中国北方人群系统性红斑狼疮患者KIR基因多态性的研究

目的:检测和分析系统性红斑狼疮患者的杀伤细胞免疫球蛋白样受体(KIR)基因的多态性, 探讨系统性红斑狼疮(SLE)从基因到临床表达的发病机制.方法:应用PCR-SSP技术检测北方62例确诊SLE患者和61例同一区域同一民族正常对照者的KIR基因位点多态性.结果:SLE 患者KIR基因表型分布较常见的为KIR3DP1、 2DL1、 2DP1、 3DL1、 2DL3及1D, 其次为2DS4、 2DL5、 3DS1、 2DS2、 2DS5和2DL2, 较低者为2DS1、 2DS3及3DP1V, SLE病例组KIR3DS1, 2DL2、 2DL5和2DL3基因型频率比对照组显著降低(P<0.01).结论:中国北方人群的SLE的发生可能与多个KIR基因等位基因有相关性.     

Real-time PCR分型法检测苏南地区汉族人群KIR基因多态性

目的利用SYBR Green I Real-time PCR分型方法检测杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-likereceptor,KIR)基因,探讨苏南地区汉族人群KIR基因的分布特点。方法应用SYBR Green I Real-time PCR法对191名苏南地区汉族非亲缘健康人群进行KIR基因分型。结果 SYBR Green I Real-time PCR法有效地进行了KIR基因分型。已知的16种KIR基因在苏南地区汉族人群均被检出。框架基因2DL4、3DL2、3DL3和假基因3DP1存在于所有受检个体中。最常见的非框架基因为2DL1、2DL3、3DL1、2DS4以及假基因2DP1。共检出33种KIR基因型,最常见的为AA1(39.27%),其次为BX2、BX4和BX8。发现仅在新加坡华人报道的罕见基因型BX331和BX337,及仅在墨西哥人群罕见的基因型BX427。结论苏南汉族人群中检测出已知的16种KIR基因,共发现33种基因型,最常见的为AA1,并见到3个罕见基因型BX331、BX337和BX427。     

桥本甲状腺炎患者杀伤细胞免疫球蛋白样受体基因多态性分析

目的 探讨杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-like receptor, KIR)基因多态性与桥本甲状腺炎(HT)的关联性.方法 选择100例散发HT患者,260例无血缘关系的正常人作为对照,提取全血基因组DNA,采用序列特异性引物聚合酶链反应(PCR-SSP)的方法,对KIR2DL1～5、KIR3DL1～3、KIR2DS1～5、KIR3DS1及KIR2DP1共15个KIR基因进行检测,其中除KIR2DS5基因外,每个基因均采用2对不同的特异性引物.结果 HT病例组中KIR2DL5基因的基因频率较对照组显著降低(0.200 vs 0.312,RR=0.64,P<0.01).结论 KIR2DL5基因频率的降低可能与HT的发病相关.     

