【作者】 梅冰；

【导师】 余平；

【作者基本信息】 中南大学 ， 免疫学， 2010， 博士

【摘要】 目的：(1)研究湖南和湖北地区汉族人群中MICA基因多态性分布特征；(2)研究MICA基因多态性与习惯性流产、不孕症的关系,并进一步探讨可能的分子机制。方法：(1)PCR-SSP法分析162例湖南地区汉族人群和193例湖北地区汉族人群MICA基因外显子2-4的多态性,并与其他人群中该基因的分布进行比较；(2)PCR-SSP方法检测56例习惯性流产患者和78例正常对照的MICA基因外显子2-4多态性分布,并进行相关性分析；(3)PCR-SSP法检测214例不孕症患者MICA基因外显子2-4多态分布,ELISA法检测患者血清抗沙眼衣原体IgG抗体,并进行相关性分析；(4)直接测序法确定HeLa细胞和U373细胞的MICA基因型；通过实时定量PCR、Western blot和流式细胞术分别从mRNA、蛋白质水平检测沙眼衣原体感染HeLa细胞和U373细胞后,不同时间点(0h、12h、24h、36h)MICA mRNA、MICA总蛋白和细胞膜表面MICA分子的变化,并通过体外细胞毒实验检测NK细胞对沙眼衣原体感染的HeLa细胞和U373细胞在不同时间点(0h、12h、24h、36h)的杀伤效应。结果：(1)湖南汉族人群中共检测到11个MICA等位基因,其中MICA*00801／02等位基因频率最高(40.4%),其次为MICA*00201／020(20.1%)和MICA*010(17.3%),MICA*019(0.3%)和MICA*031(0.3%)等位基因频率最低；湖北汉族人群中共检测到13个MICA等位基因,其中MICA*00801／02等位基因频率最高(33.7%),其次为MICA*010(18.4%)、MICA*00201／020(13.9%)和MICA*01201／02(9.6%),MICA*005(0.5%)和MICA*027(0.5%)等位基因频率最低；两个地方人群MICA等位基因分布频率与其他人群(朝鲜人、泰国人、美洲白人和非洲裔美洲人)比较,有显著性差异(P<0.05)；(2)习惯性流产患者组中共检测出8个MICA等位基因,其中MICA*00801／02等位基因分布频率较高而MICA*018等位基因分布频率最低；正常对照组中共检出9个MICA等位基因,经统计学分析,其中也是MICA*00801／02等位基因分布频率较高,而MICA*005和MICA*01201／02等位基因分布频率最低,习惯性流产患者与正常对照组之间MICA各等位基因分布频率均无显著性差异(P>0.05)；(3)有输卵管病理改变的不孕症患者中66.7%(42／63)的个体抗沙眼衣原体IgG抗体阳性,而无输卵管病理改变的不孕症患者中仅39.1%(59／151)的个体抗沙眼衣原体IgG抗体阳性(OR：3.12,95%CI：1.68-5.78,P=0.004),高水平的抗沙眼衣原体IgG抗体增加了不孕症患者输卵管发生病理改变的风险性(OR：6.88,95%CI：3.34-14.20,P=0.001)；根据抗沙眼衣原体IgG抗体是否为阳性对不孕症患者进行分组,两组中共检测到9个MICA等位基因,其中等位基因MICA*008在两组间的分布存在差异,抗沙眼衣原体IgG抗体阴性的不孕症患者组MICA*008分布频率显著高于抗沙眼衣原体IgG抗体阳性的不孕症患者组(38.1%：22.3%,P=0.0004,Pc=0.00367,OR：2.14,95%CI：1.40-3.28)；根据是否存在输卵管病理改变对不孕症患者进行分组,两组间MICA等位基因分布频率无显著性差异(P>0.05)；根据是否有输卵管病理改变对抗沙眼衣原体IgG抗体阳性的不孕症患者进行分组,两组间MICA等位基因分布频率无显著性差异(P>0.05)；(4)HeLa细胞为MICA*008纯合子,U373细胞为MICA*001纯合子；沙眼衣原体感染HeLa细胞和U373细胞0h-36h时两细胞的MICA mRNA变化趋势相同,其中0h时MICA mRNA均有低水平的表达,12h时表达水平升高,24-36h时表达水平降低；沙眼衣原体感染HeLa细胞0h-36h时MICA蛋白表达水平未降低；沙眼衣原体感染U373细胞0h-12h时MICA蛋白表达水平降低不明显,24h-36h时蛋白表达水平降低；沙眼衣原体感染HeLa细胞0h-36h时细胞膜MICA蛋白表达水平较稳定,沙眼衣原体感染U373细胞0h-12h时细胞膜MICA蛋白表达水平变化不明显,24h-36h时表达水平降低；随感染时间的延长(0h-36h),NK细胞对沙眼衣原体感染HeLa细胞的杀伤效应不断增高；NK细胞对沙眼衣原感染的U373细胞12h杀伤效应增高,24h-36h杀伤效应降低。结论：(1)湖南和湖北汉族人群MICA外显子2-4等位基因的分布频率与其他人群之间存在差异,其中MICA*00801／02等位基因分布频率均较高；(2)MICA外显子2-4等位基因多态性分布与习惯性流产之间无相关性；(3)不孕症患者抗沙眼衣原体IgG抗体水平与其输卵管病理改变相关；(4)不孕症患者MICA*008等位基因分布频率与抗沙眼衣原体IgG抗体负相关；(5)不孕症患者MICA基因的多态性分布与输卵管病理改变、沙眼衣原体感染引起的输卵管病理改变之间均无相关性；(6)感染沙眼衣原体后HeLa细胞和U373细胞MICA mRNA表达水平的变化相同,但MICA总蛋白和细胞膜MICA蛋白表达的变化不同；(7)NK细胞对感染沙眼衣原体的HeLa细胞和U373细胞杀伤效应不同。更多还原

【Abstract】 Objective:(1)To investigate the polymorphism of MICA gene in Han nationality population of Hunan province and Hubei province.(2) To investigate the associations of polymorphism of MICA gene with Recurrent Spontaneous Abortion (RSA) and infertility, and to explore further the possible molecular mechanisms on the basis of results of associations of MICA gene.Methods:(1)Samples of 162 random individuals in Hunan province and 193 random ones in Hubei province with Han nationality were genotyped by PCR-SSP to analyze the frequencies of MICA exon 2-4 alleles, which were cmoparied with the frequencies of MICA alleles of other nationalities.