河南汉族白血病与HLA-DQB1、DRB1基因相关性的研究

目的:探讨HLA-DQB1、DRB1基因与河南汉族白血病患者的关联性。方法:采用序列特异性引物聚合酶链反应(PCR-SSP)方法对52名河南汉族白血病患者和52名健康对照组进行HLA-DQB1、DRB1基因分型。结果:白血病患者组与正常对照组相比,DRB1*04等位基因频率(11.54%)明显高于对照组(3.85%),危险系数RR=3.6,χ2=4.727(P<0.05);DRB1*11等位基因频率(4.81%)明显低于对照组(12.5%),危险系数RR=0.319,χ2=4.230(P<0.05)。结论:提示河南汉族人群中,HLA-DRB1*04等位基因可能对白血病有易感作用,而HLA-DRB1*11等位基因可能对白血病有拮抗作用。     

用PCR-SSP对HLA-DQB作中分辨法分型

采用PCR-序列特异性引物技术(SSP)对HLA-DQB作中分辨法分型。该方法不仅可行,且操作简便快速,结果准确,适用于临床器官移植配型。     

1914名河北地区汉族人群HLA-DQB1基因多态性分析

目的探讨河北汉族人群人类白细胞抗原-DQB1(HLA-DQB1)基因型遗传特性。方法对1914名河北地区汉族人群献血者采用聚合酶链反应-基于测序的分型技术(PCR-SBT)进行HLA-DQB1位点高分辨基因分型,采用直接计数法计算等位基因频率,并与中国其他地区人群基因频率进行比较。结果 1914名河北汉族献血者中共检出21个HLADQB1等位基因,其中频率大于0.0500的常见等位基因共6个,分别为DQB1*03∶01、DQB1*03∶03、DQB1*06∶02、DQB1*02∶02、DQB1*06∶01和DQB1*05∶01。结论河北地区汉族人群DQB1具有丰富的多态性,基因分布有明显的地区特性,了解河北汉族人群HLA-DQB1的分布状况,有利于帮助临床寻找HLA匹配的造血干细胞无关供者,同时为我国人群群体遗传学研究等提供有价值的背景资料。     

广东汉族人群1型糖尿病与HLA-DQB1基因的相关性

目的:探讨广东汉族人群1型糖尿痛(T1DM)与HLA-DQB1等位基因的相关性.方法:选取57例T1DM患者(T1DM)和454名正常对照人群(对照组),运用PCR-SBT对HLA-DQB1等位基因进行分型.结果:在HLA-DQB1位点上,T1DM组的DQB1*0201频率显著高于对照组,而DQB1*0502的频率则明显降低,差异具有统计学意义(P<0.05).结论:HLA-DQB1*0201是广东汉族人群T1DM的易感基因,DQB1*0502则是保护基因.     

应用PCR-SSP方法分析部分中国南方汉族人群HLA-DQB1位点等位基因的多态性

目的：检测和分析部分中国南方汉族人群HLA-DQBl位点等住基因的多态性。方法：应用聚合酶链反应一序列特异性引物法(PCR-SSP)对101名健康、无血缘关系的中国南方汉族人进行HLA—DQB1分型。结果：所检测的8个HLA—DQB1靶等位基因均有检出；频率最高的为DQ2，3(7，9)，其等住基因频率(GF)为41．14％；最低的为DQ4，其GF为7．19％。结论：中国南方汉族人的HLA-DQB1等位基因的分布具有民族和地域特征，与白人、黑人和日本人的HLA-DQB1等位基因分布有差异。     

低分辨率和高分辨率HLA-Ⅱ类PCR-SSP基因分型在骨髓移植中的应用

为满足临床骨髓移植对HLA配型的要求,本研究采用先进、快速的DNA提取方法和低分辨率与高分辨率HLA-ⅡPCR-SSP分型方法,对150例临床骨髓移植供、受者进行HLA-Ⅱ类分型研究,并且对分型方法进行了改进,建立了随时间释放DNA聚合酶活性的分型方法。结果表明:DNA提取方法可以满足微量HLA-Ⅱ类DNA分型方法对DNA样本的要求,200μl全血DNA产量为4-12μg,A_(260)/A_(280)为1.7～1.9。低分辨率和高分辨率HLA-Ⅱ类PCR-SSP均具有良好稳定性、可靠性和准确性。低分辨率HLA-Ⅱ类PCR-SSP方法耗时1.5小时,适用于同胞兄弟姐妹间的骨髓移植配型。高分辨率HLA-Ⅱ类亚型PCR-SSP分型方法耗时2小时50分钟,适用于同胞兄弟姐妹间骨髓移植配型的特殊病例和无血缘关系骨髓移植供、受者间的配型。本研究的分型方法对于推动我国骨髓移植工作的开展和未来的发展均具有重要的意义。     

