【作者】 谭愈昱；

【导师】 周智广；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的：探讨湖南汉族人群杀伤细胞免疫球蛋白样受体(Killer cell immunoglobin-like receptor, KIR)基因的多态性、单倍型和基因型及其配体HLA-C基因多态性、KIR/HLA-C基因组合的特点,为进一步研究KIR基因在疾病和移植免疫中的作用提供基础数据。设计：横断面研究。方法：采用聚合酶链反应-序列特异性引物技术(polymerase chain reaction-sequence specific primer, PCR-SSP),检测429例湖南汉族健康志愿者KIR基因和HLA-C基因的分布频率,并分析KIR基因的单倍型、基因型和KIR/HLA-C基因组合特点。结果：(1)在湖南汉族人群中共检出16种KIR基因,其中框架基因KIR2DL4、3DL2、3DL3和3DP1频率为100%;KIR 2DL1、2DL3、3DL1、2DS4、2DP1基因较为常见,频率均>90%,KIR2DS1、2DL5、3DS1、2DL2、2DS2、2DS3、2DS5频率均<40%；(2)湖南汉族人群KIR基因频率分布与日本人相似,而KIR2DL2、2DS2、2DS3和2DL5频率低于高加索人群,KIR2DL3频率高于高加索人群；(3)湖南汉族人群KIR基因以A单倍型为主,A、B单倍型频率分别为69.6%、30.4%,比例约为2：1；(4)在湖南汉族人群中共发现49种KIR基因型,其中4种未见报道；KIR基因型以AA型最为常见(44.5%)；(5)湖南汉族人群HLA-C基因以HLA-C1为主,频率高达98.1%；(6)湖南汉族人群KIR2D／HLA-C基因组合以HLA-C1(+)／HLA-C2(-),KIR2DL1(+)／2DL2(-)／2DL3(+)／2DS 1(-)／2DS2(-)最为常见(23.9%)。结论：湖南汉族人群具有独特的KIR基因型,丰富的KIR、HLA-C基因多态性和KIR／HLA-C基因组合方式。目的：探讨KIR和HLA-C基因多态性与自身免疫性糖尿病的关系。设计：横断面、病例对照研究。方法：采用聚合酶链反应-序列特异性引物技术(polymerase chain reaction-sequence specific primer, PCR-SSP),检测387例湖南汉族自身免疫性糖尿病患者[180例急性起病自身免疫性糖尿病(T1ADM)患者和207例成人隐匿性自身免疫性糖尿病(LADA)患者]和性别、年龄、区域匹配的429例正常对照者(199例对照A组和230例对照组B)KIR基因和HLA-C基因的多态性,分析其与自身免疫性糖尿病遗传易感的关系。结果：(1)与相应的正常对照组比较,T1ADM组KIR2DL1 (98.9% vs.92.0%, OR=7.781,P<0.01)、3DL1(94.5% vs.86.4%, OR=2.669, P<0.01)和2DS4 (83.9% vs.70.9%,OR=2.142,P<0.01)频率增高；LADA组KIR2DL3 (99.5% vs.96.1%, OR=8.389, P<0.05)频率增高；KIR基因在湖南汉族自身免疫性糖尿病患者中的遗传易感性与其他种族的不同；(2)激活性KIR基因的总个数在T1ADM组高于其正常对照A组(2.02 vs.1.78,P＜0.05)；(3)KIR基因A、B单倍型在T1ADM组分别为69.7%、30.3%,在LADA组分别为75.4%、24.6%；A、B单倍型比例分别约为2:1,3:1;(4)T1ADM组、LADA组与相应的正常对照组比较,HLA-C(HLA-C1、HLA-C2)基因频率无差异；(5)与相应的正常对照组比较KIR/HLA-C基因组合,T1ADM组KIR2DL 1(-)／HLA-C2(-)(0.6％vs.6.0％,OR=0.087,P< 0.01).LADA组KIR2DL3(-)／HLA-C1(+)(0.5％vs.3.9％,OR=0.119,P <0.05)频率降低；(6)与正常对照A组比较KIR2D／HLA-C基因组合方式,T1ADM组HLA-C 1(+)／HLA-C2(-),KIR2DL 1(+)／2DL2(-)／2DL3 (+)／2DS 1(+)／2DS2(-)频率升高(24.4%vs.15.6%,OR=1.753,P<0.05)。结论：KIR基因多态性和KIR／HLA-C基因组合与自身免疫性糖尿病发病相关。目的：分析急性起病自身免疫性糖尿病(T1ADM)患者KIR基因与HLA-DQ或MICA基因的相互作用。设计：横断面、病例对照研究。方法：采用PCR-直接测序法(PCR-Sequencing Based Typing, PCR-SBT)对湖南汉族180例T1ADM患者和199例正常对照者的DNA样本进行HLA-DQ和MICA基因多态性检测,并分析T1ADM的HLA-DQ和MICA易感等位基因、单倍型或基因型。采用Svejgaard &Ryder检验分析KIR易感基因(KIR2DL1、3DL1或2DS4)与HLA-DQ易感单倍型或MICA易感等位基因在T1ADM中的相互作用。结果：(1)湖南汉族T1ADM患者HLA-DQ易感等位基因为DQA1*03、DQA1*0501/0503、DQB1* 0201、DQB1*0303和DQB1 *0401;保护性等位基因为DQA1*0101／0104、DQA1*0102、DQA1* 0103.DQ A1*0601、DQB1* 0301、DQB1*0502、DQB1*0601和DQB1*0602；易感单倍型为DQA1*03-DQB1*0303、DQA1*03-DQB1*0401、DQA1*05-DQB1*0201；保护性单倍型为DQA1*0601-DQB1*0301、DQA1*0102-DQB1*0601、DQA1*0102-DQB1*0602；(2)湖南汉族T1ADM患者MICA易感等位基因为MICA9,保护性等位基因为MICA5.1; MICA易感基因型为MICA4/9和MICA9/9;(3)同时携带KIR易感基因(KIR2DL1、3DL1或2DS4)和HLA-DQ易感单倍型或MICA9等位基因者T1ADM发病风险升高(OR=1.077-21.111); (4) HLA-DQ易感单倍型或MICA9等位基因在携带KIR2DL1或KIR3DL1基因者中增加T1ADM发病风险(OR=2.929-7.222); (5) HLA-DQ易感性单倍型对T1ADM的易感性强于KIR2DS4 (OR=3.750); (6) KIR3DL1或KIR2DS4基因在不携带MICA9等位基因者中可增加T1ADM发病风险(OR=3.792或2.424)。结论：KIR基因可协同增强HLA-DQ和MICA基因对T1ADM遗传易感性。目的：观察自身免疫性糖尿病患者NK细胞及其表面受体的变化,探讨NK细胞在自身免疫性糖尿病发病机制中的作用,为自身免疫性糖尿病的预测、病情监测和治疗提供思路和手段。设计：横断面、病例对照研究。