【作者】 徐金娥；

【导师】 陈子江；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 博士

【摘要】 第一部分蜕膜杀伤细胞免疫球蛋白样受体与不明原因早期复发性流产的关联性研究第一节不明原因早期复发性流产患者蜕膜杀伤细胞免疫球蛋白样受体基因多态性的研究【目的】不明原因复发性流产(unexplained recurrent spontaneousabortion,URSA)病因复杂,其中近1/2原因不明,其免疫发病机理尚不清楚。近年来,母胎界面自然杀伤细胞(NK细胞)的作用受到关注,母胎界面NK细胞亚群失衡及功能异常可能会导致自然流产等妊娠相关疾病。滋养细胞携带来自胎儿的外胚层抗原,是母胎界面唯一与母体免疫系统直接接触的胎儿细胞,绒毛滋养细胞上父源的HLA-C抗原与母体NK细胞的识别可能会影响母胎界面NK细胞的功能,进而影响妊娠结局。NK细胞的功能受其表面受体的调节,NK细胞的活化或抑制取决于活化性受体传导的活化性信号和抑制性受体传导的抑制性信号的平衡。目前研究较多的是杀伤细胞免疫球蛋白样受体(killer cellimmunoglobulin-like receptors,KIR),KIR主要存在于NK细胞和部分T细胞表面,通过与靶细胞表面的HLA配体识别,组成特异性信号传导通路(KIR/HLA-C)来调节NK细胞的功能,在调控免疫反应中发挥重要作用。编码KIR的基因在人群中具有多态性,本研究的目的是通过研究母胎界面蜕膜组织KIR基因的多态性,探讨KIR基因与不明原因早期复发性流产易感性的关系。【方法】选择2006年1月至2008年2月在山东省立医院生殖中心就诊的不明原因早期复发性流产患者36例,年龄32.34±7.22岁,孕周9.64±2.89周。选择同期在计划生育门诊行人工流产术的正常早孕妇女34例,年龄30.23±6.16岁,孕周8.61±2.11周,月经规律,无自然流产、死胎、死产史。人工流产术时留取URSA患者和正常对照组的蜕膜组织,抽提基因组DNA,序列特异性引物聚合酶链反应检测蜕膜组织14种KIR基因,分析URSA组和对照组蜕膜组织KIR基因的频率及活化性KIR基因的数目差异。【结果】①14种KIR基因在URSA组与对照组的蜕膜组织中均以不同频率表达,其中KIR2DL1,KIR2DL4,KIR3DL2,KIR3DL3的检出频率为100%;URSA组蜕膜组织中KIR2DS1基因的频率为60.27%,对照组蜕膜组织中KIR2DS1基因的频率为41.18%,两组相比,差异有显著性,P=0.03;其它KIR基因位点未发现有统计学差异。②URSA组活化性KIR基因平均数较对照组显著升高(P=0.041);URSA组中携带2个以上活化性KIR基因的个体较对照组明显增多,差异具有显著性(P=0.018)。【结论】①蜕膜组织KIR2DS1基因频率增加,抑制性、活化性KIR基因失衡,活化性KIR与胎儿滋养细胞表面相应的HLA配体结合,传递活化性信号活化NK细胞,滋养细胞植入蜕膜过程受阻,导致自然流产发生。②活化性KIR基因数目增多可能是URSA的发病原因之一。第二节不明原因早期复发性流产患者蜕膜杀伤细胞免疫球蛋白样受体mRNA的表达研究【目的】蜕膜NK细胞与滋养细胞之间存在复杂的识别机制,KIR的表达受到母胎界面微环境的调节,KIR可以增加NK细胞识别滋养细胞的能力,在胚胎种植及妊娠维持过程中发挥重要作用。KIR的表达主要由基因型决定,一个NK细胞可以同时表达KIR2D和KIR3D分子,也可以同时表达活化性和抑制性受体,但基因组仅是遗传信息的储存体,需通过转录、翻译成蛋白质,才能执行功能。URSA妇女KIR在基因水平不同于正常早孕妇女,活化性KIR基因的频率增加,但KIR基因在mRNA水平的表达是否存在差异尚不清楚。本研究目的是探讨URSA组与对照组妇女早孕子宫蜕膜组织KIR在转录水平的表达,进一步了解蜕膜KIR与URSA易感性的关系。【方法】留取URSA妇女(病例组,36例)及正常早孕妇女(对照组,34例)的蜕膜组织,Trizol一步法提取总RNA,逆转录聚合酶链反应分析KIRmRNA的表达,荧光实时定量聚合酶链反应分析KIR2DL1、2DL3 mRNA的相对表达量,分析URSA组与对照组蜕膜组织KIR mRNA表达的差异。【结果】①6种抑制性KIR mRNA在蜕膜组织中均以不同频率表达,两组的表达率均无统计学差异;其中URSA组KIR2DL2的表达率虽较对照组降低,但无显著性差异;KIR mRNA的表达率与KIR基因的表达率并不完全一致。活化性KIR mRNA未检出。②URSA组与对照组KIR2DL1 mRNA的相对表达量分别为699.37±259.78,1002.96±476.98,两组相比,差异无显著性(P=0.37);URSA组与对照组KIR2DL3 mRNA的相对表达量分别为625.93±234.81,1216.12±425.96,两组相比,差异亦无显著性(P=0.09)。【结论】①URSA患者蜕膜组织KIR2DL1、2DL2、2DL3、3DL1、3DL2、2DL4 mRNA的表达率变化可能与不明原因早期复发性流产无直接关系。②蜕膜组织KIR2DL1、2DL3 mRNA的相对表达量可能与不明原因早期复发性流产的发生无关。第二部分绒毛滋养细胞HLA-C抗原与不明原因早期复发性流产的关联性研究第一节绒毛滋养细胞HLA-C基因与不明原因早期复发性流产的相关性研究【目的】随着生殖免疫学研究的深入,发现30%—40%URSA的发生与HLA有关,URSA与HLA的关系已成为生殖免疫研究的热点。在妊娠过程中,滋养细胞携带来自胎儿的外胚层抗原,是母胎界面唯一与母体免疫系统直接接触的胎儿细胞,滋养细胞表达绎典的MHCⅠ类分子HLA-C,母系和父系的HLA-C等位基因在滋养细胞表面均有表达,因此,绒毛滋养细胞上父源的HLA-C抗原与母体NK细胞KIR的结合可能会影响母胎界面NK细胞的功能,进而影响妊娠结局。HLA-C属于经典的HLA-Ⅰ类基因,具有高度多态性。