内蒙古蒙古族人群杀伤细胞免疫球蛋白样受体基因簇多态性研究

目的 研究中国蒙古族人群杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-like receptors,KIR)基因的携带频率、KIR基因型及其遗传规律.方法 采集90名内蒙古农牧区蒙古族个体的血样,应用序列特异性引物聚合酶链反应(polymerase chain reaction-sequence specific primer,PCR-SSP)方法分别扩增KIR基因簇16个基因,依据实验结果计算各基因的携带频率并查询样本的基因型,和已报道的24个其他人群的相关数据进行主成分分析、计算Nei氏遗传距离并绘制遗传树.结果 (1)蒙古族人群KIR 2DL2、2DS2携带率高于蒙古利亚人,低于高加索人.(2)蒙古族KIR基因单倍型AA为37.78%,高于高加索人,低于蒙古利亚人.(3)系统进化树显示蒙古利亚人和高加索人分别聚类,蒙古族人群则介于两者之间.结论 蒙古族其成因似与受到高加索人和蒙古利亚人的双重影响有关,表现为介于高加索人和蒙古利亚人之间的中间形式.

Abstract：

Objective To investigate the killer cell immunoglobulin-like receptor (KIR) gene frequencies and genotypes distributions in the Inner Mongolian population. Methods Ninety genomic DNA samples were extracted from blood samples of randomly chosen Mongolian individuals. Gene-specific PCR amplification was used to identify genes present or absent for 16 KIR loci. KIR genotype distributions were obtained and compared to that of 24 populations published in literatures using principal component analysis by SAS8.0 software. Genetic tree was constructed by the calculate Nei's genetic distance. Results (1) The frequency of KIR 2DL2,2DS2 in Mongolian individual is higher than that in north Mongoloid and less than that in Caucasian. (2) Haplotype AA was identified in 37.78% of individuals, which is higher than that in north Mongoloid and lower than that in Caucasian. (3) Mongolian was considered between north Mongoloid and Caucasian by principal component and genetic tree analysis. Conclusion Mongolian might be affected by the north Mongoloid and Caucasian, and showed intermediate between the two populations.     

C型凝集素受体CLEC-2的研究进展

C型凝集素样受体-2(CLEC-2)是一个非经典型的C型凝集素受体,主要表达在血小板、中性粒细胞等细胞表面,在马来西亚蝮蛇蛇毒蛋白rhodocytin和跨膜唾液酸糖蛋白poloplanin等相关配体的刺激下,能通过细胞质尾部的YXXL结构域形成二聚体,从而激活syk依赖的信号传导.最近有很多研究显示:CLEC-2在血小...     

新型胶原样凝集素的研究进展

胶原样凝集素作为脊椎动物C-型凝集素超家族内的成员,以含有胶原样结构域及糖识别结构域为其显著特征.胶原样凝集素除了“经典”成员甘露聚糖结合凝集素(MBL)、表面活性蛋白A(SP-A)和SP-D外,还发现肝胶原样凝集素1(CL-L1)、肾胶原样凝集素1(CL-K1)和胎盘胶原样凝集素1(CL-P1).在机体固有免疫系统中,这些“新”型胶原样凝集素除了同“经典”胶原样凝集素一样发挥着十分重要的作用外,它们还具有其独特的生理功能.本文综述了这些新发现的胶原样凝集素的分子结构、组织分布以及生物学功能等方面的研究进展.     