方法：选择我院内分泌科收治的53例自身免疫性糖尿病患者(35例T1ADM患者和18例LADA患者),健康体查筛选的28例正常对照者,采用流式细胞仪技术检测T细胞亚群、NK细胞的比例和NK细胞表面受体(KIR和NKG2D)的表达,采用MTT方法检测IL-2孵育前后NK细胞的活性,比较、分析自身免疫性糖尿病患者NK细胞数目和活性及其表面受体表达的变化。结果：(1)与相应的正常对照组比较,T1ADM组CD3+T细胞比例升高(70.5%vs.64.6%,P<0.05),NK细胞比例下降(13.8%vs.20.1%,P＜0.01);(2)T1ADM组和LADA组NK细胞活性低于相应的正常对照组(30.9%vs.47.1%,P<0.01；39.8%vs.49.2%，P<0.05),与相应的LADA组及其亚组比较,T1ADM组(30.9%vs.39.8%,P<0.01)、新发(33.2%vs.40.9%,P<0.05)和长病程T1ADM组(28.1%vs.38.6%,P<0.05)NK细胞活性降低；(3)IL-2孵育后T1ADM组和LADA组NK细胞活性均增强(43.2%vs.30.9%,47.4%vs.39.8%,P<0.05); T1ADM组和其新发组NK活性的增长差值分别大于LADA组和LADA新发组(9.9%vs.7.4%,P<0.05；12.9%vs.8.0%,P<0.05)；(4)与正常对照A组比较,T1ADM组KIR2DL1(10.2%vs.6.2%,P＜0.05)和NKG2D (96.9% vs.95.2%, P<0.05)表达升高。结论：(1)自身免疫性糖尿病患者外周血中CD3+T细胞比例升高,NK细胞的比例和活性下降,其中T1ADM患者较LADA患者存在更严重的NK细胞免疫异常；(2)IL-2可改善自身免疫性糖尿病患者NK细胞的活性,且其对早期T1ADM患者的改善能力高于LADA患者；(3)T1ADM患者NK细胞表面受体KIR2DL1和NKG2D表达升高,可能与T1ADM的发病机制相关。更多还原

【Abstract】 Objective:To characterize the gene polymorphism, haplotypes, and genotypes of killer cell immunoglobin-like receptor (KIR), the gene polymorphism of HLA-C and the KIR/HLA-C gene compatibility in Hunan Han population. The obtained results will be used as basic data for further study in its linkage with disease and transplantation immunity.Design:Cross-sectional study.Methods:The method of polymerase chain reaction-sequence specific primer (PCR-SSP) was used to detect KIR and HLA-C genotypes in 429 healthy individuals of Hunan Han population. Then the distribution and characteristics of the haplotypes, genotypes of KIR genes and the KIR/HLA-C gene compatibility were analyzed.Results:(1)16 KIR genes were detected in Hunan Han population, of which the frequencies of framework genes KIR2DL4,3DL2,3DL3, and 3DP1 were 100%. KIR2DL1,2DL3,3DL1,2DS4 and 2DP1 genes were common with frequencies more than 90%, while the frequencies of KIR2DS1,2DL5,3DS1,2DL2,2DS2,2DS3,2DS5 were lower than 40%. (2) In Hunan Han population, the distribution of KIR genes frequencies were similar to Japanese, but the frequencies of KIR2DL2, 2DS2,2DS3 and 2DL5 were lower than Caucasian, the frequencies of KIR2DL3 was higher than Caucasian. (3) The KIR genes gave priority to A haplotype in Hunan Han population, the frequencies of A and B haplotype were 69.6% and 30.4% with a ratio of 2:1. (4) 49 KIR genotypes were found in the Hunan Han population,4 of which had not been reported. AA was the main genotype of KIR gene (44.8%) (5) HLA-C1 was dominant in Hunan Han population, with a frequency of 98.1%. (6)HLA-C1(+)/HLA-C2(-), KIR2DL1(+)/2DL2(-)/2DL3(+)/2DS1 (-)/2DS2(-) was the most common KIR2D/HLA-C gene compatibility in Hunan Han population (23.9%).Conclusion:Distrubution of KIR genes, HLA-C genes and KIR/ HLA-C gene compatibility show rich polymorphism, with distinctive KIR genotypes in Hunan Han population. Objective:To investigate the gene polymorphisms of Killer cell immunoglobin-like receptor (KIR) and HLA-C in autoimmune diabetes.Design:Cross-sectional and case control study.Methods:The method of polymerase chain reaction-sequence specific primer (PCR-SSP) was used