HLA 抗原的DNA 分型

HLA系统定位于6p2l.3, 是人体内最复杂的遗 传多态性系统,在免疫调控过程中发挥重要作用。 HLA I类基因区域包括HLA-A, B, C, E, F, G基因 和11个假基因“‘’。HLA一A, B和C基因座编码A, B 和C抗原a链(44kd),它通过第三个功能区与受痊于 第15号染色体的月,微球蛋白链(12kd）作非共价结 合构成杭原分子,其多态性取决于。链的第一、二功能 区。HLA-E, F和G基因编码I类样产物,其功能未 明。I类抗原分布于任何有核全』饱表面,主要参与提 供外来伉原给T细胞毒细胞「’“’。HLA II类基因座在 HLA-D区,主要包括HLA-DR, DQ和DP三个}2 区。DR亚区有6个基因座：DRA, DRB1, DPB2, DRB3, DRB4和DRB5, 其中DRB2为假基因;DQ 亚区有4个居因座：DQA1, DQB1, DQA2和DQB2, 其中DQA2和DQB2未知有产物表达;DP亚区也 有4个基因座：DPA1, DPB1, DPA2和DPB2,其 中DPA2和DPB2为假基因。此外,还有DO和DN 两个新亚区,各具有DOB和DNA（注意勿与脱氧核塘 核酸的缩写DNA相混淆）,分别产生DO(链和DNa 链「‘’。11类抗原HLA-DR, DQ和DP存在于B淋巴 细胞、巨噬细胞和激活的T淋巴细胞表面, 由。链 (32-35kd）和R链(28-30kd）非共价组成。。链无 多态性或多态性程度不高,R链的第一功能区呈现高 度多态性。11类抗原参与提皇外来伉原给T辅助细 胞,后者在识别外来伉原的同时识别HLA 11类坑 原「,,’。据1987年第十届国际组织相容性试验专题会 议报道,HLA系统检出148种抗原特异性,其中A基 因座24种,B基因座52种,C基因座11种,D区26 种,DR亚区20种,DQ亚区9种和DP亚区6种。 HLA-A, B, C, DR和DQ抗原系由特异性同种异 体抗血清检出,HLA-D和DP抗原则由纯合分型细胞 (HTC)和预致敏淋巴细胞分型(PLT）方法检出,两者 是以混合淋巴细胞培养(MLC）方法为基础的。近年 发展用蛋白质化学方法研究HLA系统抗原多态性L77o 自1975年Southern印迹法〔247间世后,运用HLA系 统基因探针通过分析限制性片段长度多态性(Restriction Fragment Length Polymorphism, RFLP）在DNA. 水平上进行分型。八十年代中期开始,聚合酶链反应 (Polymerase Chain Reaction, PCR）技术的创立为分 子生物学发展开辞了一个新起点。许多研完Hl.h系 统的实验室竞相运用PCR技术,结合等位基因特异的 寡核昔酸（Allele-specific Oligonucleotide, ASO)或 顺序特异的寡核普胶(Sequence-specific Oligonucleotide, SSO)探针对HLA系统进行寡核昔酸分型,2_3 也有直接利用PCR扩增产物作RFLP分沂进行DNA 分型「'6,,"' + O DNA分型方法适用于任何有核细袍, 显示的结果与血清学或细胞学分型结果高度对应,而 且能够发现血清学或细抱学方法不能检测的额外特异 性,在理论上有助于进一步研究HLA系统基因的结构 和功能。对一些无法作常规分型的病例如裸露淋巴细 胞综合征、严重联合免疫缺乏症等,DNA分型叮解决 骨髓移植供受体配型问题‘哪’。     