急性移植物抗宿主病中Ly49A+T淋巴细胞的分布及其调节

目的 :探讨急性GVHD模型中小鼠杀伤性细胞抑制性受体Ly4 9A在造血系统T淋巴细胞亚群的表达及CsA、IL 4、IL 15对Ly4 9A表达的调节 ;方法 :BALB c、C5 7BL 6、CB6F1间脾细胞和骨髓细胞混合移植 ,建立急性GVHD模型 ,利用FCM双色免疫荧光检测 ,动态观察植入后不同时间造血器官T淋巴细胞亚群中Ly4 9A表达情况 ;体外T淋巴细胞培养观察IL 4、IL 15对Ly4 9A表达的调节 ;结果 :①Ly4 9AB6 在B6、F1小鼠的外周血、脾脏和骨髓细胞中均有表达 ,但在胸腺中基本无表达 ;②aGVHD中CD3 Ly4 9A 及CD8 Ly4 9A T细胞在移植早期增加 ,1w后迅速减低 ,至移植后 5w开始升高 ,并可超过移植前 ;CsA明显下调Ly4 9A变化幅度 ;③IL 4、IL 15均有上调脾CD3 Ly4 9A 细胞群的作用 (P =0 0 0 1)。结论 :Ly4 9A T细胞主要出现在外周淋巴器官 ;供者Ly4 9A T细胞在改变的MHC背景环境下 ,趋向于对宿主的免疫耐受 ,抑制性受体表达增加 ;骨髓的造血微环境可影响骨髓Ly4 9A T细胞比例的变化 ;一些细胞因子可以调节抑制性受体表达率     

C型凝集素受体CLRs与固有免疫

固有免疫系统的抗原提呈细胞(APC)主要包括巨噬细胞(MΦ)、树突状细胞(DC)和B细胞［1］.DC是目前发现的功能最强的APC,广泛分布于脑以外的全身各脏器,在体内行使“哨兵”职责,其主要功能是摄取、加工处理并提呈抗原,从而行使有效的宿主防御和免疫记忆功能.     

中国恒河猴DNGR-1分子C型凝集素样结构域在大肠杆菌系统表达

目的获得恒河猴C型凝集素结构域家族9的A成员(Clec9A)基因编码区序列;体外表达CLEC9A的C型凝集素样结构域(CTLD)。方法利用Clec9A基因特异引物,反转录PCR扩增中国恒河猴Clec9A基因编码区,亚克隆至pGEM-T载体,获得pGEM-T-DNGR质粒并进行测序。构建CLEC9A分子的CTLD区原核表达质粒pET-32a-CTLD,转化BL21(DE3),IPTG诱导重组蛋白表达,并用SDS-PAGE和Western blot法对目的蛋白进行鉴定。结果 PCR扩增得到726 bp的Clec9A编码区全长,测序和序列比对发现恒河猴CLEC9A编码区的核苷酸序列与人和小鼠的同源性分别为95.0%和73.1%,推测的氨基酸同源性分别为91.7%和57.3%。体外表达获得相对分子质量(Mr)约32 000的CTLD融合蛋白,Western blot方法鉴定发现其与人DNGR-1分子具有相似的抗原性。结论中国恒河猴Clec9A基因与人的高度相似并能编码与人CTLD抗原性相似的蛋白。     

自然杀伤细胞活化性受体的研究进展

自然杀伤细胞（NK）是机体固有免疫系统的重要效应细胞,其不仅能杀死病毒感染细胞和肿瘤细胞,还参与调节固有免疫应答和适应性免疫应答.NK细胞对靶细胞的杀伤效应取决于NK细胞抑制性受体和活化性受体与其配体相互作用的整合,而NK细胞不杀伤正常组织是因为抑制性受体对HLA-Ⅰ类分子的优势识别.NK细胞抑制性受体研究比较成熟,近几年NK细胞活化性受体研究进展很快.     

不同剪切力对内皮细胞表面凝集素样氧化低密度脂蛋白受体1表达的影响

目的 研究不同剪切力对内皮细胞表面凝集素样氧化低密度脂蛋白受体1(LOX-1)表达的影响.方法 体外培养人脐静脉内皮细胞,将其种植于载玻片上,放入流室系统分别施加以不同的剪切力.按施加剪切力的不同分为实验组和对照组:实验组分为低剪切力组(4.2 dyne/cm2,n=5)、中等剪切力组(8.4 dyne/cm2,n=5)和高剪切力组(15.0 dyne/cm2,n=5);对照组(n=5)静态条件下培养,不施加剪切力.剪切力加载1、2、4、8 h后RT-PCR测定各组内皮细胞LOX-1 mRNA的表达.结果 低剪切力(4.2 dyne/cm2)作用下,内皮细胞LOX-1表达随时间延长而增加.与对照组比较,中等剪切力(8.4dyne/cm2)作用2、4、8 h内皮细胞LOX-1的表达水平均降低[2 h:0.2346±0.0397比0.2948±0.0128,4 h:0.1747±0.0585比0.2985±0.0204,8 h:0.0256±0.0067比0.2968±0.0175,均P＜0.05].高剪切力(15.0dyne/cm2)作用下,血管内皮细胞LOX-1表达一直维持在极低水平.结论 不同剪切力可以影响内皮细胞的LOX-1表达水平.     