根据HLA—C分子al螺旋第77、80位氨基酸的不同,将HLA—C多态性分子分为两组:第一组为HLA—Cwl、Cw3、Cw7、Cw8及其他相关分子,第80位氨基酸为天冬酰胺,可被NKⅡ类细胞(表达KIR2DL2)识别而不被杀伤;第二组为HLA—Cw2、Cw4、Cw5、Cw6和其他相关分子,它们的第80位氨基酸残基为赖氨酸,可以被NKⅠ类细胞(表达KIR2DL1)识别而不被杀伤。由于HLA-Cw抗原比HLA-A、HLA-B要弱10倍,传统血清学方法在检测HLA-Cw方面有很大的局限性,采用DNA分型和测序方法可以准确的进行HLA-Cw分型。为探讨绒毛滋养细胞HLA-C基因是否参与不明原因早期复发性流产的发病,本研究采用DNA测序法对URSA患者绒毛滋养细胞HLA-C基因进行分析,探讨其与URSA发病的相关性,为不明原因早期复发性流产的病因分析和临床治疗提供理论基础。【方法】人工流产术时留取URSA患者(36例)及正常早孕妇女(34例)的绒毛组织,提取基因组DNA,采用美国ABI公司BigDye Terminator v3.1测序试剂盒,按照使用说明在ABI Prism 3100测序仪上测序,分析绒毛滋养细胞HLA-C1、HLA-C2基因的频率,探讨HLA-C1、HLA-C2基因与URSA发病的相关性。【结果】①两组基因HLA-C1、HLA-C2在绒毛组织中呈不平衡表达,以HLA-C1基因占明显优势。HLA-C1基因在URSA组与对照组的频率分别为66.67%,81.03%,两组相比差异无显著性(P=0.657);HLA-C2基因在URSA组与对照组的频率分别为33.33%,19.07%,两组相比差异有显著性(P=0.007)。②对照组妇女HLA-C1/C1基因型占65.52%,HLA-C1/C2基因型占31.03%,1例HLA-C2/C2基因型,HLA-C1/C1基因型占明显优势;URSA患者中HLA-C1/C1基因型为33.33%,HLA-C1/C2基因型占66.67%。URSA组的HLA-C1/C1基因型的频率明显低于对照组,差异有显著性(P=0.039);而URSA组的HLA-C1/C2基因型的频率明显高于对照组,差异亦有显著性(P=0.001)。【结论】URSA患者HLA-C1/C1基因型的频率降低、HLA-C1/C2基因型增加,导致单倍型HLA-C1的频率降低,HLA-C2基因频率升高。HLA-C1、HLA-C2失衡可能是URSA发病的免疫遗传学因素之一。第二节绒毛滋养细胞HLA-C抗原的表达与不明原因早期复发性流产的相关性研究【目的】在母胎界面,滋养细胞执行着复杂的生物学功能,如果这些功能的调控机制紊乱,将导致多种病理性妊娠的发生。妊娠中绒毛滋养细胞HLA-C在表达水平上的研究尚未见有报道,本研究拟通过分析绒毛滋养细胞HLA-C抗原的表达,进一步探讨绒毛滋养细胞HLA-C抗原与不明原因早期复发性流产易感性的关系。【方法】留取URSA患者(36例)及正常早孕妇女(34例)的绒毛组织,Trizol一步法提取总RNA,荧光实时定量PCR法分析绒毛滋养细胞HLA-C1mRNA、HLA-C2 mRNA的相对表达量,免疫印迹法分析绒毛滋养细胞HLA-C蛋白的表达水平。【结果】①URSA组绒毛滋养细胞HLA-C1 mRNA的相对表达量为59.39±13.43,对照组HLA-C1 mRNA的相对表达量为95.43±28.78,两组相比差异有显著性(P=0.016);URSA组绒毛滋养细胞HLA-C2 mRNA的相对表达量为68.22±18.73,对照组HLA-C2 mRNA的相对表达量为48.33±17.30,两组相比差异无显著性(P=0.321)。②URSA组绒毛滋养细胞HLA-C1 mRNA的表达率为41.43%,对照组绒毛滋养细胞HLA-C1 mRNA的表达率为50%,两组相比差异无显著性(P=0.289);URSA组绒毛滋养细胞HLA-C2 mRNA的表达率为58.57%,对照组绒毛滋养细胞HLA-C2 mRNA的表达率为50%,两组相比差异亦无显著性(P=0.330)。③URSA组与对照组绒毛滋养细胞HLA-C蛋白的相对表达值分别为1.32±0.19,1.45±0.21,两组相比差异无显著性(P=0.640)。【结论】①绒毛滋养细胞HLA-C抗原表达失衡,HLA-C1 mRNA表达降低,可能是引起URSA发病的免疫因素之一。②绒毛滋养细胞HLA-C总蛋白的表达可能与URSA的发病无关。更多还原

【Abstract】 SECTIONⅠASSOCIATION OF KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTOR GENES AND THEIR EXPRESSION WITH EARLY UNEXPLAINED RECURRENT SPONTANEOUS ABORTIONPART 1 Association of Killer Cell Immunoglobulin-like Receptor Genes in Decdiua and Early Unexplained Recurrent Spontaneous AbortionOBJECTIVES Killer cell immunoglobulin-like receptors(KIR) are expressed on the surface of natural killer(NK) cells and some of T Lymphocytes.NK cell receptors have been suggested have a high-level infiltration with stromal cells and more importantly with invading trophoblast.But their function at the fetomaternal interface remain unknown.To study the relationship between the