负载肝癌抗原的人树突状细胞生物学特性的研究

目的　探讨负载肝癌抗原的树突状细胞 (DC)对自身CIK细胞的诱导增殖能力和杀伤能力 .方法　从人外周血分离获得单核细胞 ,体外经重组细胞因子 (GM -CSF)、白细胞介素 - 4 (IL - 4 )、肿瘤坏死因子α(TNF -α)培养获得DC ,电镜观察其形态 ,流式细胞仪检测其表面抗原 ,混合淋巴细胞反应检测负载肝癌抗原DC诱导CIK细胞增殖活性 ,检测激活的CIK对肝癌细胞的杀伤作用 .结果　经体外培养 ,从外周血分离获得的大量成熟DC ,检测表明高表达DC的抗原标志 ,负载抗原的DC具有很强的激发同种CIK增殖的能力 ,激活的CIK对肝癌细胞系具有很强的杀伤作用.结论　实验结果为肝癌的临床免疫治疗提供了理论基础 .     

树突状细胞负载肝癌抗原肽疫苗体外诱导特异性免疫学反应的研究

目的：研究树突状细胞(dendritic cell，DC)负载肝癌抗原肽EPVTKAEML体外诱导特异性CTL的能力及其抑癌效果。方法：用顺序特异引物聚合酶链反应技术(PCR—SSP)选择HLA—B7表型供者，从脾组织中分离、培养DC-EPVTKAEML特异性CTL。用^51Cr释放法检测CTL的杀伤活性，并用抗HLA-1分子单抗(mAb)进行杀伤抑制实验。结果：找到4例HLA-B7杂合子供者，用DC负载HLA-B7限制的抗原肽EPVTKAEML可诱导特异性CTL反应，对肝癌细胞HHCC有较强的杀伤作用。结论：DC负载抗原肽EPVTKAEML在体外可诱发较强的特异性免疫反应。     

肝癌细胞冻融抗原负载树突状细胞瘤苗的制备与活化

目的: 探讨制备肝癌树突状细胞(DCs)瘤苗和体外诱导DCs活化的优化方法。方法: 取健康人新鲜血50mL, Ficoll密度梯度离心分离外周血单个核细胞(PBMC), 制备肝癌细胞冻融抗原, 采用不同因子组合培养PBMC, 诱导和激活DCs, A组: rhGM-CSF+IL-4; B组: rhGM-CSF+IL-4+冻融抗原负载;C组:rhGM-CSF+IL-4+TNF-α; D组: rhGM-CSF+IL-4+TNF-α+冻融抗原负载。结果: 各组均诱导出DCs, 并高表达CD11c和CD54, 冻融抗原可明显上调CD86、CD80、HLA-DR表达和IL-12 p40分泌(P<0.01), TNF-α促进CD86表达的作用更显著, 但对IL-12分泌无影响, 依次用TNF-α诱导和冻融抗原负载可进一步促进CD86表达和IL12 p40分泌(P<0.05)。CD54和HLA-DR双标记免疫细胞化学染色显示, 各组DCs大多为CD54⁺。表达HLA-DR的DCs均为CD54⁺HLA-DR⁺, 其中, A组CD54⁺HLA-DR⁺细胞数量最少, D组最多, 平均每个细胞HLA-DR的表达水平也与此对应。结论: 采用rhGM-CSF、IL-4诱导的未成熟DCs, 依次使用TNF-α刺激与肝癌细胞冻融抗原修饰可有效促进DCs的活化与成熟。     

人外周血树突状细胞的研究现状

人外周血树突状细胞(DC)是一种独特的细胞,其形态、组织化学、免疫细胞化学反应和超微结构均与单核细胞不同。无吞噬能力,也无单核细胞、淋巴细胞和NK细胞所特有的标志。它是T细胞免疫反应的主要辅助细胞。并参与多种自身免疫疾病、免疫缺陷病、肿瘤和某些炎症反应的病理过程。观察外周血中DC的质和量的变化,将为了解这些疾病的发生、发展和预后提供有价值的参数。     

