乙型肝炎病毒抗原诱导T细胞受体Vβ基因亚家族特异性扩增与多样性

目的对乙型肝炎疫苗注射前后及ＨｅｐＧ２２２１５上清ＨＢｓＡｇ、ＨＢｅＡｇ感染淋巴细胞，Ｔ细胞受体（ＴＣＲ）Ｖβ基因亚家族表达特征与多样性，为研究识别机制提供依据。方法采用ＲＴ－ＰＣＲ－Ｓｏｕｔｈｅｒｎ印迹扫描。结果乙肝疫苗注射后Ｖβ６、１４增高，ＨｅｐＧ２２２１５上清感染后Ｖβ６、１５增高。结论Ｖβｂ为两种抗原共同识别亚家族。Ｖβ１４、１５为各自的特异性识别亚家族     

乙肝疫苗免疫前后人TCRVβ基因亚家族取用表达的研究

目的：研究Ｔ细胞受体（ＴＣＲ）Ｖβ基因亚家族在免疫后的表达特征。方法：运用ＲＴ－ＰＣＲ及Ｓｏｕｔｈｅｒｎ　ｂｌｏｔ技术对乙肝疫苗免疫前后健康成年人ＴＣＲ　Ｖβ１－２０基因亚家族的表达水平迸行了半定量研究。结果：Ｖβ６、Ｖβ１４基因亚家族的表达增高；ＶＢ４、ＶＢ９基因亚家族的表达下调。结论：免疫后诱导产生了ＨＢｓＡｇ特异的Ｔ细胞应答，该结果为制定预防和治疗ＨＢＶ感染的免疫策略提供了新的依据。     

T细胞受体Vβ基因在识别HSV—2过程中的表达水平和特点

本课题主要研究Ｔ细胞受体（ＴＣＲ）识别抗原后Ｖβ基因发生重排，ｍＲＮＡ表达水平改变。用紫外线处理的单纯疱疹病毒－２型（ＨＳＶ－２）、ＨＳＶ－２及ＰＨＡ分别感染或刺激正常人的ＰＢＬｓ，培养４～６天后，提取ｍＲＮＡ，并采用ＲＴ－ＰＣＲ、Ｓｏｕｔｈｅｒｎ杂交发现识别ＨＳＶ－２的ＴＣＲＶβ２、６、７、８基因在体外选择性扩增。在采用ＨＳＶ－２攻击皮肤病患者发作期、缓解期以及再发作期的ＰＢＬｓ时，发现ＴＣＲＶβ基因表达随着病程变化而改变，尤其Ｖβ７亚家族表达水平显著高于其它亚家族基因，这显示了ＴＣＲＶβ基因的变化是恒定特异性的。     

广西不同人群庚型肝炎病毒感染状况的调查

目的 了解庚型肝炎病毒（ＨＧＶ）感染在广西柳州地区不同人群中的感染状况，并比较静脉毒瘾者与健康体检者ＨＧＶ，乙型肝炎病毒（ＨＢＶ）、丙型肝炎病毒（ＨＣＶ）与ＨＧＶ共同感染的特点，方法 应用酶联免疫法（ＥＬＡ）检测抗－ＨＧＶ，抗－ＨＣＶ，ＨＢＶＭ（ＨＢｓＡｇ，ＨＢｓＡｂ，ＨＢｅＡｇ，ＨＢｅＡｂ，ＨＢｃＡｂ），并对抗－ＨＧＶ阳性者随机抽取２０例（１９％）进一步用逆转录套式ＰＣＲ（ＰＴ－ｎＰＣＲ）法检测     

通过受体介导法将核酶特异导入肝细胞以阻断HBV蛋白表达的初步研究

应用半乳糖末端糖蛋白受体（ＡＳＧＰ－Ｒ）介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的转染和细胞表面转铁蛋白受体（Ｔｆ－Ｒ）介导的内吞作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｆ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒（ＨＢＶ）ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞并发挥作用，通过酶联免疫吸附法（ＥＬＩＳＡ）检测细胞培养液中的乙型肝炎表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ），评价核酶在细胞水平对ＨＢＶ抗原表达的阻断作用。结果表明当核酶质粒ｐＣＭＶ－Ｒｉｐｃ与ＨＢＶ抗原表达质粒ｐＵＣ－２ＨＢＶ共转染ＨｅｐＧ２细胞时，核酶对ＨＢｓＡｇ和ＨＢｅＡｇ表达的抑制率分别为５５．２９％和６８．７３％。     