江苏7地市汉族人群HLA-DQB1等位基因多态性分析

目的分析来自中华骨髓库江苏分库7地市汉族人群HLA-DQB1基因多态性。方法采用聚合酶联反应-直接测序分型(PCR-SBT)技术,对2010年1月—2017年12月中华骨髓库江苏分库4973名志愿者的HLA-DQB1位点进行基因分型,分别计算7地市DQB1基因频率并比较分析。结果江苏汉族人群共检测出24种DQB1等位基因,其中徐州16种,淮安18种,盐城19种,扬州19种,镇江20种,南京18种,常州13种。常见的DQB1等位基因分布在徐州为:DQB1~*06∶01、03∶01、06∶09等;淮安:DQB1~*03∶03、03∶01、06∶01等;盐城:DQB1~*02∶02、02∶01、06∶09等;扬州:DQB1~*02∶02、03∶03、06∶01等;镇江:DQB1~*03∶03、02∶02、03∶01等;南京:DQB1~*03∶03、03∶01、06∶01等;常州:DQB1~*03∶03、03∶01、06∶01等。结论徐州、淮安、盐城、扬州的DQB1基因多态性更偏向我国北方,常州的DQB1基因多态性更偏向我国南方,而镇江、南京介于南方、北方之间。由此为研究江苏汉族人群基因遗传学及DQB1与疾病相关性提供了重要的基础资料。     

血小板抗原基因分型及配型的研究与初步应用

目的 对患者进行血小板抗原基因分型及配型的研究，以探讨临床血小板输注的安全、有效性。方法 应用聚合酶链序列特异性引物技术（PCR－SSP），对南京地区55例患者HPA1－5系统等位基因进行分型，并为其进行血小板基因配型输注。结果 经血小板基因配型的吻合组血小板输注效果明显优于未经配型的对照组。结论 对患者进行血小板抗原基因分型及配型可提高血小板输注的安全、有效性。     

HLA-DRB1基因分型在移植配型及亲子鉴定中的应用

应用聚合酶链反应－序列持异性引物（ＰＣＲ＿ＳＳＰ）方法对南京地区汉族人群ＨＬＡ－ＤＲＢ１位点基因特异性及骨髓移植配型、亲子鉴定等进行了分析。ＤＲＢ１基因频率范围在０．００３３～０．１８３３之间，以ＤＲ９１ＤＲ４占多数；骨髓移植通过配型移植成功３例，２例存活情况良好；ＤＲＢ１位点排除亲子关系５例，其中２例为ＨＬＡ－ＤＲＢ１单独排除亲缘关系。本法具有操作简单、快速、结果可靠的特点，不仅适用于移植配型、     

中国汉族人群鼻咽癌患者HLA-DQB1等位基因多态性分析★

背景:目前开展HLA-DQB1基因与鼻咽癌疾病相关性研究一方面受昂贵的进口试剂所制约,另一方面单纯做HLA-DQB1第2外测序分型,模棱两可,结果较少。目的:采用HLA-DQB1基因第2至第3外显子双向测序分型检测技术方法。方法:纳入286份中国汉族健康人群血液样本,46例鼻咽癌患者样本,使用Gentra DNA提取试剂制备基因组DNA,取100份健康人群样本作为平行对照组进行基因分型,选择及自行设计HLA-DQB1基因第2至3外显子扩增引物及测序引物,探索最适合PCR扩增及测序反应条件。采用商品化HLA-DQB1基因单向测序分型试剂盒作对照。结果与结论:100份平行对照组样本与HLA-SBT测序分型结果一致。286例健康个体中共检出13种等位基因,27例样本的等位基因为纯合子。鼻咽癌人群共检出DQB1等位基因11种,5例样本的等位基因为纯合子。鼻咽癌患者DQB1*02等位基因频率高于对照组,但所有等位基因频率比较,差异无显著性意义(χ2<3.84,P>0.05)。未发现HLA-DQB1基因与鼻咽癌有相关性。说明采用的中国汉族人群HLA-DQB1基因测序分型方法具有操作方便、结果稳定和低成本消耗等优势。     