(2) MICA exon 2-4 alleles were genotyped in 56 RSA patients and 78 healthy controls through PCR-SSP method, and the association was analyzed at the same time.(3)MICA exon 2-4 alleles were genotyped in 214 infertile women recruited through PCR-SSP method. Anti-Chlamydia trachomatis IgG antibodies were determined by ELISA method. The associations between prevalence of Anti-Chlamydia trachomatis IgG antibodies,tubal pathology and MICA allele polymorphism were further analyzed. (4) The genotypes of HeLa cell and U373 cells were determined by sequencing method. MICA mRNA, total MICA protein and cell surface MICA protein of HeLa and U373 cells were analyzed at different time points (0h、12h、24h、36h) after Chlamydia trachomatis infection through quantity realt-ime PCR, Western blotting and flow cytometry, respectively. Cytocity of NK cell to HeLa and U373 cells was measured at different time points (0h、12h、24h、36h) after Chlamydia trachomatis infection through in vitro cytocity method.Results:(1)11 alleles were found in Han nationality population of Hunan province, among which MICA*00801/02 (40.4%) was the dominant allele. The others were MICA*00201/020 (20.1%)and MICA*010(17.3%),etc.The rarest alleles were MICA*019 (0.3%)and MICA*031 (0.3%).In Han nationality population of Hubei province,13 alleles were determined, among which MICA*00801/02 was the dominant MICA allele, accounted for 33.7%.The others were MICA*010 (18.4%), MICA*00201/020(13.9%), MICA*01201/02(9.6%), respectively. The rarest alleles were MICA*005 (0.5%)and MICA*027 (0.5%).MICA allele frequencies were significantly different between Hunan and Hubei Han nationality population and ones from Korean, Thais,Caucasian and African American populations (P<0.05).(2) 8 MICA alleles were detected in RSA group.MICA*00801/02 is the dominant MICA allele, and the rarest allele is MICA*018.9 MICA alleles were found in normal control group.MICA*00801/02 is also the dominant MICA allele, and the rarest allele is MICA*005 and MICA*01201/02.Allele frequencies of MICA exon 2-4 in RSA patients was not significantly different from ones in control group (P>0.05).(3) Women with tubal infertility more often had antibodies to Chlamydia trachomatis(66.7% versus 39.1%;OR 3.12,95% CI 1.68-5.78,P=0.004) than infertile women without tubal pathology. High level of anti-Chlamydia trachomatis IgG antibodies increased the risk of severe tubal pathology (OR 6.88,95% CI 3.34-14.20, P=0.001).The prevalence of MICA alleles were assessed and compared between infertile women with and without anti-Chlamydia trachomatis IgG antibodies.There were 9 MICA alleles found in two groups.Among them, The frequency of the MICA*008 allele was significantly higher in infertile patients without anti-Chlamydia trachomatis IgG antibodies than ones with anti-Chlamydia trachomatis IgG antibodies (38.1% versus 22.3%,P=0.0004,Pc=0.00367,OR=2.14,95%CI:1.4-3.28).The