半乳糖凝集素-3(Gal-3)在卵巢癌细胞系中的表达及其对NK细胞杀伤活性的影响

目的:探究半乳糖凝集素-3(Gal-3)在卵巢癌细胞系中的表达及其对NK细胞杀伤活性的影响及机制.方法:Western blot、流式细胞术及ELISA检测Gal-3在正常卵巢上皮细胞IOSE80与上皮性卵巢癌细胞系Hey、ES2及与SKOV3中的表达;分离纯化健康志愿者外周血中NK细胞,流式细胞术进行鉴定;采用人重组...     

NK受体KIR生物学功能的研究进展

杀伤细胞免疫球蛋白样受体(Killer Ig-like receptor,KIR)为免疫球蛋白超家族成员,表达于NK细胞和某些T细胞亚群。KIR包括抑制型和激活型受体,其通过识别表达于靶细胞上的人类白细胞抗原1(HLA-Ⅰ)类分子,调节NK细胞的杀伤活性,在自身免疫性疾病、妊娠、移植等生理病理过程中起重要的作用。本文综述了近年来关于KIR研究的最新进展,主要包括KIR的结构、其特异性配体、KIR介导的信号传导和KIR的生物学功能。     

Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor reportoire

Subsets of mouse natural killer (NK) cells express receptors encoded by the Ly49 gene family that recognize allelic determinants on major histocompatibility complex (MHC) class I molecules. Recognition of self class I molecules typically inhibits NK cell lytic function. The presence of NK cell subsets expressing receptors which are able to discriminate class I alleles raises the possibility that there exist mechanisms to coordinate the NK cell receptor repertoire with the class I molecules of the host. In the present study, we determined the effects of class I gene expression on the frequencies of NK cells expressing three different Ly49 receptors defined by monoclonal antibodies. We show here an MHC-dependent skewing of NK cell subsets expressing multiple Ly49 receptors with specificity for self MHC. The results provide the first evidence that the frequencies of NK cells expressing different Ly49 receptors are determined by the host's MHC molecules. The results also extend previous findings that MHC class I expression influences the cell surface levels of each Ly49 receptor, suggesting an additional mechanism by which MHC molecules may influence the effective specificity of NK cells. Models to account for self tolerance and MHC-controlled repertoire differences are discussed.     

Expression of regulator of G protein signalling proteins in natural killer cells, and their modulation by Ly49A and Ly49D

The small GTPase accelerators regulator of G protein signalling (RGS) proteins are important regulators of proximal signalling from G protein coupled receptors. Although natural killer (NK) cells express a number of G-protein coupled receptors, expression of RGS proteins has not been investigated. We analysed the expression of RGS proteins in rat NK cells, and detected mRNA for RGS1, RGS2, RGS5, RGS8, RGS16, and RGS18. Interestingly, when we included a panel of different leucocyte subsets, we found that RGS8 was selectively expressed by NK cells. NK cells are under control of both activating and inhibitory receptors and, utilizing a xenogeneic system where the mouse activating Ly49D or inhibitory Ly49A receptors were transfected into the rat RNK-16 cell line, the potential regulation of RGS proteins by single NK cell receptors was studied. We found that ligation of Ly49D led to a rapid and transient increase in message for RGS2, while Ly49A ligation up-regulated RGS2, RGS16, and RGS18 mRNA. Both receptors also induced a prolonged increase in RGS2 endogenous protein levels. These findings suggest that RGS proteins may be influenced by or involved in NK cell receptor events, suggesting a crosstalk between G-protein coupled receptors and NK cell receptors.     

