8.
[30]Sageshima N,Ishitani A,Omura M,et al.Necrotic feature of the trophoblasts lacking HLA-G expression in normal and pre-eclamptic placentas[J].Am J Reprod Immunol.2003,49(3):174-82.
[31]Christman JK.5-azacytidine and 5-aza-2-deoxycyfdine as inhibitors of DNA methylation:mechanistic studies and their implications for cancer therapy[J].ONcogene,2002,21:5483-5495.
[1] Emmer Pm,Steegers EA,Kerstens HM,et al.Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies[J]. Hum Reprod.2002,17(4): 1072-80.
[2] Hviid TV, Larsen LG, Hogh AM and Bzorek M.HLA-G expression inplacenta in relation to HLA-G genotype and polymorphisms[J]. Am JReprod Immunol. 2004,52:212-217.
[3] Ingman WV,Robertson SA.Defining the actions of transforming growth factor beta in reproduction[J].Bioessays.2002,24:904-914.
[4] LeMaoult J, Le Discorde M, Rouas-Freiss N,et al.Biology and functions of human leukocyte antigen-G in health and sickness[J] .Tissue Antigens.2003, 62:273-284.
[5] Joanna LJ,Peter RS,Lawrence WC.The isolation and characterization of a population of extravillous cytotrophoblast progenitors from first trimester human placenta[J] .Human Reproduction,2007,22(8):2111-2119.
[6] Creput C, Le Friec G, Bahri R,et al.Detection of HLA-G in serum and graft biopsy associated with fewer acute rejections following combined liverkidney transplantation: possible implications for monitoring patients[J].Hum Immunol [J]. 2003,64:1033-1038.
[7] Ishitani A, Sageshima N, Lee N, et al.Protein and peptide binding suggest unique and interacting functional roles for HLA-E, expression F, and G in maternalplacental immune recognition[J]. J Immunol.2003,171:1376-1384.
[8] Rebmann V, Busemann A, Lindemann M,et al.Detection of HLA-G5 secreting cells[J] .Hum Immunol.2003,64:1017-1024.
[9] Philippe LB,Valerie M.HLA-G and pregnancy [J] Journal of Reproduction and Fertility,1997,2:7-13.
[10]Rabot M,Tabiasco J,Polgar B,et al.HLA class I/NK cell receptor interaction in early human decidua basalis:possible functional consequences.Chem Immunol Allergy.2005;89:72-83.
[11]王炎秋,陈士岭,邢福祺。HLA-G蛋白在滋养层细胞的表达及意义[J]。第一军医大学学报,2005,25(12):1488-1490。
[12]Chrysoula,Dosiou,Linda C.Natural killer cells in pregnancy and recurrent pregnancy loss:Endocrine and immunologic perspectives[J].Endocr Rev.2005,26(1):44-62;
[13]Kuhbav GO,Critchley HO,Bowen JM,et al.Prolactin induces ERK,phosphorylation in epithelial and CD56+ natural killer cells of the human endometrium[J].J Clin Endocrinlo Metab.2002,87:2329-2335;
[14]Borrego F,Kabat J,Kim DK,etal.Structure and function of major histocompatibility complex(MHC)class Ⅰ specific receptors expressed on human natural killer(NK)cells[J].Mol Immunol,2002,38(9):637-660.
[15]Menier C,Riteau B,Carosella ED,et al.MICA triggering signal for NK cell tumor lysis is counteracted by HLA-Gl-mediated inhibitory signal[J].Int J Cancer.2002,100:63-70.
[16]Sala FG,Del Moral PM,Pizzato N,et al.The HLA-G~*0105N null allele induces cell surface expression of HLA-E molecule and promotes CD94/ NKG2Amediated recognition in JAR choriocarcinoma cell line[J].Immunogenetics,2002,4:238-242.
[17]Hunt JS,Petroff MG,McIntire RH,et al.HLA-G and immune tolerance in pregnancy[J].FASEB J.2005,19(7):681-93.
[18]Rieger L,Hofmeister V,Probe C,et al.Th_1 and Th_2 like cytokine production by first trimester decidual large granular lymphocytes is influenced by HLA-G and HLA-E[J].Mol Hum Reprod,2002,8(3):255-261.