expression of the susceptiblity to killer-immunoglobulin-like receptors in decidua and early unexplained recurrent spontaneous abortion(URSA). METHODS Decidual samples were obtained from the patients(n=36) who suffered from unexplained recurrent spontaneous abortion and the normal pregnant women(n=34).Specific sequence primer PCR(PCR-SSP) was used to determine the individual KIR genotypes in women experiencing URSA and controls.RESULTS①A higher prevalence of activating KIR genes was seen in patients experiencing URSA than in controls.Among women experiencing URSA,the gene express frequence of KIR2DS1 was more prevalent(60.27%vs 41.18%,P=0.03) compared with controls.②Increased numbers of activating KIR genes was observed in patients with URSA(P=0.041).CONCLUSIONS The increased frequencies of KIR2DS1 may be one of the reasons of URSA.A genetic imbalance between activing and inhibitory KIR genes might confer susceptibility to the occurrence of pregnancy loss.PART 2 Expression of Killer Cell Immunoglobulin-like Receptor Genes in Decdiua and Early Unexplained Recurrent Spontaneous AbortionOBJECTIVES Decidual natural killer(NK) cells express killer cell immunoglobulin-like receptors(KIRs),which bind to ligands on trophoblast cells. This interaction appears to block NK cytotoxicity against trophoblast cells.In this study,we investigated the expression of mRNA of KIRs in decidua of women with early unexplained recurrent spontaneous abortion(URSA).METHODS The decidual samples taken via uterine cucrretting from 36 patients experiencing URSA and 34 healthy controls.The expressions of KIR2DL2、2DL4、3DL1、3DL2 mRNA in decidual tissue were carried out by the RT-PCR.The relative level of KIR2DL1 and 2DL3 mRNA were studied by real time quantitive RT- PCR.RESULTS①The frequencies of inhibitory KIRs mRNA did not differ between women with URSA and normal controls.None of the activating KIRs mRNA was detected.However,there was an decrease in transcripts of KIR2DL2 in women with URSA when compared to normal controls.②The relative level of the KIR2DL1 mRNA did not show significant differences between women with URSA and nomal controls(699.37±259.78 vs 1002.96±476.98,P=0.37).And also no significant differences in KIR2DL3 mRNA was observed between two groups(625.93±234.81 vs 1216.12±425.96,P=0.09).CONCLUSIONS The expression of KIR mRNA may not explain the pathogenesis of URSA.SECTIONⅡASSOCIATION OF HUMAN LEUKOCYTE ANTIGEN-C MOLECULES ON TROPHOBLAST CELLS AND EARLY UNEXPLAINED RECURRENT SPONTANEOUS ABORTIONPART 1 Association of Human Leukocyte