The activating rat Ly49s5 receptor responds to increased levels of MHC class Ib molecules on Listeria monocytogenes-infected enteric epithelial cells

We have investigated whether rat Ly49 receptors can monitor Listeria-infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC-I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria-infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC-I molecules of the RT1(u) haplotype, but not with mAb OX18 reacting with classical MHC-I molecules in this haplotype. Similar responses were observed against IFN-γ-treated cells that also upregulated their expression of MHC-I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC-I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN-γ in response to Listeria-infected CC531 cells, and that this was not limited to Ly49-expressing cells, since similar levels of IFN-γ production were observed in Ly49(+) and Ly49(-) NK cell subsets. Therefore, NK cells may recognize Listeria-infected cells through both MHC-I-dependent and -independent innate immune receptor systems.     

Expression of the Ly49A gene in murine natural killer cell clones is predominantly but not exclusively mono-allelic

The Ly49 family of natural killer (NK) cell receptors are major histocompatibility complex (MHC) class I-specific inhibitory receptors that are distributed to overlapping NK cell subsets. Extending earlier studies of polyclonal NK cell populations, we have employed an analysis of short-term NK cell clones from Ly49A heterozygous mice, to demonstrate that the Ly49A gene is usually expressed from one or the other allele in each Ly49A⁺ cell. However, we also detected a small percentage of clones that expressed both Ly49A alleles. The possibility that the colonies exhibiting bi-allelic Ly49A gene expression had been inoculated with more than one cell was ruled out by parallel analysis of clones isolated from mixtures of NK cells from Ly49A homozygous mice. The frequency of bi-allelic Ly49A⁺ clones suggested that the two Ly49A alleles in an NK cell are chosen for expression independently. These data are consistent with the proposal that mono-allelic Ly49A gene expression may arise as a consequence of a stochastic Ly49 gene activation mechanism. Analysis of Ly49A⁺ clones from MHC-different mice demonstrated that class I-deficient mice harbored a greater number of bi-allelic Ly49A⁺ cells than did H-2^d mice, which express a Ly49A ligand. Although the numbers were insufficiently large for a clear assignment, H-2^b mice may harbor an intermediate number of bi-allelic Ly49A⁺ NK cells. The effects of MHC expression on the prevalence of bi-allelic Ly49A⁺ cells suggest that an MHC-dependent education process modifies the Ly49 repertoire.     

MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells

Using appropriate conditions natural killer (NK) cells can be cultured from the liver and thymus of day 14 fetal mice. These fetal NK cells are phenotypically and functionally indistinguishable from adult NK cells with the exception that they lack measurable expression of all of the Ly49 molecules that can currently be detected with antibodies. Despite this, they preferentially kill tumor cells and blast cells deficient in the expression of major histocompatibility complex class I molecules, although the degree of discrimination is usually weaker than that shown by adult NK cells and varies depending on the particular combination of effector and target cells used. Polymerase chain reaction analysis revealed that although fetal NK cells are severely deficient in the expression of mRNA for Ly49A, B, C, D, G, H, and I they express high levels of Ly49E mRNA, raising the possibility that Ly49E may have an important and special function in the early development of the NK lineage.     

Ly49Q ligand expressed by activated B cells induces plasmacytoid DC maturation

Ly49Q, a type II C‐type lectin expressed on mouse plasmacytoid DC (pDC), contains a single carbohydrate recognition domain in its extracellular region and an ITIM in its cytoplasmic domain. We have identified the MHC class I molecule H‐2K^b as a Ly49Q ligand, confirming prior reports. Although H‐2K^b is expressed on essentially all hematopoietic cells, we found that only CpG‐stimulated B cells were able to activate Ly49Q. This discovery correlated with our finding that although H‐2K^b forms clusters on CpG‐activated B cells, it is diffusely expressed on resting B cells. Furthermore, CpG‐stimulated, but not resting, B cells up‐regulated co‐stimulatory molecules on pDC. This finding was confirmed by the fact that binding by anti‐Ly49Q mAb to Ly49Q led to pDC maturation in vitro. Our results suggest that clustered H‐2K^b on activated B cells act as ligands for Ly49Q and induce pDC maturation in vitro.     