高渗盐水输入对低氧条件下失血性休克家兔内分泌功能的影响

本实验采用放免法观察了低氧条件下失血休克前后和输液治疗后的１４项内分泌激素改变。实验结果表明，低氧条件下复合急性失血性休克的血浆中ＡＮＰ，β－ＥＰ，ｋＰＧＦ１　，ＴＸＢ２，ＲＤＮ，ＡⅡ，ＡＬＤ，ＴＴＳＨ，ＧＨ等多种激素水平明显增高，输入高渗盐水或高盐液体后能使ＡＮＰ，β－ＥＰ，ＴＸＢ２，ＲＥＮ，ＡⅡ，ＡＬＤ，Ｔ３等增高的激素水平明显降低，同时使血浆６ｋＰＧＦ１α水平明显增高，也能使降低的ＣＳ和ＡＣ     

抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达

目的 尝试将１株来源于人源性噬菌体抗体库的抗ＨＢｓＡｇ人Ｆａｂ,转换成完整的抗ＨＢｓＡｇ人ＩｇＧ。方法 采用重叠ＰＣＲ方法,在抗ＨＢｓＡｇ人Ｆａｂ和Ｖκ的ＶＨ基因的５’端接上人工合成的人κ链的前导序列,构建表达完整ＩｇＧ１的真核表达载体,转染ＧＨＯ（ＤＨＦＲ＾－）细胞,用ＥＬＩＳＡ、ＲＴ－ＰＣＲ和免疫印迹检测抗ＨＢｓＡｇ人ＩｇＧ的表达。通过ＤＨＦＲ／ＭＴＸ体系及金属离子诱导提高Ｉｇ表达。结果：获得     

人巨细胞病毒感染对肾移植受者血清sIL－2R水平的影响

用ＰＣＲ检测ＨＣＭＶ－ＤＮＡ，ＥＬＩＳＡ法检测ＨＣＭＶ－ＩｇＭ及ＩｇＧ，以诊断肾移植受者ＨＣＭＶ感染。用双抗体夹心法ＥＬＩＳＡ检测６５例肾移植受者血清ｓＩＬ－２Ｒ水平，结果表明：ＨＣＭＶ感染后宿主血清ｓＩＬ－２Ｒ水平明显增高（Ｐ＜０．０１），且ＨＣＭＶ疾病组ｓＩＬ－２Ｒ增高程度大于无症状感染组（Ｐ＜０．０１）；６例原发性ＨＣＭＶ感染者ｓＩＬ－２Ｒ水平与ＩｇＭ水平呈正相关（ｒ＝０．９９０８），提示随感染程度增加，血清ｓＩＬ－２Ｒ水平随之增高，还发现血清ｓＩＬ－２Ｒ水平与Ｃ９４／ＣＤ８比值是负相关（ｒ＝－０．９７８９），说明ＨＣＭｖ感染后ｓＩＬ－２Ｒ水平增高与Ｔ细胞亚群改变有关，反之也说明ｓＩＬ－２Ｒ增高程度可表明体内免疫抑制状态。对于ＨＣＭＶ感染后血清ｓＩＬ－２Ｒ水平增高的机理有待进一步探讨。     