to detect the gene polymorphisms of KIR and HLA-C in 387 T1ADM patients [180 acute onset autoimmune diabetes(T1ADM) patients and 207 latent autoimmune diabetes in adults (LADA)patients] and 429 healthy controls (199 control A group and 230 control B group) who matched for sex, age in Hunan Han population.Results:(1)Compared with the corresponding controls, the frequencies of KIR2DL1 (98.9% vs.92.0%,OR=7.781, P<0.01), 3DL1(94.5% vs.86.4%, OR=2.669, P<0.01) and 2DS4 (83.9% vs. 70.9%, OR=2.142, P<0.01) increased in T1ADM patients; the frequency of KIR2DL3 (99.5% vs.96.1%, OR= 8.389, P<0.05) increased in LADA patients; The genetic susceptibility of KIR genes in Hunan Han patients with autoimmune diabetes was different from other races. (2) The total number of activating KIR genes in T1ADM patients was higher than their controls (2.02 vs.1.78, P<0.05). (3) The frequencies of A and B haplotype of KIR gene were 69.7% and 30.3% in T1ADM patients, 75.4% and 24.6% in LADA patients. The ratio between A and B haplotype were about 2:1 and 3:1 respectively. (4)There are no differences in frequencies of HLA-C genes (HLA-C1, HLA-C2) between T1ADM or LADA patients and their corresponding controls. (5) The frequencies of KIR2DL1(-)/HLA-C2(-) (0.6% vs.6.0%, OR= 0.087, P< 0.01) in T1ADM patients and KIR2DL3(-)/HLA-C1(+) (0.5% vs.3.9%, OR=0.119,P<0.05) in LADA patients were lower than their corres-ponding controls. (6) Compared with the controls, the frequency of HLA-C1(+)/HLA-C2(-),KIR2DL1(+)/2DL2(-)/2DL3(+)/2DS1(+)/2DS2(-) (24.4% vs.15.6%, OR=1.753, P<0.05) increased in T1ADM patients.Conclusion:The KIR gene polymorphism and KIR/HLA-C gene compatibility are associated with the susceptivity of autoimmune diabetes. Objective:To analyze the interactive effects between KIR gene and HLA-DQ or MICA gene in acute onset autoimmune diabetes (T1ADM).Design:Cross-sectional and case control study.Methods:All DNA samples of 180 T1ADM patients and 199 healthy controls of Hunan Han were typed for the polymorphisms of HLA-DQ and MICA genes with PCR sequencing-base typing (PCR-SBT) method. After analyzed the susceptible alleles, haplotypes or genotypes of HLA-DQ and MICA gene, Svejgaard& Ryder test was performed to investigate the interactive effects between susceptible KIR gene (KIR 2DL1,3DL1 or 2DS4) and susceptible HLA-DQ haplotypes or suscep-tible MICA alleles.Results:(1) The susceptible alleles of HLA-DQ gene in Hunan Han were DQA1*03, DQA1*0501/0503, DQB1* 0201, DQB1*0303and DQB1*0401. The protective alleles were DQA1*0101/0104, DQA1 *0102, DQA1* 0103, DQ A1*0601, DQB1* 0301, DQB1*0502, DQB1 *0601 and DQB1*0602. The susceptible haplotypes were DQA1*03-DQB1*0303, DQA1*03-DQB1*0401 and DQA1*05-DQB1*0201. The protective haplotypes were DQA1*0601-DQB1*0301, DQA1*0102-DQB1*0601 and DQA1*0102-DQB1*0602. (2) The susceptible allele of MICA in Hunan Han was MICA9 and the protective was MICA5.1. The susceptible genotypes were MICA4/9 and MICA9/9. (3) The risk of T1ADM increased in ones who carried susceptible