CCR2-64Ⅰ在中国南方14个少数民族群体中的分布

为调查HIV-1感染相关等位基因CCR2-64Ⅰ在我国南方14个少数民族群体的频率和多态性分布, 从上述人群外周血中抽提基因组DNA, 采用PCR和PCR-RFLP等方法进行基因分型。在791例调查对象中, 636例是野生纯合子基因型, 104例为杂合子基因型, 51例为突变纯合子基因型。上述各群体等位基因型的分布符合Hardy-Weinberg平衡。14个民族群体的平均突变基因频率为13.6％, 等位基因频率范围分布在1.6％~30.3％之间, 14个民族群体之间突变基因频率具有显著差异(P<0.05)。广西壮族群体CCR2-64Ⅰ突变基因频率最低, 为1.6％, 云南的六库傈僳族频率最高, 为30.3％。12个群体的突变基因频率均低于中国汉族健康群体, 南方3个少数民族群体基因突变频率显著低于西南11个少数民族群体, 该突变基因在艾滋病发病过程中的影响值得进一步深入研究。     

T、B淋巴细胞联合免疫缺陷615-SCID导入近交系小鼠的培育及其一般生物学特性的研究

目的　建立T、B淋巴细胞联合免疫缺陷同源导入近交系 6 15 SCID小鼠。方法　利用经典遗传学的杂交 互交方法 ,将SCID小鼠携带的T、B淋巴细胞联合免疫缺陷基因 (scid基因 ) ,导入我国自己培育的 6 15近交系小鼠中。育种过程中采用白细胞计数分类及ELISA检测Ig含量方法 ,筛选每个M循环中的纯合子 ;再用蛋白质和同工酶电泳方法 ,分析纯合子位于 11个染色体上的 2 5个生化标记基因位点。结果　第 7个M循环中 ,所得纯合子与 6 15小鼠的 2 5个生化标记基因位点完全一致 ,表明M7循环的纯合子具有 6 15小鼠的全部标记基因。结论　M6、M7循环的纯合子小鼠表达scid基因 ,具有放射敏感性和T、B淋巴细胞联合缺陷的特征 ,且T细胞比例显著低于亲代SCID小鼠     

ENU诱变获得一例巨结肠症小鼠及其突变基因的染色体定位

采用化学诱变剂乙酰基亚硝基脲(N-ethyl-N-nitrosourea,ENU)获得一种可遗传的腹部白斑突变杂合子小鼠,将该杂合子小鼠互交后获得具有花斑表型的突变纯合子小鼠,进一步研究表明突变纯合子小鼠表现为严重的巨结肠表型。肠全标本乙酰胆碱酯酶组织化学染色显示突变纯合子小鼠巨结肠段神经节细胞缺失。为定位该基因,利用微卫星标记(B6×D2)对F1互交获得的F2突变纯合子小鼠进行基因组扫描,初步将该突变基因定位于小鼠第14号染色体。     

HLA分型方法进展

传统的HLA分型方法主要是血清学和细胞学方法,分型准确性较差,且不能进行亚型分析。随着PCR技术及基因芯片技术等分子生物学技术的出现、发展,HLA基因分型得到了迅速的发展,各具特点的HLA分型技术不断出现,使HLA的分型更加准确、精细、简便、实用,为临床推广应用打下了基础。     

随机扩增多态性对白色念珠菌分型的估价

利用随机扩增多态性(RAPD-PGR)的方法对48株白念珠菌Candida albicans(Robin)Berkh进行了分析。由初步试验中,随机选用了18种引物,筛选出OPA-14引物,该引物扩增的带型清晰可辨,不同菌株之间扩增的带数6—12条不等,共有6条主带。扩增片段的长度粗略估计在300—2000bp左右。除个别菌株的带型相同以外,大多数菌株之间呈多态性分布,其带型的数目和扩增的片段存在差异。对简单的DNA制备方法,以及RAPD-PCR在临床应用和流行病学调查中的可行性进行了初步探讨。     