Search for non-relative donor by the Russian register of bone marrow donors

AIM: To select maximally HLA compatible donor for hematological patients who need transplantation of bone marrow from non-relative donor. MATERIALS AND METHODS: 75 patients with hematological malignancy were observed. All of them have indications to non-relative transplantation of the bone marrow. Methods of polymerase chain reaction with sequence-specific primers and classic microlymphocytotoxic test were used. RESULTS: Typing of HLA antigens of class I and alleles of class II loci enabled search for non-relative donor for transplantation of bone marrow in accordance with the requirements of the European Federation of Immunogenetics. Most of the patients (86.6%) had at least one potential HLA-A, -B, -DR compatible donor. Half of the patients had potential donors typed at the allele level by class II loci. This diminishes time of HLA compatible donor selection. CONCLUSION: DNA typing enables the search for the non-relative donors meeting modern requirements. This allowed 5 non-relative bone marrow transplantations.     

HLA typing and transplantation

Clinical trial of organ transplantation was renal transplantation by Voronoy at 1936. The discovery of HLA in the 1950s was one of the most important new findings in the area of transplantation. Nowadays, developing HLA genotyping methods, the serum analysis does not use for donor and recipient HLA typing but for cross-matching test. Because each of HLA genotyping methods has its merits and demerits, it is important to choice right methods for avoiding type error. PCR-Luminex method using fluorescence microsphere was developed for high-resolution HLA-A, HLA-B and HLA-DRB1 genotyping in the Japanese population. This genotyping method allows to define all the possible combinations of alleles at each loci existing in Japanese at the four-digital level. In hematopoietic stem cell transplantation, to match high resolution level of HLA between donor and recipient lead an improvement of recipient's survival. In organ transplantation, removed organ has to be so immediately transplanted into recipient that no time is left for HLA genotyping. In order to have good survival of transplanted organ, HLA, cytokine promoter lesion and immunoglobulin like receptor genotyping might be helpful. We focused on this review at HLA genotyping, especially new SSO methods.     

Minor histocompatibility antigen-specific cytotoxic T cell lines, capable of lysing human hematopoietic progenitor cells, can be generated in vitro by stimulation with HLA-identical bone marrow cells

Recipient-antidonor alloreactivity before HLA genotypically identical bone marrow transplantation (BMT) between donor-recipient pairs that are negative in the mixed lymphocyte reaction (MLR), the cell-mediated lympholysis (CML) assay, and the lymphocyte crossmatch was not detectable in the majority of cases, using recipient peripheral blood lymphocytes (PBL) collected before BMT as responder cells and donor PBL as stimulator cells. However, when donor bone marrow mononuclear cells (BMMNC) instead of PBL were used as stimulator cells, we could detect donor-specific alloreactivity in 7 of 10 HLA genotypically identical donor-recipient pairs. To demonstrate that this alloreactivity was minor histocompatibility (mH) antigen specific and not directed against HLA class I splits or variants, two cytotoxic T lymphocyte (CTL) lines were tested in further detail against phytohemagglutinin (PHA) blasts from pairs of HLA genotypically identical siblings positive for the HLA class I restriction molecule. Both CTL lines recognized mH antigens, as illustrated by the differential recognition of PHA blasts of one of the two siblings from several pairs. The potential role of these mH antigen-specific CTLs in bone marrow graft rejection was demonstrated by the mH antigen-specific growth inhibition of hematopoietic progenitor cells from the original bone marrow donor and from HLA class I restriction molecule-positive individuals who expressed the mH antigens on their PBL and BMMNC. Our assay can be used in HLA genotypically identical BMT to detect a recipient-antidonor response, directed against cellularly defined mH antigens expressed on donor HPC, BMMNC, and PBL, before transplantation.     

The first-year costs of establishing an unrelated bone marrow donor registry

The cost of therapies like bone marrow transplantation has been an important consideration for several decades. Bone marrow transplantation is becoming increasingly accepted as an effective treatment for hematologic disorders, including acute nonlymphocytic leukemia. To find suitable donors, bone marrow donor registries are being developed. The first-year costs of establishing an unrelated bone marrow donor registry are reported here. First-year costs are largely due to personnel costs and HLA typing charges. The cost per registrant decreases over time, but further decreases due to economies of scale are limited by the continued fixed requirement for HLA typing. Data are presented by separating costs into six unique categories, thereby allowing other blood centers to estimate start-up costs based on our experience.     