[19]Varla-Leftherioti M.The significance of the women's repertoire of natural killer cell receptors in the maintenance of pregnancy[J].Chem Immunol Allergy. 2005,89:84-95.
[20] Shiroishi M,Tsumoto K,Amano K et al.Human inhibitory receptors Ig-like transcript 2(ILT2) and ILT4 compete with CD8 for MHC class I binding andbind preferentially to HLA-G.Proc. Natl. Acad.Sci. 2003,100:8856-8861;
[21] Morales PJ,Pace JL,Platt JS,et al.Placenta cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype.J. Immunol.2003,171:6215-6224;
[22]Contini P,Ghio M,Poggi A,et al.Soluble HLA-A,B,G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation.Eur. J. Immunol.2003,33:125-134;
[23]Kanai T,Fujii T,Unno N,et al.Human leukocyte antigen-G expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood.Am J Reprod Immunol.2001,45(2):94-99;
[24]Fournel S,Aguerre GM,Huc X,et al.Cutting edge:soluble HLA-G1 triggers CD95/CD95 ligand mediated apoptosis in activated CD8+ cells by interacting with CD8[J] J Immunol,2000,164(12):6100-6104.
[25] Forte P,Pazmany L,Matter UB,et al. HLA-Ginhibits rolling adhesion of activated human NK cells on porcine endothelial cells[J].J Immunol,2001,167(10):6002— 6008.
[26]Rukavina D,Vince GRoles of agtokines and immune cell at the interface—a workshop report[J].Placenta,2000,21:97-98.
[27] Pfeiffer KA, Rebmann V ,Ven K.Soluble histocompatibility antigen levels in early pregnancy after IVF[J]. Hum Immunol.2002,61:559-564.
[28] Emmer Pm,Steegers EA,Kerstens HM,et al.Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies[J].Hum Reprod.2002 ,17(4):1072-80.
[29]Hviid TV,Hylenius S,Rorbye C,et al.HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels[J]. Immunogenetics.2003,55(2):63-79.
[30] Hviid TV, Hylenius S, Lindhard A,et al.Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome[J]. Tissue Antigens.2004,64:66-69.
[31] Patel RN, Quack KC, Hill JA,et al.Expression of membraneboundHLA-G at the maternal-fetal interface is not associated with pregnancy maintenance among patients with idiopathic recurrent pregnancy loss[J]. Mol Hum Reprod. 2003, 9: 551-557.
[32] Hviid TV,Hylenius S,Rorbye C,et al. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels[J].Immunogenetics. 2003,55(2):63-79.
[33] Pfeiffer KA,Fimmers R,Engels G,et al. The HLA-G genotype is potentially associated with idiopathic recurrent spontaneous abortion[J].Mol Hum Reprod. 2001,7(4):373-378.
[34] Barakonyi A,Kovacs KT,Miko E,et al. Recognition of nonclassical HLA class I antigens by gamma delta T cells during pregnancy. J Immunol.2002 Mar 15;168(6):2683-8.
[35] Sageshima N,Ishitani A,Omura M,et al. Necrotic feature of the trophoblasts lacking HLA-G expression in normal and pre-eclamptic placentas.Am J Reprod Immunol. 2003 Mar;49(3): 174-82.
[36] Varla-Leftherioti M.The significance of the women's repertoire of natural killer cell receptors in the maintenance of pregnancy. Chem Immunol Allergy.2005;89:84-95.
[37] Yie SM, Li LH, Li YM,et al.HLA-G protein concentrations in maternal serum and placental tissue are decreased in preeclampsia[J]. Am J Obstet Gynecol. 2004,191:525-529.
[38]Hylenius S, Nybo Andersen AM, Melbye M,et al.Association between HLA-G genotype and risk of pre-eclampsia: a case-control study using family triads[J]. Mol Hum Reprod.2004,10:237-246
[39] Nagamatsu T, Fujii T, Yamashita T,et al.Hypoxia does not reduce HLA-G expression on extravillous cytotrophoblasts[J]. J Reprod Immunol. 2004, 63: 85-95.
[40] Hviid TV,Christiansen OB,Johansen JK,et al.Characterization of a new HLA-G allele encoding a nonconservative amino acid substitution in the alpha3 domain (exon4) and its relevance to certain complications in pregnancy[J].Immunogenetics,2001,53(1):48-53.