人白细胞抗原G3在稳定转染细胞细胞膜上的表达

人白细胞抗原G(HLA G)是一种在母婴耐受中起主要作用的非经典的HLA Ⅰ类分子 .其中HLA G3结构简单 ,仅具有α1结构域、穿膜区及胞浆区 ,其是否在细胞表面表达尚存在争议 .为了建立HLA G3稳定转染细胞株并确定其在细胞内的定位 ,采用RT PCR法从 9周人胎盘组织中获得了HLA G3的cDNA ,并构建到真核表达载体pcDNA3中 ,将所获得的真核表达质粒pcDNA G3转染至HLA Ⅰ (- )细胞株K5 6 2 ,经G4 18筛选后获得稳定转染的细胞株K5 6 2 G3.利用RT PCR、Western印迹检测方法证明在K5 6 2 G3细胞株中 ,HLA G3在mRNA水平和蛋白水平上均有表达 .进一步利用免疫荧光标记技术 ,证明HLA G3能够在转染细胞细胞膜上表达 .结果表明 ,稳定转染细胞株中HLA G3蛋白能够定位表达在细胞膜     

利用DHPLC研究我国几个地方绵羊品种黑素细胞刺激素受体基因单核苷酸多态性

利用测序及变性高效液相色谱(DHPLC)研究了蒙古羊、哈萨克羊、滩羊和藏绵羊黑素细胞刺激素受体（MSHR）基因单核苷酸多态性（SNP）。结果表明,在扩增片断长度为415bp范围内存在一个T317C突变,DHPLC可检测到该突变并被证明是一种高通量且简便的筛选方法。通过两次DHPLC可确定两个杂合子和一个纯合子,第一次DHPLC可迅速检测出由于形成异源双链而呈肩峰的杂合子（TC）,但不能区分两个均呈单峰的纯合子（TT或CC）。第二次DHPLC将未知纯合子与已知序列的纯合子混合后进行,通过判定单峰或肩峰即可推断未知纯合子的基因型。所研究的4个绵羊群体在该突变位点均处于Hardy-Weinberg平衡状态。蒙古羊与哈萨克羊较近的遗传亲缘关系以及滩羊与藏绵羊较近的遗传亲缘关系与线粒体DNA的研究结果一致。     

与拟除虫菊酯抗性相关的烟粉虱钠通道基因突变及其检测

通过RT-PCR克隆了烟粉虱Bemisia tabaci (Gennadius) 南京种群(B-生物型)的钠离子通道结构域ⅡS4-6 cDNA片段,证实了与拟除虫菊酯抗性相关的是位于第925位亮氨酸到异亮氨酸的突变(L925I),并建立了L925I突变的PASA检测技术。与SUD-S敏感品系相比,2002年采自南京棉花上的烟粉虱种群对氯氰菊酯具有77倍的抗性,用氯氰菊酯对该种群进行多次筛选后,该种群对氯氰菊酯的抗药性提高到227倍。PASA检测结果表明筛选后的南京种群中100%个体都具有L925I突变(61.1%的个体为L925I突变纯合子,38.9%的个体为杂合子),而未筛选的南京种群只有75%个体具有L925I突变(35%个体为L925I突变纯合子,40%的个体为杂合子,25%的个体为野生型)。该结果表明了烟粉虱钠离子通道L925I突变与对拟除虫菊酯抗性密切相关。还讨论了烟粉虱对拟除虫菊酯抗性的代谢机理。     

我国大陆主要少数民族HLA多态性聚类分析和频率分布对中华民族起源的启示

本文是1991年11月6—13日横滨第11届国际组织相容性会议(IHWC)协作科研中我国主要少数民族(苗族、布依族、蒙古族、满族、回族、藏族、维吾尔族)和南北汉族样本HLAⅠ、Ⅱ、Ⅲ类抗原多态性分析的联合报告。聚类分析表明,苗、布依与南方汉族聚类,蒙、满、回、藏与北方汉族集群,提示中华民族包含南北两大群体。维吾尔族非常疏远地共聚于高加索人种。高加索人种起源的HLA抗原基因频率(A3、B8等)由西向东又由北向南递减。东南亚蒙古人种起源的HLA抗原基因频率(B46等)由南向北递减。HLA抗原基因频率的这种梯度分布反映我们祖先自远古史前时期以来的不断迁移和相互融合的过程。本文讨论了中华民族包含南北两大群体这一事实对于中华民族起源的启示。     