中国人群HLA-Cw、KIR2D受体基因多态性分析

目的 分析中国南、北方两个汉族人群、一个蒙古族人群的人类白细胞抗原-Cw(human leucocyte antigen-Cw,HLA-Cw)遗传多态性;进一步分析南、北方汉族人群杀伤细胞免疫球蛋白样受体2D(killer immunloglobulin-like receptor 2D,KIR2D)基因的遗传多态性、与HLA-Cw的组合特点.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)检测湖南地区112名汉族人群、内蒙古地区98名汉族人群、内蒙古地区83名蒙古族人群HLA-Cw基因、第80位密码子(Lys80、Ash80)多态性;检测两个汉族人群KIR2DL 1/2/3、KIR2DS 1/2基因分布.结果 (1)湖南地区汉族人群与内蒙古地区汉族、蒙占族人群在HLA-Cw等位基因、第80位密码子的频率差异均有非常显著的统计学意义(P＜0.001),而内蒙古地区汉族、蒙古族人群之间上述频率差异无统计学意义(P＞0.05).(2)南、北方两个汉族人群间,5个KIR2D基因频率、各基因型频率差异无统计学意义(P＞0.05).(3)两个汉族人群均以Asn80/Asn80,2DL1+/2DL2-/2DL3+/2DS1-/2DS2-组合模式最为常见(45/112、29/98);其次为Asn80/Asn80,2DL1+/2DL2-/2DL3+/2DS1+/2DS2-(18/112,16/98)和Asn80/Lys80,2DL1+/2DL2-/2DL3+/2DS1-/2DS2-(11/112,17/98).Lys80/Lys80,2DL1+/2DL2-/2DL3+/2DS1-/2DS2-组合模式的频率差异有统计学意义(1/112,8/98;Fisher's P=0.0134),其余11种组合模式在两个人群间的频率差异均无统计学意义(P＞0.05).结论 提供了中国南方湖南地区、北方内蒙古地区正常汉族人群的HLA-Cw、5个KIR2D受体基因多态性数据、蒙古族人群HLA-Cw DNA分型数据;提示我国南、北方汉族人群在HLA-Cw第80位密码子、KIR2D受体基因的组合层面上可能存在着以抑制性信号通路为优势的共同特点.     

尿毒症患者NK1细胞表面受体KIR基因的表达

目的 调查人类自然杀伤(NK)细胞表面免疫球蛋白样受体KIR在尿毒症患者的表达.方法 采用PCR-SSP完成55例尿毒症患者KIR基因分析.结果 尿毒症患者表达16个KIR基因,2DL4、3DL2、3DL3、3DPI基因频率1.00,2DL1、2DL3、3DL1、2DS4基因频率0.73～0.87,2DL2、2DS1、2DS2、2DS3、2DS5基因频率0.11～025,2DL5、3DS1、2DP1基因频率0.30～0.41.对尿毒症患者与部分亚洲地区人群的分布频率比较分析中发现,KIR-2DL4、3DL1、3DL2,3DL3、2DS4(GF 0.73～1.00)和KIR-2DL2、2DL5、2DS1、2DS3、2DS5、3DS1(GF0.11～0.34)与韩国人、日本人、南亚人之间相差＜0.20;KIR-2DL1、2DL3、2DS2与韩国人、日本人、南亚人群之间相差≥0.20,2DL1低于日本人,2DS2低于南亚人,2DL3高于南亚人.结论 55例尿毒症患者普遍带有结构基因2DL4、3DL2、3DL3、3DP1,其次为2DL1、2DL3、3DL1、2DS4,较罕见为2DL2、2DS1、2DS2、2DS3和2DS5;显示尿毒症患者中14个KIR功能基因频率与亚洲其他人群KIR基因频率接近,但是KIR基因多态性与上述不同种族之间存在分布差异.     

四川凉山地区彝族家系KIR单体型分析

目的了解四川凉山地区彝族家系KIR单体型的基因组成情况。方法采用多重PCR-SSP的方法对89个四川凉山地区彝族家系共364人的16个KIR基因位点进行基因分型;根据KIR基因型结合家系分析推导KIR单体型。对部分样本中某些不能确定拷贝数的KIR基因进行荧光定量PCR分析,再结合家系分析得到其KIR单体型。结果 89个四川凉山地区彝族家系中发现22种KIR基因型,21种单体型,其中3种A单体型,18种B单体型;并发现了2种较为独特的KIR单体型。结论四川彝族家系有其独特的KIR单体型种类,其基因组成具有以下规律:2DL1、2DP1总是和3DP1同时出现;有2DS1时则2DS4缺失,反之亦然;2DS2总是和2DL2同时出现。     

浙江汉族人群KIR2DL3基因遗传多态性

目的探讨浙江汉族人群KIR2DL3基因的多态性分布情况。方法采用PCR测序分型方法进行KIR2DL3等位基因分型。首先合成引物特异性扩增KIR2DL3基因的两个片段,然后对KIR2DL3基因第4、5、7、8和9外显子进行测序分析,根据多态性位点格局判断样本的KIR2DL3等位基因情况。结果样本中共检测到两种等位基因,其中KIR2DL3*001等位基因占77%。结论PCR测序分型方法可有效检测KIR2DL3等位基因。     