Ly49 receptors: evolution,genetic diversity,and impact on immunity

Natural killer (NK) cells express cell surface receptors that recognize class I major histocompatibility complex (MHC-I) molecules to distinguish between healthy and unhealthy cells. The multigenic and polymorphic nature of the MHC-I genes has influenced the convergent evolution of similarly polymorphic and diversified NK cell receptor families: the C-type lectin-like Ly49 receptors in mice, and the killer cell immunoglobulin-like receptors (KIRs) in humans. Although structurally distinct, both receptor families have similar functions in terms of MHC-I recognition and downstream signal transduction, and they regulate multiple aspects of NK cell biology during development and after maturation as fully differentiated and functionally competent cells. The Ly49 gene locus has undergone rapid, lineage-specific expansions and contractions resulting in multiple distinct haplotypes of variable gene number, allelic diversity, and MHC-I ligand specificity. This in turn has influenced the type and degree of Ly49 receptor expression on NK cells, and their contribution to immunity in different mouse strains. In this review, we have attempted to describe the evolutionary processes that have shaped strain-specific Ly49 receptor repertoires, and their impact on NK cell functions during health and disease.     

Evolution of the Ly49 and Nkrp1 recognition systems

The Ly49 and Nkrp1 loci encode structurally and functionally related cell surface proteins that positively or negatively regulate natural killer (NK) cell-mediated cytotoxicity and cytokine production. Yet despite their clear relatedness and genetic linkage within the NK gene complex (NKC), these two multi-gene families have adopted dissimilar evolutionary strategies. The Ly49 genes are extremely polymorphic and evolutionarily dynamic, with distinct gene numbers, remarkable allelic diversity, and varying MHC-I-ligand specificities and affinities among different murine haplotypes. In contrast, the Nkrp1 genes have opted for overall conservation of genomic organization, sequences, and ligand specificities, with only limited and focused allelic polymorphism. Possible selection pressures driving such varied evolution of the two gene families may include disequilibrium from ligand co-inheritance, pathogen immunoevasin strategies, flexibility in host counter-evolution mechanisms, and the prevalence and dynamics of inherent repetitive elements.     

Differential Expression of the Ly49GB6, but Not the Ly49GBALB,Receptor Isoform during Natural Killer Cell Reconstitution after Hematopoietic Stem Cell Transplantation

Inhibitory natural killer (NK) cell receptors specific for major histocompatibility complex class I (MHC-I) molecules include Ly49 receptors in mice and killer immunoglobulin-like receptors (KIR) in humans. The “licensing” or “arming” models imply that engagement of these receptors to self MHC-I molecules during NK cell development educates NK cells to be more responsive to cancer and viral infection. We recently reported that hematopoietic stem cell transplantation (HSCT) induced rapid and preferential expansion of functionally competent Ly49G⁺, but not other Ly49 family, NK cells independent of NK cell licensing via Ly49–MHC-I interactions. We now extend these studies to evaluate expression of the two Ly49G receptor isoforms Ly49G^B6 and Ly49G^BALB, using mice with different MHC-I haplotypes that express one or both of the isoforms. NK cells from CB6F₁ (H-2^bxd) hybrid mice express two different alleles for Ly49G receptor, Ly49G^B6 and Ly49G^BALB. We found that CB6F₁ mice had more Ly49G^B6+ NK cells than Ly49^BALB+ NK cells, and that only Ly49G^B6+ NK cells increased in relative numbers and in Ly49G mean fluorescence intensity values after HSCT similar to the B6 parental strain. We further observed that Ly49G⁺ NK cells in BALB/c (H-2^d) and BALB.B (H-2^b) mice, which have the same background genes, recover slowly after HSCT, in contrast to Ly49G⁺ NK cells in B6 (H-2^b) recipients. The difference in expression of Ly49G^B6 relative to Ly49G^BALB was linked to differences in the activity of the Pro1 promoter between the two alleles. Thus, we conclude that the Ly49G^B6 receptor dominates Ly49G expression on NK cells after HSCT in strains in which that allele is expressed. The data suggest that Ly49 allelic polymorphism within a particular Ly49 family member can differentially affect NK cell recovery after HSCT depending on the background genes of the recipient, not on the MHC-I haplotype.