comparison of MICA alleles was also made between infertile women with or without tubal pathology.The allele distribution in infertile women with tubal pathology was similar to the distribution in women without tubal pathology. No statistical significant differences were found in the MICA allele frequencies for tubal pathology (P>0.05).Though a significant association was clearly observed between high levels of antibodies against anti-Chlamydia trachomatis antibodies and tubal pathology, no significant differences were found in the MICA allele frequencies between anti-Chlamydia trachomatis IgG antibody positive infertile women with or without tubal pathology (P>0.05).(4) HeLa cells were MICA*008 homozygote, and U373 cells MICA*001 homozygote. After Chlamydia trachomatis infection, the changes of MICA mRNA in HeLa and U373 cells were similar, which was expressed at low level at Oh time point, increased significantly highly at 12h time point, and slowly decreased during the period from 24h to 36h. After Chlamydia trachomatis infection, total MICA protein of HeLa cells never obviously decreased during the whole infection period. However, in Chlamydia trachomatis infected U373 cells, total MICA protein ascended only a little during the period from Oh to 12h time point, then began to decrease from 24h to 36h time point. After infection, MICA molecules on the HeLa cell membrane surface never obviously decreased. MICA molecules on the U373 cell membrane surface were changed unobviously during the period from Oh to 24h, but they were obviously down-regulated during 24h to 36h time points.Cytocity of NK cell to HeLa cells infected by Chlamydia trachomatis was improved gradually during the whole infection period. But this is not true in infected U373 cells.At 12h time point after infection, the cytocity of NK cell to U373 cell increased a little, then began to step down from 24h to 36h time point. Conclusions:(1)MICA exon 2-4 allele frequencies were significantly different between Han nationality population of Hunan and Hubei provinces and ones from other races.MICA*00801/02 was the dominant allele in Han nationalty population in both Hunan and Hubei province.(2) The polymorphism of MICA exon 2～4 was not associated with RSA. (3)Anti-Chlamydia trachomatis IgG antibodies were associated with tubal pathology of infertile women. (4) MICA*008 allele was significantly reverse associated with the anti-Chlamydia trachomatis IgG antibodies in infertile women.(5)No statistical significant differences were found between polymorphism of MICA allele frequencies,tubal pathology and tubal pathology induced by Chlamydia trachomatis infection.(6) The effects of Chlamydia trachomatis infection on MICA mRNA of HeLa and U373 cells were similar, while its effects on total MICA proteins and membrane MICA proteins of two cells different. (7) The discrepancy of cytocity of NK cell to Chlamydia trachomatis infected HeLa and U373 cells was found.更多还原