Antigen-C Molecules Genes on Trophoblast Cells and Early Unexplained Recurrent Spontaneous AbortionOBJECTIVES To investigate the relationship between the genotype of human leukocyte antigen(HLA)-C molecules on trophoblast cells and early unexplained recurrent spontaneous abortion(URSA).METHODS Trophoblast samples were obtained from the patients(n=36) who suffered from early recurrent spontaneous abortion and the normal pregnant women(n=34).Genotype were detected for HLA-C1 and HLA-C2 by sequence based typing.We used two-tailed Fisher’s exact test to compare variables between groups.RESULTS①The haplotype of HLA-C1 showed the highest frequencies in both groups.There was no significant differences in the frequencies of HLA-C1 molecules on trophoblast cells between two groups(66.67%vs 81.03%,P=0.657).But there was higher frequencies of HLA-C2 molecules in the patients than in the controls(33.33%vs 19.07%,P=0.007).②The significant differences were observed in genotype HLA-C1C1(65.52%vs 33.33%,P=0.039) and HLA-C1C2(31.03%vs 66.67%,P=0.001) between control and URSA groups.CONCLUSIONS The increased frequences of HLA-C2 genes were resulted from the imbalance of HLA-C1 and HLA-C2 on the trophoblast cells.The increased frequences of HLA-C2 genes might confer susceptibility to the occurrence of pregnancy loss.PARTⅡAssociation of Human Leukocyte Antigen-C Molecules Expression on Trophoblast Cells and Early Unexplained Recurrent Spontaneous AbortionOBJECTIVES To study the relationship between the expression of the susceptiblity to human leukocyte antigen(HLA)-C molecules on trophoblast cells and early unexplained recurrent spontaneous abortion(URSA).METHODS Trophoblast samples were obtained from the patients(n=36) who suffered from early unexplained recurrent spontaneous abortion and the normal pregnant women(n=34).The level of HLA-C1 mRNA and HLA-C2 mRNA on trophoblast cells were measured by the real time PCR method,and the expression of HLA-C protein was detected by Western immunoblotting method. RESULTS①The relative levels of the HLA-C1 mRNA were significant lower in the patients(59.39±13.43) than in the normal pregnants(95.43±28.78), P=0.016.And the relative levels of the HLA-C2 mRNA did not show differences between two groups(68.22±18.73 vs 48.33±17.30,P=0.321 ).②The expression of HLA-C protein was similar between two groups(1.32±0.19 vs 1.45±0.21,P=0.640).CONCLUSIONS The expression of human leukocyte antigen(HLA)-C1 mRNA on trophoblast cells might confer to the pathogenesis of early unexplained recurrent spontaneous abortion.更多还原