乙型肝炎病毒新的免疫逃避株的S基因序列分析

目的分析乙型肝炎患者体内ＨＢＶＳ基因的变异情况及对乙型肝炎的免疫预防的现实意义。方法采用巢式聚合酶链反应（ＰＣＲ）、Ｍ１３噬菌体克隆和核苷酸序列分析方法对一例乙型肝炎免疫失败儿童患者体内ＨＢＶＳ基因序列进行分析。结果发现该患儿所感染ＨＢＶ的Ｓ基因上有一个重要的点突变,即第５５１位碱基由野生型的Ａ变为Ｇ。该突变使乙型肝炎表面抗原（ＨＢｓＡｇ）１３３位氨基酸由甲硫（ＡＴＧ）变为缬（ＧＴＧ）。这一氨基酸替换恰恰发生在ＨＢｓＡｇ中和性α抗原决定簇区段（ａａ１２４～ａａ１４７）内。结论鉴于该患者接种乙型肝炎疫苗,其血清呈抗－ＨＢｓ阳性和ＨＢｓＡｇ阴性,推测其体内的ＨＢＶ为一个新的疫苗诱导的免疫逃避株     

HepG2和HepG2.2.15细胞对凋亡剌激的耐受性

目的阐明ＨＢＶ对感染细胞凋亡的影响。方法ＨｅｐＧ２及其转染ＨＢＶ的ＨｅｐＧ２２１５细胞培养中,加ＭＴＸ、ＡｃｔＤ或去血清培养,以ＦＣＭ检测细胞凋亡率。结果ＨｅｐＧ２．２．１５细胞加ＭＴＸ后２４小时和４８小时,凋亡率分别为１０８％和１３３％,加ＡｃｔＤ后分别为１６８％和３７７％,去血清后第４天及第６天,分别为１３２％和１４８％;而ＨｅｐＧ２细胞加ＭＴＸ后２４小时和４８小时,凋亡率则为１２６％和６５３％,加ＡｃｔＤ后分别为４４５％和８９７％,去血清后第４天和第６天凋亡率为１９８％和２８８％。结论在上述条件下,ＨｅｐＧ２２１５细胞较ＨｅｐＧ２细胞耐受凋亡;ＨＢＶ可能抑制肝癌细胞凋亡。     

Immunological function of HLA-DQw6 molecules expressed in DQw6 transgenic C57BL/6 mice

HLA-DQw6 transgenic C57BL/6 mice (DQw6-B6) were utilized to define the role of HLA-DQw6 gene product in immune-recognition. To investigate the responsiveness of lymph node cells from C57BL/6 mice (B6) or DQw6-B6 in response to DQw6 molecules expressed in the DQw6-B6, in vitro secondary MLR was performed. The lymph node cells from B6 proliferated in response to spleen cells from DQw6-B6, whereas those from DQw6-B6 did not. These results suggested that DQw6-B6 acquired tolerance to DQw6 molecules. The difference of T cell repertoire between B6 and DQw6-B6 was investigated using mAbs directed against T cell receptor V beta regions, V beta 3, V beta 5, V beta 6, V beta 8, V beta 11 and V alpha 3.2. Although the proportion of V beta 5+ CD8+ T cells and V beta 6+ CD4+ T cells were increased in the B6 anti-DQw6-B6 MLR T cell line, there was no significant difference in the proportion of peripheral T cells expressing each V alpha or V beta region between B6 and DQw6-B6. Both CD4+ and CD8+ long term-cultured T lymphocyte cell lines were generated from lymph node cells of B6 stimulated in vitro by irradiated spleen cells from DQw6-B6. The CD4+ T cell line proliferated in response to spleen cells of DQw6-B6 or L cell transfectant expressing HLA-DQw6 molecules and these responses were completely inhibited by either anti-DQ or anti-CD4 monoclonal antibodies (mAbs) but not by anti I-Ab mAbs. The CD8+ T cell line lysed splenic cells activated by lipopolysaccharide (LPS) from DQw6-B6 spleen cells but not from B6. The CD8+ T cell line also exhibited a cytotoxicity to splenic LPS blast cells from backcross progenies between (DQw6-B6 x DBA1) F1 and DBA1, only when target cells expressed both HLA-DQw6 molecules and H-2b. These observations indicated that recognition of HLA-DQw6 by the CD8+ cytotoxic T cell line was restricted by H-2b. The cytolytic activity of the CD8+ T cell line was inhibited by either anti-CD8 or anti-H-2Db mAbs but not by anti-HLA-DQ nor anti-H-2Kb mAbs. These results show that HLA-DQ transgene products expressed in DQw6-B6 can induce xenogeneic MLR in both CD4+ and CD8+ T cells. The CD4+ T lymphocytes recognize the HLA-DQw6 molecule itself whereas the CD8+ cytotoxic T lymphocytes recognize the HLA-DQw6 gene product in the context of H-2Db.     