Genomic and Phylogenetic Analysis of the Human CD1 and HLA Class I Multicopy Genes

The human CD1 proteins belong to a lipid-glycolipid antigen-presenting gene family and are related in structure and function to the MHC class I molecules. Previous mapping and DNA hybridization studies have shown that five linked genes located within a cluster on human chromosome 1q22-23 encode the CD1 protein family. We have analyzed the complete genomic sequence of the human CD1 gene cluster and found that the five active genes are distributed over 175,600 nucleotides and separated by four expanded intervening genomic regions (IGRs) ranging in length between 20 and 68 kb. The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Some L1 sequences have acted as receptors for other subtypes or families of retroelements. Alu molecular clocks that have evolved during primate history are found distributed within the HLA class I duplicated segments (duplicons) but not within the duplicons of CD1. Phylogeny of the alpha3 domain of the class I-like superfamily of proteins shows that the CD1 cluster is well separated from HLA class I by a number of superfamily members including MIC (PERB11), HFE, Zn-alpha2-GP, FcRn, and MR1. Phylogenetically, the human CD1 sequences are interspersed by CD1 sequences from other mammalian species, whereas the human HLA class I sequences cluster together and are separated from the other mammalian sequences. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. In contrast to the HLA class I genomic structure, the human CD1 duplicons are smaller in size, they lack Alu clocks, and they are interrupted by IGRs at least 4 to 14 times longer than the CD1 genes themselves. The IGRs seem to have been created as "buffer zones" to protect the CD1 genes from disruption by transposable elements.     

TagSNP approach for HLA risk allele genotyping of Saudi celiac disease patients: effectiveness and pitfalls

Background: Celiac disease (CD) is a genetically complex autoimmune disease which is triggered by dietary gluten. Human leukocyte antigen (HLA) class II genes are known to act as high-risk markers for CD, where >95% of CD patients carry (HLA), DQ2 and/or DQ8 alleles. Therefore, the present study was conducted to investigate the distribution of HLA haplotypes among Saudi CD patients and healthy controls by using the tag single nucleotide polymorphisms (SNP).Methods: HLA-tag SNPs showing strong linkage value (r²>0.99) were used to predict the HLA DQ2 and DQ8 genotypes in 101 Saudi CD patients and in 103 healthy controls by using real-time polymerase chain reaction technique. Genotype calls were further validated by Sanger sequencing method.Results: A total of 63.7% of CD cases and of 60.2% of controls were predicted to carry HLA-DQ2 and DQ8 heterodimers, either in the homozygous or heterozygous states. The prevalence of DQ8 in our CD patients was predicted to be higher than the patients from other ethnic populations (35.6%). More than 32% of the CD patients were found to be non-carriers of HLA risk haplotypes as predicted by the tag SNPs.Conclusion: The present study highlights that the Caucasian specific HLA-tag SNPs would be of limited value to accurately predict CD specific HLA haplotypes in Saudi population, when compared with the Caucasian groups. Prediction of risk haplotypes by tag SNPs in ethnic groups is a good alternate approach as long as the tag SNPs were identified from the local population genetic variant databases.     

Evidence for heterosis in the HLA system

The number of persons with homozygous HLA haplotypes in several groups of South American Indians was 39% less than that expected assuming unmodified equilibrium. In a subpopulation of 122 persons whose parents' HLA constitutions were known, there were 56% fewer homozygous persons than expected. This deficit was widely distributed in different haplotypes and different tribal groups.     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