负载不同形式肝癌抗原的树突状细胞抑瘤功能的比较

目的：探讨不同形式的肝癌抗原修饰的树突状细胞(DC)的抑瘤功能。方法：分别用肝癌H22冻融抗原、H22小分子抗原肽和Hsp70-H22抗原肽复合物修饰DC；用MTT比色法分析DC激活的CTL对H22细胞的杀伤能力，并用RT-PCR法测定脾脏T细胞中IFN-γ mRNA的表达水平；用不同修饰的DC免疫小鼠，观察其对H22肝癌的生长抑制作用。结果：单独的H22肝癌抗原肽修饰的DC不能激活CTL。Hsp70-H22肽复合物修饰的DC激活CTL的能力强于H22肝癌冻融抗原修饰的DC，对H22细胞的杀伤率分别为47．3％和18．3％。各组T细胞中IFN-γ表达水平的变化与杀伤率的变化相一致。用H22肝癌冻融抗原和Hsp70-H22肽复合物修饰的DC免疫小鼠后，均可抑制H22细胞生长，但后者的抑制作用更强，成瘤率仅40％。其他各组的成瘤率均为100％。结论：Hsp70-H22肽复合物是一种DC的强致敏物．可通过激活CTL、诱导CD4^ T细胞分化成Th1型细胞而参与肝癌的免疫排斥。     

树突状细胞负载肺癌细胞抗原的方法研究

目的:建立自体热休克凋亡肺癌细胞抗原制备、树突状细胞(DC)体外诱导、以及抗原负载方法, 为制备DC肿瘤疫苗提供技术基础.方法:采用酶消化法从手术切除的肺癌新鲜组织获得单细胞悬液, 热休克后用白桦脂酸诱导其凋亡制备成细胞抗原;采用血细胞分离机采集外周血单个核细胞(PBMC), 贴壁法获得单核细胞, 经GM-CSF与IL- 4体外诱导成未成熟树突状细胞(imDC);负载细胞抗原后制备成DC肿瘤疫苗, 并对DC疫苗的形态、数量及表型特征进行分析.结果:(1)肿瘤细胞抗原得率:(13.68±1.30)×106/g组织;平均凋亡率:(93.79±2.31)%;(2)imDC平均得率为(9.37±0.83)×106/(118.77±12.56)×106个PBMC, 活率>95%;imDC表型分析:CD11c CD14-、 CD11c HLA-DR 、 CD11c CD80 、 CD11c CD83 、 CD11c CD86 表达率分别为(87.45±3.42)%、 (87.16±1.08)%、 (2.80±1.52)%、 (5.64±1.56)%、 (5.11±1.09)%;(3)DC疫苗平均得率为(5.75±0.69)×106/(9.37±0.83)×106个imDC, 活率>95%, DC疫苗表型:CD11c CD14-、 CD11c HLA-DR 、 CD11c CD80 、 CD11c CD83 、 CD11c CD86 表达率分别为(92.73±1.21)%、 (89.35±2.31)%、 (86.80±1.23)%、 (69.53±7.21)%、 (94.92±1.48)%. 结论:该方法稳定、安全、可靠, 可制备出具有成熟DC表型的肿瘤疫苗.     

人外周血树突状细胞对LAK细胞杀伤活性的影响

从人外周血中分离出树突状细胞，体外观察了其对ＬＡＫ细胞活性的影响。发现：５×１０２～１×１０４／ｍｌ树突状细胞对ＬＡＫ细胞活性起增强作用，而５×１０４～１×１０５／ｍｌ树突状细胞却抑制ＬＡＫ杀伤活性。这说明人外周血树突状细胞对ＬＡＫ细胞活性起双相性调节作用。     

树突状细胞负载肝癌可溶性抗原后的免疫应答

目的 用负载肝癌可溶性抗原的DC诱导肝癌特异性T细胞。方法 在体外用GM－CSF和IL－4诱导健康人外周血单核细胞，使其分化为高纯度树突状细胞（DC）。用负载人肝癌细胞株SMMC－7721可溶性抗原的DC诱导自身淋巴细胞。结果 诱导后淋巴细胞增殖指数大于1．5，表面CD56分子表达下降，CD3^ T/CD4^ T和CD3^ T/CD8^ T细胞比例增加，以CD3^ T/CD4^ T细胞比例增加最为明显。CD4／CD8比例由诱导前的0．84增加为1．04，活化前后γδ比例没有改变。活化后的淋巴细胞不但可杀伤SMMC－7721细胞，同时还可不同程度的杀伤其它3株肝癌细胞。另外，诱导7d的DC可不同程度的抑制4株肝癌细胞和胃癌细胞。结论 实验结果为肝癌DC疫苗的研究提供了理论基础。     