吉林省汉族154例正常人HLA-Cw及KIR基因型频率的分析

目的:了解中国人HLA-Cw与KIR的识别方式,为今后研究其在疾病中的作用提供理论基础.方法:对154例吉林汉族无偿献血者,以PCR-SSP技术进行KIR和HLA-Cw基因分型.根据HLA-Cw与KIR识别方式,分析个体HLA-Cw与相应激活性或抑制性KIR受体的识别情况.结果:吉林汉族人群中,HLA-C2Lys80+ 2DL1的组合频率最高为27.27％,其次分别HLA-C1Asn80+2DL2/2DL3为68.83％,2DS2+HLA-C1Asn80为9.74％,2DS1+HLA-C2Lys80为9.74％,分别有72.08％和21.43％盼观察对象表达KIR2DL1、KIR2DS1而无相应HLA-Cw表达;21.43％的个体表达KIR2DS1而不表达HLA-Cwlys-KIR2DL1配对.2.60％的个体表达KIR2DS2而不表达HLA-Cwasn-KIR2DL2/L3配对.结论:在吉林汉族人群中,抑制性HLA-Cw-KIR受配体对表达高于活化性受配体对,约20％的个体单独表达激活性HLA-Cw-KIR受配体对.     

KIR在NK-92MI及K562细胞中的基因型及表达谱分析

目的:分析杀伤细胞免疫球蛋白样受体(KIR)在NK-92MI及K562细胞中的基因型及表达谱, 探索KIR受体的表达调控规律.方法:采用PCR、 RT-PCR及分子克隆测序技术, 检测K562及NK-92MI细胞中KIR2DL1、 2DL2、 2DL3、 2DL4、 2DL5、 2DS1、 2DS2、 2DS3、 2DS4、 2DS5、 3DL1、 3DL2、 3DL3、 3DS1、 2DP1、 3DP1的基因型, 及KIR2DL1、 KIR2DL2、 KIR2DL3、 KIR3DL1、 KIR2DS1、 KIR2DS3及KIR2DS2/4受体mRNA的表达.结果:K562细胞携带KIR2DL1、 2DL2、 2DL3、 2DL4、 2DL5A、 2DS2、 2DS4*003-006、 2DS5、 3DL1、 3DL2、 3DL3基因, 但不表达KIR.NK-92MI细胞携带KIR2DL1、 2DL2、 2DL3、 2DL4、 2DS2、 2DS4*001/002、 2DS4*003-006、 3DL1、 3DL2、 3DL3, 但只表达KIR2DL1、 KIR2DL3及KIR2DS2/4.结论:K562细胞及NK-92MI细胞均携带KIR2DL1、 2DL2、 2DL3、 2DL4、 2DS2、 2DS4*003-006、 3DL1、 3DL2、 3DL3.NK-92MI细胞表达KIR受体, K562细胞不表达上述KIR, KIR呈细胞特异性表达.     

慢性粒细胞白血病患者杀伤细胞免疫球蛋白样受体(KIR)基因研究

目的探索CML患者的KIR（killer cell immunoglobulin-like receptors）基因的多态性与正常人群是否存在差异。方法应用PCR-SSP技术进行KIR基因的检测。结果共检出目前已知的18个KIR基因,在检测的全部个体中,3DL3,3DL2及2DL4出现频率均为100%。在CML的研究中发现KIR2DL1基因频率（0.509）比正常对照组（1.000）显著降低,KIR2DL1与CML呈现强负相关P〈0.001,与CML负相关的KIR基因尚有3DS1,2DL5和3DL1,P值分别等于0.021,0.029,0.031。其他KIR基因位点频率未发现统计学差异。结论CML患者具有其独特KIR基因位点频率的特点。     

Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments

Detection of killer-cell immunoglobulin-like receptors (KIR) genes by polymerase chain reaction with sequence-specific primers (PCR-SSP) led in 1997 to the discovery that human genomes diverge largely in the KIR they encode. While only a few KIR genes are conserved in all humans, most individuals lack several those genes, which tend to associate in diverse haplotypic combinations. The PCR-SSP technique, updated to detect the more recently identified KIR genes and alleles, is still used widely to analyze the diversity of human populations, and to study the influence of KIR-gene variability on human health. Several published PCR-SSP methods for KIR genotyping, although simple and robust, have the drawback of relying on the amplification of DNA fragments spanning 0.5-2.0 kbp, which tends to fail in low-quality DNAs. Valuable collections of DNAs often include such poor quality samples, which lead to loss of data and resources. Even worse, undetected falsely negative or positive reactions may result in erroneous gene frequencies and in odd gene combinations. To address those problems, we have redesigned our previously published KIR genotyping method so that it produces short amplicons (less than 200 bp for most genes). This modification minimizes amplification failures, thus conferring greater consistency and reliability to KIR genotyping. In addition, the new PCR-SSP method detects recently described alleles of several KIR genes, and allows for discrimination between the major structural variants of KIR2DS4 and KIR3DP1 without increasing the number of reactions.     

Killer cell immunoglobulin-like receptor (KIR) genes in 4 distinct populations and 51 families in mainland China

In this study, we investigated the killer cell immunoglobulin-like receptor (KIR) genes and HLA-C1/C2 dimorphism in 819 healthy, unrelated individuals composed of two southern Chinese Han populations (Hunan Han and Guangdong Han) and two northern Chinese populations (Inner Mongolia Han and Inner Mongolia Mongol), using polymerase chain reaction-sequence-specific priming (PCR-SSP) method. Fifty-one Chinese families were used to determine KIR haplotypic configuration. Our data showed that KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP1 genes were present in all of the 819 individuals. However, KIR2DL4 and KIR3DP1 genes were not detected in two members of a northern Chinese family. None of the KIR genes showed significant difference between the four populations. Thirty-five different KIR gene profiles were identified, one of which has not been previously reported in the Allele Frequencies KIR database. Eleven distinct KIR haplotypic configurations were determined through family analysis. Individuals with KIR2DLl and KIR2DL3 genes but lacking KIR2DSl and KIR2DS2 genes, coupled with HLA-C1 (Asn(80)) homozygosity, predominated in each population. At least one known inhibitory KIR-HLA pair was detected in each individual. The findings shown here are valuable for future studies of the potential role of KIR genes as well as KIR-HLA interaction in disease susceptibility in related ethnic groups.     

Diversity distributions of killer cell immunoglobulin-like receptor genes and their ligands in the Chinese Shaanxi Han population

In the present study, 17 killer cell immunoglobulin-like receptors (KIR) genes and KIR ligands (human leukocyte antigen [HLA] -A and -B) were detected by using a polymerase chain reaction-sequence-specific primer (PCR-SSP) method in 104 unrelated healthy Han individuals living in Shaanxi province, China. The observed carrier frequencies of the 12 KIR genes ranged from 0.14 to 0.96. KIR2DL4, 3DL2, 3DL3, 2DP1 and 3DP1 were found to be present in every individual. A total of 51 different KIR gene profiles were identified, in which 11 gene profiles exclusively belonged to the study population. Neighbor-joining phylogenetic tree between the studing population and its neighboring ethnic groups was constructed using the observed carrier frequencies of 13 KIR loci. The phylogenetic tree shows that the Shaanxi Han population, Han populations in different regions, Yi, Japanese, and Koreans were in the same cluster. KIR/HLA relationships show that KIR3DS1(-)/3DL1(+)/Bw4(+) was the most common association in the population. In conclusion, the present study findings reveal the high polymorphism of KIRs in the Shaanxi Han population, demonstrate the KIR/HLA association in the study population, and enrich the KIR and HLA gene resources. The obtained KIR data will further the understanding of genetic relationships among populations in different geographic areas, and assist in answering questions regarding KIR/HLA relationships.     