人胎脾交错突细胞对T,B淋巴细胞发育影响的免疫组织化学研究

用免疫酶单重和双重染色研究人胎儿脾连续切片中交错突细胞（ＩＤＣ）与Ｔ,Ｂ淋巴细胞的定位关系及ＨＬＡ－ＤＲ表达。结果表明,Ｓ－１００阳性树突状细胞为ＩＤＣ,多数表达ＨＬＡ－ＤＲ。９－１２周的胎脾中就可见到散在分布的ＩＤＣ。１３－１６周胎脾中ＩＤＣ开始定位于白髓的Ｔ细胞集落内和周缘,及Ｂ细胞集落的周边。在上述区域ＩＤＣ常与Ｔ细胞形成ＩＤＣ－Ｔ细胞聚合体。在脾的发育过程中,ＩＤＣ不仅与Ｔ,Ｂ淋巴细胞在分布上关系密切,而且可与这两类细胞形成突起－胞体、胞体－胞体的连接。提示,胎儿脾中ＩＤＣ与Ｔ,Ｂ细胞的迁移,定位及功能成熟过程有密切联系。     

广东丹霞山国家级自然保护区 蝙蝠物种多样性调查

于2017年至2018年对广东丹霞山国家级自然保护区的翼手目动物进行调查,主要采用日栖息地与夜栖息地、捕食区网捕等调查方法,共调查到翼手目5科13属23种,其中蝙蝠科(Vespertilionidae)9属15种,菊头蝠科(Rhinolophidae)1属4种,蹄蝠科(Hipposideridae)1属2种,假吸血蝠科(Megadermatidae)1属1种,犬吻蝠科(Molossidae)1属1种。从区系组成来看,以东洋界为主(19种),其次为广布种(3种),古北界仅1种。从栖息类型上看,分为洞栖型、树栖型及建筑物栖息型,以洞栖型为主(15种),建筑物栖息型其次(12种),树栖型最少(5种),但其中9个物种的栖息地类型同时包含了上述3种栖息类型中的2种。本研究在广东丹霞山发现中国蝙蝠新分布记录1种,为卡氏伏翼(Hypsugo cadornae);而中印鼠耳蝠(Myotis indochinensis)为中国分布的再次确认。     

HLA linkage and B14, DR1, BfS haplotype association with the genes for late onset and cryptic 21-hydroxylase deficiency.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) has been established to be an HLA-linked, recessive monogenetic disease. However, two nonclassical forms of 21-OH-def have also been described: "cryptic" 21-OH-def, which has been shown to be HLA-linked, and "late onset" 21-OH-def, for which the status of linkage to HLA has been less certain. We now describe studies of eight additional unrelated probands with symptomatic, "late onset" 21-OH-def, and conclude that this form is also HLA-linked. Both "late onset" and "cryptic" 21-OH-def are highly associated with the same HLA antigens and markers (HLA-B14, HLA-DR1, and Bf type S) in individuals from different ethnic and geographical backgrounds. Since both "late onset" and "cryptic" 21-OH-def appear to occur in individuals with one classical 21-OH-def (21-OHCAH) allele who in addition have another 21-OH-def allele, as well as in individuals who appear to be homozygous for variant 21-PH-def alleles, and since both late onset and cryptic 21-OH-def appear to occur in the same families, our data suggest that these syndromes may represent different clinical expressions of similar or identical nonclassical 21-OH-def alleles.     

Family studies of hereditary hemochromatosis in Denmark and the Faroe Islands

Summary Pedigree studies were performed based on one Faroese and four Danish probands with overt idiopathic hemochromatosis (IH). The study consisted of HLA typing and determination of biochemical iron status indicators (serum transferrin saturation, serum ferritin). In total, 130 persons were evaluated. The screening identified 6 homozygous (h/h) subjects with preclinical IH, 46 heterozygous (h/n), and 8 normal (n/n) subjects, while 39 subjects were classified as normal or heterozygous (n/h?). One family demonstrated both a homozygous x heterozygous as well as a heterozygous x heterozygous mating. Recombination between the HLA region and IH locus occurred possibly in three subjects in three different families. The significance of detailed screening in families with probands with IH is discussed.     

Supportive haemotherapy in bone marrow transplantation

The intensive supportive haemotherapy which has to be used in bone marrow transplantation is discussed, taking mainly into account platelet transfusions. Ways to avoid alloimmunizations against platelet antigens, especially HLA-ABC antigens, are shown (use of HLA-AB homozygous donors or of cross-reacting groups).