Induction of minor histocompatibility antigen-specific T-helper but not T-cytotoxic response is dependent on the source of antigen-presenting cell

We studied the accessory cell requirements for triggering in vivo primed human major histocompatibility complex class I- and class II-restricted T cells specific for minor histocompatibility antigens. We compared the antigen-presenting capacities of peripheral blood lymphocytes (PBLs) and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs), both derived from the same donor, to induce minor histocompatibility antigen-specific cytotoxic and proliferative T cells. PBLs and EBV-LCLs were equally effective as antigen-presenting cells to trigger cytotoxic-T-cell responses specific for minor histocompatibility antigens, some of which were reactive with B cells only. In contrast, a clear difference was found between the capacities of the two antigen-presenting cell types to induce minor histocompatibility antigen-specific T-helper-cell responses. PBLs as antigen-presenting cells could induce T-helper-cell lines reactive against minor histocompatibility antigens presented on PBLs, on EBV-LCLs, or on both cell types as stimulator cells. Unexpectedly, however, EBV-LCLs as antigen-presenting cells in all instances failed to induce T-helper-cell responses specific for minor histocompatibility antigens presented on PBLs or on both PBLs and EBV-LCLs as stimulator cells and could only trigger T-helper cells directed against B-cell-specific minor histocompatibility antigens. Our findings indicate a dichotomy in the capacity of EBV-LCLs to present minor histocompatibility antigens in the induction versus the effector phse of the in vitro T-helper-cell response. Furthermore, the results show different in vitro accessory cell requirements for major histocompatibility complex class I- and class II-restricted T-cell responses specific for human minor histocompatibility antigens.     

Self-nonself discrimination and repertoire selection of human T cells differentiated in an HLA-semiallogeneic environment following bone marrow transplantation for severe combined immunodeficiency.

We have analyzed allorecognition, HLA restriction and T cell receptor (TcR) diversity in an HLA-heterozygous (HLA-DRw6,7) severe combined immunodeficiency (SCID) patient whose T cell system had been repopulated by HLA-homozygous (HLA-DRw6) paternal T cells following T cell-depleted bone marrow transplantation (BMT). Donor origin of T cells and host origin of antigen-presenting cells (APC) in peripheral blood and BM is shown by HLA typing of separated cell populations and two-color immunofluorescence using an anti-HLA monoclonal antibody (mAb). Peripheral blood lymphocytes (PBL) from the chimeric patient proliferate normally against PHA, anti-TcR/CD3 mAb, pooled allogeneic PBL, and also against the recall antigen (Ag) tetanus toxoid and purified protein derivative of tuberculin (PPD) following immunization, suggesting recognition by donor (DRw6) T cells of Ag presented by host (DRw6,7) APC. PPD-specific cytotoxic T lymphocytes generated in vitro from patient PBL post-BMT display specific cytotoxicity against targets expressing DRw6 and DR7, but not against DR-mismatched targets, suggesting that HLA restriction of Ag recognition may occur through determinants expressed by the host and not by the donor. Donor T cells differentiated in the HLA-semiallogeneic host show specific proliferative and cytotoxic responses against HLA-mismatched stimulators, but not against stimulators taken from the host, expressing the host-specific HLA-haplotype, or expressing the host-specific HLA-DR7 antigens. Compared to T cells directly taken from the donor, differentiation of donor T cells in the host is associated with a significant decrease of T cells expressing TcR V beta 5 and V alpha 2 determinants, while no differences in the abundance of of TcR V beta 6, V beta 8 and V beta 12 subsets were noticed. We conclude that allorecognition, major histocompatibility complex (MHC) restriction and TcR diversity generation of human T cells can be modulated through differentiation in an MHC-different environment, as had been previously shown to be the case in murine model systems.     