KIR gene (KIR2DL1, 3DL1 or 2DS4) and HLA-DQ haplotypes or MICA9 (OR=1.077-21.111). (4) Susceptible HLA-DQ haplotypes or MICA9 increased the risk of TIADM in ones who carried KIR2DL1 or KIR3DL1 (OR= 2.929-7.222). (5) The susceptible effects of HLA-DQ haplotypes was stronger than that of KIR2DS4 (OR=3.150). (6) KIR3DL1 or KIR2DS4 increased the risk of T1ADM in ones who without MICA9 (OR= 3.792 or 2.424).Conclusion:KIR genes have a synergistic action on the susceptible effects of HLA-DQ or MICA gene in T1ADM. Objective:To observe the variances of NK cells and its surface receptors in autoimmune diabetes patients, and to explore the role of NK cells in the pathogenesis of T1ADM, in order to offer some ideas and means for the forecast, monitor and therapy of autoimmune diabetes.Design:Cross-sectional and case control study.Methods:53 T1ADM patients (35T1ADM patients and 18 LADA patients) from the patients admitted to the department of endocrinology in our hospital,28 healthy controls from health-check screening were included in the study. The percentages of T cell subsets and NK cells and the expression of surface receptors (KIR and NKG2D) of NK cell were detected by flow cytometry. The activities of NK cells before and after incubated with IL-2 were detected by the assay of MTT. We compared and analyzed the variances of NK cells and its surface receptor in autoimmune patients.Results:(1) Compared with the corresponding controls, the propor-tion of CD3+ T cells increased (70.5% vs.64.6%, P<0.05), while the proportion of NK cell decreased (13.8% vs.20.1%, P<0.01) in T1ADM patients.(2)The NK cell activity of T1ADM patients and LADA patients was lower than the corresponding controls (30.9% vs.47.1%, P<0.01; 39.8% vs.49.2%, P<0.05). The NK cell activity of T1ADM patients (30.9% vs.39.8%, P<0.01), whose recent-onset subgroup patients (33.2% vs.40.9%, P<0.05) and long course subgroup patients (28.1% vs. 38.6%, P<0.05) were lower than the corresponding LADA patients; (3) After incubated with IL-2, the NK cell activities of T1ADM patients and LADA patients were enhanced (43.2% vs.30.9%,47.4% vs.39.8%, P< 0.05). The growth of NK cell activity in T1ADM patients and its recent-onset subgroup patients were higher than the corresponding LADA patients(9.9% vs.7.4%, P<0.05,12.9% vs.8.0%, P<0.05). (4) Compared with the control A group, the expression of KIR2DL1 (10.2% vs.6.2%, P <0.05), NKG2D(96.9% vs.95.2%, P<0.05) increased in T1ADM patients.Conclusion:(1) The proportion of CD3+ T cells increase, while the proportion and the activity of NK cells decrease in the peripheral blood of autoimmune diabetes patients. The NK cell-mediated immunity abnormality of T1ADM patients is more severe than LADA patients. (2)IL-2 can improve the NK cell activity of autoimmune diabetes patients, and the effect is more obvious in the early stage of T1ADM than LADA. (3)The expression of the surface receptors of NK cell (KIR2DL1 andNKG2D) increase in T1ADM, which may play a role in the patho-genesis of T1ADM.更多还原