人外周血树突状细胞的分离与鉴定

应用细胞分层剂密度梯度离心辅以细胞贴壁和花环沉降法，分离和纯化人外周血树突状细胞，通过Ｅｔ花环形成细胞测定，荧光染色镜检和ＦＡＣＳ分析表明，分离的细胞纯度可达４０％￣６０％，残留的其他细胞多为ＮＫ细胞，单核细胞和Ｔ细胞，ＦＡＣＳ分析还显示：所分离的细胞表面ＭＨＣⅡ类抗原的阳性率达９７．３４％，扫描电镜观察可见独特的细胞形态，将此类细胞加入体外淋巴细胞增殖反应体系则呈现明显促进作用，提示：分离的细胞     

甲氧基聚乙二醇修饰人红细胞血型抗原的研究

红细胞输注是输血医学的主体，化学材料甲氧基聚乙二醇（mPEG）与红细胞膜共价结合后，可遮蔽红细胞表面血型抗原，使红细胞血型抗原呈阴性，即形成通用型红细胞，对临床输血具有重要意义。研究了mPEG修饰对红细胞血型抗原、膜流动性的影响；红细胞被修饰程度；mPEG与红细胞结合的稳定性；mPEG在体内的代谢情况。结果表明，mPEG能遮蔽红细胞上的血型抗原；mPEG与红细胞结合稳定；mPEG修饰红细胞的比率随浓度增高而增大；mPEG在小鼠体内24h后基本被代谢，且在体内无蓄积。mPEG修饰的红细胞为解决临床急用稀有血型和配型困难（如自身溶血性贫血，AIHA）等问题提供了有意义的参考。     

Further Evidence for HL-A Antigens Present on Adult Peripheral Red Blood Cells

Two antigens of the HL–A system, W17 and W28, are demonstrated on adult peripheral red blood cells. Their implications for blood group serology are discussed.     

Generation of Dendritic Cells from Peripheral Blood Adherent Cells in Medium with Human Serum

Dendritic cells (DC) provide an effective pathway for presenting antigens to T cells, both self-antigens during T-cell development and foreign antigens during immunity. As such, these cells may be promising adjuvants for immunotherapy. Thus, it is important to establish simple and fast method(s) to generate sufficient numbers of human DC in medium free of calf serum so that the cells can be used for both experimental and clinical purposes. In this report, we used peripheral blood adherent cells, without laborious cell purification or depletion, as the starting population and cultured them in medium supplemented with granulocyte/macrophage colony-stimulating factor and interleukin-4. Substantial numbers of cells with the phenotypical and functional characteristics of immature DC were obtained in a 7-day culture. We then compared DC cultured in medium supplemented with either fetal calf serum or pooled human ABRh⁺ serum and found no difference in cell yields and in their ability to stimulate alloreactive T cells or to present soluble antigens to T cells. Irradiated cells were less efficient than non-irradiated cells in antigen presentation and stimulation of T cells. Finally, we have examined DC with or without additional tumour necrosis factor-α treatment and found that antigen-pulsed mature cells could as efficiently present antigen to T cells as did immature cells. This method is suitable for the generation of DC in studies of large clinical materials.     

Hepatitis B Virus Antigens in Peripheral Blood Mononuclear Cells during the Course of Viral Infection

We studied the expression of surface (HBsAg) and core (HBcAg) proteins of hepatitis B virus (HBV) on the surface of peripheral blood mononuclear cells (PBMC) from HBV-infected patients. A total of 122 patients with different liver viral diseases was analyzed by indirect immunofluorescence with monoclonal antibodies. The 35 patients with HBV chronic active hepatitis (CAH) and 38 of 60 patients with acute hepatitis B (63%) expressed HBsAg on the PBMC. No expression was detected on the cells from both normal and HBV-unrelated viral hepatitis control groups. Serial follow-up of patients with acute hepatitis B showed that HBsAg expression by PBMC tended to be undetectable 4 months after the onset of the disease and at the same time the clinical improvement was evident. Cell cultures of EBV-transformed B lymphocytes were established from PBMC of HBV-infected patients; immunoelectron microscopy demonstrated the HBsAg on the cellular membrane. One-third of HBV-infected patients who were studied showed the expression of HBcAg by PBMC. HBcAg was detected in patients with acute hepatitis B at the early stage of infection. The cells of these patients also expressed HBsAg in PBMC. In CAH patients, a positive association was observed between the expression of HBcAg and the presence of serum HBeAg.     