T cell reactivity to human insulinoma cell line (CM) antigens in patients with type 1 diabetes.

Autoimmune (type 1) diabetes mellitus results from a progressive destruction of insulin secreting beta cells operated by T lymphocytes in pancreatic islets. Circulating autoreactive T cells to specific beta cell antigens are detected in patients with type 1 diabetes. To date, several beta cell autoantigens have been identified in this disease (GAD, IA-2, 38kD secretory protein, insulin, ICA69 etc.), however, it is possible that also other unidentified self molecules contribute to trigger beta cell autoimmunity. In this study we used the human insulinoma cell line CM as source of beta cell antigens to detect reactive T lymphocytes in patients with type 1 diabetes mellitus. This cell line has been previously shown to express a number of recognized beta cell antigens. Since the expression of several beta cell antigens is affected by glucose stimulation we tested two preparations of CM cells cultured under different conditions containing low (0.8 mM) and high glucose concentration (11 mM). T cell proliferation was measured using cells from 32 patients with type 1 diabetes (19 of recent onset and 13 at 3 to 22 months from diagnosis) and 27 age-matched control subjects. A significant increase in T cell proliferation to CM cells grown in high glucose conditions (11 mM) (p < 0.05) was found in type 1 diabetic patients compared to controls. No significant differences were observed when using CM cells cultured at the low glucose concentration. Furthermore, the response to both extracts of CM cells was independent of disease duration (p = 0.6 for both CM cells cultured at 0.8 and 11 mM glucose). These data indicate that T cell reactivity to homogenates of CM cells is detectable in patients with type 1 diabetes and suggest that this human insulinoma cell line is an interesting potential source of beta cell material for immunological studies of autoimmune diabetes.     

Characterization of a monoclonal anti-beta 2-microglobulin antibody and its use in the genetic and biochemical analysis of major histocompatibility antigens.

A monoclonal anti-beta 2-microglobulin (BBM.1 antibody) was produced by cell fusion between the mouse myeloma, P3-X63-Ag8, and spleen cells from a BALB/c mouse immunized with Molt 4, a human T cell line. BBM.1 antibody was fully inhibited by soluble beta 2-microglobulin and purified HLA-A, B antigens and reacted with human-mouse somatic cell hybrids only if they had chromosome 15 and expressed human beta 2-microglobulin. It was cytotoxic in complement-dependent lysis and of the IgG class. BBM.1 and a monoclonal anti-HLA-A, B, C glycoprotein antibody, W6/32 (Barnstable, C. J. et al., Cell 1978. 14:9.), were used to quantitate relative amounts of beta 2-microglobulin and HLA-A, B, C glycoproteins on different human cell types. Thymocytes and the Molt 4 cell line showed a considerable excess of beta 2-microglobulin over HLA-A, B, C glycoproteins, as measured by W6/32 reactivity. B cell lines, peripheral blood lymphocytes, fibroblasts, a HeLa cell derivative, and HSB2, another T cell line, had equal amounts. Immunological cross-reactions between HLA-A, B, C antigens and beta 2-microglobulin and their homologues in other species were detected with the BBM.1 and W6/32 antibodies. The W6/32 antigenic determinant appears to be more highly conserved than that recognized by the BBM.1 antibody.     

肝细胞提取物生物学活性研究

为深入研究肝细胞提取物的生物学活性,以正常人肝细胞株、肝癌细胞株、肝星状细胞株及原代培养的肝星状细胞为研究靶细胞,观察在不同浓度肝细胞提取物作用下,上述细胞的增殖活性(MTT法)、细胞凋亡活性、细胞外基质成分透明质酸(HA)产生水平及I型胶原基因启动子活性的影响,同时利用流式细胞术比较正常成人外周血单个核细胞(PBMC)在肝细胞提取物作用下,活性T淋巴细胞(CD4+/CD69+)的变化。结果发现肝细胞提取物对正常人肝细胞系(张氏肝细胞)具有明确刺激增殖作用,而对原代培养的肝星状细胞具有抑制增殖作用;肝星状细胞株HSC-T6的研究中,肝细胞提取物具有抑制其细胞外基质成分HA产生、抑制α1(I)型胶原基因启动转录的活性。60μg/ml的肝细胞提取物对肝癌细胞株Hep G2具有明确的诱导凋亡作用。正常成人PBMC经肝细胞提取物作用后活性T淋巴细胞(CD4+/CD69+)数量显著上升。研究表明,肝细胞提取物具有促进肝细胞再生、抑制肝纤维化发生和抗肿瘤作用,并首次发现具有免疫调节作用。     