Generation of leukemia-reactive cytotoxic T lymphocyte clones from the HLA-identical bone marrow donor of a patient with leukemia.

Allogeneic bone marrow transplantation (BMT) has been associated with a graft-vs.-leukemia (GVL) reactivity. Since T cell depletion of the bone marrow graft has decreased the risk of graft-vs.-host disease (GVHD), but has been associated with higher rates of leukemia relapse, GVL reactivity is probably caused by donor-derived T lymphocytes. Previously, we demonstrated that minor histocompatibility (mH) antigen-specific cytotoxic T lymphocyte (CTL) clones, generated from patients after BMT, are capable of major histocompatibility complex-(MHC) restricted lysis of (clonogenic) myeloid leukemic cells. Here, we investigated whether donor-derived leukemia-specific CTL clones can be generated in vitro, before BMT, using irradiated leukemic cells from a patient with acute myeloid leukemia as stimulator cells, and peripheral blood or bone marrow from the HLA genotypically identical sibling donor as responder cells. Several CTL lines were generated that showed specific lysis (> 50%) of the recipient leukemic cells in a 51Cr-release assay. Two of these CTL lines were cloned by limiting dilution in the presence of the irradiated recipient cells. Multiple leukemia-reactive, HLA class I and II-restricted clones with various specificities could be established. These alloreactive, antileukemic CTL clones may cause GVL reactivity after BMT, and may be used as adjuvant immunotherapy in the treatment of leukemia.     

急性白血病患者HLA半倍相合骨髓移植后供者源复发2例

为了探讨半倍相合骨髓移植后急性白血病患者的复发情况，对2例接受半倍相合骨髓移植后出现白血病复发的急性白血病患者进行了外周血、骨髓检查、骨髓细胞形态观察及HLA基因型和染色体核型分析。结果发现。2例原发病分别为急性淋巴细胞白血病（普通细胞型）和急性巨核细胞白血病，且2例白血病细胞均有染色体畸变或癌基因突变、活化，2例骨髓移植后完全供者源造血重建，但2例复发后白血病细胞均来源于供者源，血型和HLA分型均为供者型，复发后2例诊断为急性淋巴细胞白血病（B细胞型）和急性巨核细胞白血病。这一结果提示，供者源复发可能与移植后应用大剂量免疫抑制剂有关，化疗、放疗以及受者本身造血微环境异常也可能参与了二次白血病的发生过程。异基因造血干细胞移植后供者源复发可能是研究人白血病发生的适宜模式。     

大鼠胚胎期体内诱导免疫耐受对抗移植物排斥反应的研究

目的 探讨大鼠胚胎期诱导免疫耐受能否取代HLA配型而用于造血干细胞移植.方法 妊娠期SD大鼠胚胎卵黄囊内注射宿主Wistar大鼠外周血单个核细胞,诱导免疫耐受,取成熟子代雄性大鼠作为供体,对接受致死量照射后的宿主雌性大鼠进行骨髓移植,以假诱导耐受移植组、单纯照射移植组及单纯照射组为对照,观察大鼠生存期及精神状态、摄食、摄水、皮毛特点、活动等一般情况以及行组织病理检查移植物抗宿主病(GVHD)情况;骨髓移植后第40天,对供体及受体大鼠进行自体皮肤移植及供受鼠相互间皮肤移植,观察皮肤植活情况.结果 接受诱导免疫耐受供体骨髓移植的宿主鼠,生存期明显长于对照组(30 d生存率分别是86.7%、6.7%、0%、0%),病理检查未见GVHD发生.而在假诱导移植耐受组中,则明显可见GVHD的发生.诱导耐受移植组供体及宿主间皮肤移植成活率明显高于非相关鼠皮肤移植(分别为84.6%及0%).结论 胚胎期诱导免疫耐受可跨越HLA屏障,从而为造血干细胞移植和实体器官移植提供良好供者.