人血管平滑肌细胞种植构建工程生物血管的实验研究

了解猪血管去细胞后平滑肌细胞种植情况,为猪血管用于血管组织工程提供资料。取猪颈动脉,生物酶预处理猪血管,在自行设计制作的新型动力性生物反应器中,用原代培养的人平滑肌细胞种植在去细胞血管基质材料内,HE染色及银染检测平滑肌细胞种植效果。结果表明生物酶预处理血管后,HE染色及银染检测可见血管腔平滑肌细胞形态正常,沿血管长轴分布,提示经生物酶预处理的猪血管人平滑肌细胞能成功种植,可望构建实用的组织工程血管。     

Distribution of HL-A Antigens on Blood Cells

The presence of HL—A antigens on various blood cells was determined by absorption techniques with the absorbed antisera being tested in the lymphocyte cytotoxicity test for loss of antibody activity.
To overcome the risk of contamination by lymphocytes, methods for the preparation of pure cell suspensions of platelets, granulocytes and erythrocytes were evolved, giving high yields of cells.
The following antigens were detected on platelets and granulocytes, but not on erythrocytes either fresh or papainized: HL—A1, HL—A2, HL—A3, HL—A9, HL—A10 and HL—A11 of the first series, and HL—A5, HL—A7, HL—A8, HL—A12, HL—A13, W5, W14, W15, W17, W18, W22, W27 and possibly W10 of the second series.
The distribution of antigens on the various blood cells using cells from the same donor was also investigated, and it was found that the same HL—A antigens were present on platelets, granulocytes and lymphocytes.     

Epstein Barr Virus Transformation of Peripheral Blood B Cells Secreting Antibodies Reactive with Cell Surface Antigens

The natural structuring of the immune system is responsible for the functional physiological state of the body. The development of natural autoantibodies involved in homeostasis relies on the ability to distinguish between exposed/masked and altered/non-altered self antigens. The objectives of this article were to address the relationships between antigen and autoantibodies against serum amyloid A (SAA), define SAA protein concentrations in 219 blood donor (BD) sera and determine their autoantibody levels and search for possible clinical associations with autoimmune and thrombotic diseases. Just recently, an increasing number of reports have indicated significantly decreased levels of autoantibodies against pro-inflammatory molecules, such as anti-TNF-alpha, anti-IL-6, or anti-CRP found in diseased conditions, as compared to healthy donors, or even to less severe disease conditions. In accord with this line of thought, our data indicate a predominant presence of anti-SAA autoantibodies in healthy BDs (above 95% as tested by the immunoblot analysis, n = 41). Using ELISA, high levels of anti-SAA antibodies were confirmed with a median OD = 0.996 for the BD group (n = 219). This suggests that anti-SAA antibodies might have a physiological role in homeostasis and/or the innate immune system and could actually be a part of the natural antibody repertoire. Significantly, lower median levels were found in patients with arterial thrombosis. Based on 219 BD sera, we could establish a new median value of 20 μg/ml for SAA antigen and a cut-off value of 114.7 μg/ml (97.5th percentile). Significantly, higher concentrations of SAA were observed for antiphospholipid syndrome, rheumatoid arthritic, and SLE patients.     

Expression of Human Placental Cell Surface Antigens on Peripheral Blood Lymphocytes and Lymphoblastoid Cell Lines

The expression of human placental cell surface antigens was examined in cells of lymphoid origin, including peripheral blood lymphocytes and cultured lymphoblastoid cells of bone marrow or thymus derivation. A select group of this. defined set of surface antigens was detected on all three cell preparations. The most remarkable observation was the conspicuous absence of three subunits previously demonstrated to be present on all human cell surfaces examined to date. Antiserum directed against several placental components prevents adhesion and spreading of cell which grow attached to surfaces. These results suggest a role for these three glycoproteins in mediating cellular adhesion.     

人外周血树状突细胞在体外对LAK杀伤活性的影响

本文采用三因素不同水平的析因设计，研究了人外周血树状突细胞在体外对ＬＡＫ抗肿瘤作用的影响。结果显示，３种浓度的树状突细胞均能增强ＬＡＫ的杀伤活性（Ｐ＜０．００１），以中等浓度的树状突细胞作用最为明显。加入ＩＬ－２后，树状突细胞对ＬＡＫ抗肿瘤活性的增强程度进一步提高。