人类杀伤细胞免疫球蛋白样受体基因PCR-SSP分型方法的建立及其应用

目的建立PCR-SSP方法检测人类杀伤细胞免疫球蛋白样受体（KIR）基因。方法参照K／R基因数据库，采用计算机软件设计一系列特异性引物建立PCR-SSP方法，以人类生长激素基因特异性引物作为内对照，以Dynal公司的KIR PCR-SSP分型试剂盒作为验证。结果本实验所设计的KIR特异性引物可检测出所有K／R基因，扩增产物条带清晰，特异性强。结论本实验建立的KIR PCR-SSP方法结果准确，具有可行性。     

上海地区汉族人群KIR2DL5基因多态性及单元型分析

KIR2DL5基因是杀伤细胞免疫球蛋白样受体 (KIR )单元型B的特征性标志 ,至少有 4个变异体 ,其中 2DL5A 0 0 1和2DL5B 0 0 3是表达型 ,统称为KIR2DL5 1;2DL5B 0 0 2和 2DL5B 0 0 4是不表达型 ,统称为KIR2DL5 2。本文用序列特异性引物PCR法 (PCR SSP )分析了KIR2DL5表达型KIR2DL5 1和不表达型KIR2DL5 2在上海地区正常汉族人群的分布及其在KIR单元型的组成。结果发现 (1)在 87名健康汉族人和 14个家族中表达型KIR2DL5 1和不表达型KIR2DL5 2基因频率分别为0 1977和 0 0 4 11;(2 )KIR2DL5基因变异体在不同单元型中分布不同 ,有 5种不同组合 ,其中单元型 1、 2、 3、 4、 11、 12、15和 16不含KIR2DL5及其变异体 ;单元型 5和 14只含有KIR2DL5 1;单元型 13和 17只含有KIR2DL5 2 ;单元型 6含有单一KIR2DL5 1或二者兼有 ;单元型 8和 9同时含有KIR2DL5 1和KIR2DL5 2。     

A novel real-time PCR method for KIR genotyping

Genes encoding killer cell immunoglobulin-like receptors (KIRs) are variable among individuals. Sequence-specific primer-directed polymerase chain reaction (PCR) amplification (PCR-SSP) and sequence-specific oligonucleotide hybridization of the PCR-amplified products (PCR-SSO) are the methods currently used to characterize the diversity of KIR gene content. Both these methods include time-consuming post-PCR analyses. Here, we developed a real-time PCR method that identifies the presence or absence of 16 KIR genes during PCR and avoids post-PCR analyses. This method is specific, sensitive, shortens the turnaround time compared with the conventional PCR-SSP and PCR-SSO methods, and it can be easily adapted for automation.     

上海地区汉族人群杀伤细胞免疫球蛋白样受体基因多样性及单倍型组合研究

目的 分析上海地区汉族人群杀伤细胞免疫球蛋白样受体(killer Ig-like receptors，KIR)基因多态性及单倍型，为进一步研究不同人群KIRs与某些疾病的相关性提供线索。方法 采用序列特异性引物PCR法对87名无关汉族健康志愿者进行KIR基因低分辨率检测和单倍型分析。结果 (1)共检出Xv、KIRlD在内的18个KIRs基因。3DL3、2DL4、3DL2和3DLl存在于所有个体；较常见为2DL3、Z、2DLl、X；其次为2DS4、1D、2DL5、2DSl、3DSl、2DS5；2DS2、2DL2、2DS3、Xv频率较低。(2)共检出13种单倍型，最常见的是单倍型2。(3)共检出18种基因型．以AJ(2．2)、AF(1．2)型最常见，其次为AH(5，2)、AI(1，5)和AG(1，1)。另外有5种基因型FZl(2，9或6，16)、FZ2(1，16或2，15)、FZ3(6，17)、FZ4(4，13)和FZ5(2，6)，目前在白人中尚未发现。结论 上海地区汉族人群有其独特的KIRs基因频率、单倍型频率和基因型频率分布。