Abstract：

Objective To investigate whether the fetal immune tolerance induction could replace the HLA typing for hematopoietic stem cell transplantation. Methods Immune tolerance of SD rats was induced by injecting host Wistar rats peripheral blood mononuclear cells into yolk sac of the embryo, afterward the mature male offsprings were used as donor. The host female receipients received lethal dose irradiation and bone marrow transplantation (BMT). The Wister rats transplanted with bone marrow from donor and unrelated SD rats as well as the rats which received radiation alone were used as control. The survival, histopathologically GVHD, the mental status, food and water intake, coat characteristics, activities were observed. Forty days after BMT, autologous and allogenous skin transplantation between donor and receipient rats was performed to observe the engraftment of solid organ. Results The survival of the rats received bone marrow grafts from the immune tolerant donor was significantly longer than that of control groups ( 30 day survival rates were 86. 7% , 6. 7% , 0% , and 0% respectively), and there was no histopathologically GVHD observed, while in the sham group, the manifestations of GVHD was clearly visible. The skin engraftment rate between the host and the immune tolerant donor was significantly higher than that among non-related rats ( 84. 6% and 0% respectively) . Conclusion The induction of immune tolerance in embryo can overcome the HLA barrier and provide a good donor for hematopoietic stem cell and solid organ transplantation.     

Major histocompatibility restriction of antigen recognition by T cells in a recipient of haplotype mismatched human bone marrow transplantation.

Immune T cells proliferate in response to antigen that is recognized in association with self-Ia determinants. T cells from a patient with severe combined immunodeficiency that has been successfully reconstituted with haplotype-mismatched, maternal bone marrow were studied in an attempt to understand the development of Ia restriction of antigen recognition in man. All the patient's T cells were of maternal origin as determined by HLA typing. The patient received a series of three immunizations with tetanus toxoid (TT) antigen between the 6th and 14th week posttransplant. TT-specific T cell lines were established from the patient's peripheral blood at 6 and 8 mo posttransplantation and were maintained in culture in the presence of irradiated monocytes from the patient, TT antigen, and interleukin-2. HLA typing of the two T cell lines revealed them to be exclusively of donor origin. Both T cell lines could proliferate to TT in the presence of monocytes derived from either the patient's mother or father. In contrast, a TT-specific T cell line obtained from the patient's mother proliferated to TT in the presence of autologous monocytes, but not in the presence of monocytes derived from the patient's father. Studies using monocytes from a panel of HLA-typed donors indicated that the patient's T cell lines proliferated to TT in the presence of monocytes that expressed the paternal DR antigen (HLA-DR4) inherited by the patient but not in the presence of monocytes that expressed the paternal DR antigen (HLA-DR1) not inherited by the patient or in the presence of monocytes bearing irrelevant DR antigens. Monocytes that expressed either one of the two maternal DR antigens (HLA-DR3 and DR5) could support the proliferation of the patient's T cell lines in response to TT antigen. HLA typing of the patient's monocytes at 6 mo post-transplant revealed only recipient HLA-DR antigens (HLA-DR3 and DR4). At 12 mo posttransplant, the patient's monocytes expressed recipient HLA-DR antigens as well as the non-shared HLA-DR5 antigen of donor origin. The results of the present study indicate that T cells of human bone marrow chimera recognized antigen in the context of Ia determinants of recipient origin. The apparent recognition of antigen by the chimera's T cells in the context of donor Ia determinants that were not shared with the recipient is discussed.     

未成熟树突状细胞诱导半相合骨髓移植免疫耐受的实验研究

目的探讨未成熟树突状细胞(imDC)诱导半相合骨髓移植(HBMT)免疫耐受的疗效。方法用雄性SD大鼠及雌性Wistar大鼠建立HBMT动物模型,取供鼠外周血诱导培养imDC,实验组(n=10):接受HBMT+供鼠imDC输注;对照组(n=10)只接受HBMT;观察比较2组受鼠的急性移植物抗宿主病(aGVHD)发生率及其严重程度。结果 1)实验组受鼠的aGVHD发生率为60%(6/10)明显低于对照组的100%(10/10)(P<0.05);2)实验组受鼠的临床aGVHD评分为(2.7±1.0)分,明显低于对照组的(6.9±1.5)分(P<0.05);3)实验组受鼠小肠aGVHD的发生率44.4%(4/9)明显低于对照组的100%(10/10)(P<0.05)。结论 imDC可降低大鼠HBMT后aGVHD的发生率并减轻大鼠HBMT后aGVHD的严重程度。