HSV-2感染人外周血淋巴细胞后TCRVβ基因片段的选择性扩增

T细胞受体（TCR）Vβ基因 1—20亚家族在识别和杀伤病毒及肿瘤抗原方面各自有着重要的作用。本研究采用单纯性痢疾病毒（HSV-2）感染正常人的外用血淋巴细胞（PBLs）后发现TCR Vβ2,6,7,8基因在体外选择性扩增,而用 HSV-2攻击生殖器单纯疱疹性皮肤病患者不同病程时期的 PBLs时发现 TCRVβ基因的表达水平随病程不同而改变。结果提示Vβ7,8在识别HSV-2时呈选择性扩增而限制 HSV-2的增殖。     

Neonatal tolerance to MIs-1a determinants: deletion or anergy of V beta 6+ T lymphocytes depending upon MHC compatibility of neonatally injected cells.

Recent investigations in mice revealed that natural immunological tolerance to endogenous minor lymphocyte-stimulating locus 1a (MIs-1a) antigen correlates primarily with deletion of MIs-1a-specific V beta 6+ T lymphocytes in the thymus. Similar mechanisms account for acquired tolerance in some instances since the neonatal injection of MIs-1a-expressing MHC compatible cells in neonatal mice within the first 24 h of life causes clonal deletion of V beta 6+ T cells. Here we demonstrate that V beta 6+ T cells are not deleted in mice neonatally treated with MIs-1a spleen cells expressing allogeneic H-2 molecules. However, when such non-deleted V beta 6+ T cells were tested in vitro, no interleukin 2 (IL-2) secretion or proliferation was observed after MIs-1a stimulation. This non-responsive state could be overcome by addition of exogenous IL-2, consistent with the fact that V beta 6+ cells enlarged and expressed IL-2 receptors upon MIs-1a stimulation. Furthermore, the same neonatally treated mice showed in vitro functional unresponsiveness of cytotoxic T cells but not of IL-2-secreting cells specific for the tolerated allogeneic MHC antigens. Taken together, our data indicate that neonatal tolerance to MIs-1a can be accomplished by either clonal deletion or clonal anergy, and that it does not necessarily correlate with tolerance to MHC determinants.     

Cell components required for deletion of an autoreactive T cell repertoire

T cells become tolerant to self antigens during their development in the thymus. Clonal deletion of thymocytes bearing T cell receptor (TcR) which recognize self antigens is a major mechanism for generating tolerance. In the present study we have used allogeneic bone marrow (BM) chimeras, prepared with various combinations of mouse strains and focusing especially on expressions of I-E molecules and Mls-1a antigens on the cell surface, to investigate both immunohistochemically and by flow cytometry the cell components that contribute to the clonal deletion of T cells positive for V beta 6 TcR. The V beta 6 TcR expression is strongly associated with T cell recognition of both I-E and Mls-1a antigens. We found that I-E+ cells derived from donor BM (and thus not of recipient lineage) represented a primary requirement for deletion of Mls-1a-reactive thymocytes which bear V beta 6 TcR. Immunohistochemical analysis revealed that the donor-derived I-E+ cells were distributed mainly to the thymic medulla and that the V beta 6+ cells were eliminated from the thymic medulla between 2 and 3 weeks following BM transplantation. In contrast, Mls-1a+ cells of either donor or recipient origin might be responsible for the deletion, even though cortical epithelial cells appeared not to express Mls-1a antigens.