B7瘤苗与DC瘤苗诱导的抗L615白血病的免疫作用

目的 研究Ｂ７瘤苗的负载了肿瘤细胞膜抗原的ＤＣ瘤苗在小鼠Ｌ６１５白血病模型中诱导抗白血病免疫的作用。方法 通过观察小鼠的存活率和生存时间,研究两种瘤苗在动物体内诱导免疫预防保护的能力;通过体外细胞毒实验和细胞增殖实验,检测两种瘤苗在体外诱导的Ｔ细胞特异性杀伤活性及对特异性抗原刺激的增殖活性。结果 在体内,两种瘤苗免疫的小鼠对随后活瘤细胞的攻击均有保护作用,ＤＣ瘤苗效果更佳;同时还发现输注免疫保护鼠     

双顺反子人B7—1、IFN—γ共表达载体修饰的卵巢癌瘤苗免疫反应的研究

目的：研究B7－1、IFN－γ双基因修饰的卵巢癌瘤苗的免疫效果。方法：构建了双顺反子人B7－1、IFN－γ共表达逆转录病毒载体，用其修饰果癌原代细胞，制备成肿瘤疫苗，体外刺激自体淋巴细胞，以MTT法进行杀伤抑制试验。对SCID小鼠进行人免疫重建，于皮下接种双基因修饰的卵巢上皮癌细胞系3AO，观察肿瘤的皮瘤情况。结果：体外试验证明人B7－1、IFN－γ基因修饰的卵巢癌疫苗可诱导肿瘤特异性杀伤活性。体内试验观察到双基因修饰的卵巢癌细胞系成瘤性下降。结论：B7－1、IFN－γ双基因修饰的肿瘤细胞可诱导很强抗肿瘤免疫反应，为联合免疫基因修饰的卵巢癌瘤苗的制备提供了一定的借鉴。     

细胞因子对白血病细胞B7-1基因表达及诱导外周血T细胞活化的影响

目的：观察γ干扰素（ＩＦＮγ）、白细胞介素（ＩＬ）１０、ＩＬ１２对转染Ｂ７１白血病细胞Ｂ７１基因表达及其诱导健康人外周血单个核细胞（ＰＢＭＣ）产生ＩＦＮγ、ＩＬ２的影响。方法：用逆转录聚合酶链反应（ＲＴＰＣＲ）和酶联免疫吸附反应（ＥＬＩＳＡ）方法检测ＩＦＮγ、ＩＬ２基因表达和蛋白质合成。结果：发现ＩＦＮγ、ＩＬ１２增强ＰＢＭＣ产生ＩＬ２、ＩＦＮγ，ＩＬ１０抑制其产生，同时发现ＩＦＮγ、ＩＬ１２增强转染Ｂ７１白血病细胞Ｂ７１基因表达，ＩＬ１０抑制其表达。结论：细胞因子ＩＦＮγ、ＩＬ１０、ＩＬ１２影响转染Ｂ７１白血病细胞诱导ＰＢＭＣ产生ＩＦＮγ、ＩＬ２，其可能是通过影响Ｂ７１基因表达实现的     

石腊包埋癌组织瘤苗（PETV）对荷瘤615小鼠的治疗作用

应用石蜡包埋的Ｃａ７６１－ＦＰ８／Ｌ小鼠移植性乳腺癌组织制备的瘤苗，对荷瘤（Ｃａ７６１－ＦＰ８／Ｌ）的６１５近交系小鼠的治疗作用进行了研究，结果显示， ＰＥＴＶ治疗后小鼠的存活期明显延长，肿瘤的重量明显小于对照组，肺转移率也较对照组降低，应用ＰＥＴＶ配合选择性去除Ｔｓ细胞的小剂量ＣＴＸ增强免疫应答反应的细胞因子ＩＮＦａ－２ｂ的可以进一步提高疗效。作者认为，ＰＥＴＶ应用范围广、可长期保存抗原等优点，     

负载肝癌肿瘤抗原的B淋巴细胞诱导特异性抗肿瘤免疫的实验研究

目的:探讨体外经小鼠肝癌细胞不同抗原致敏的CD40配体活化的B淋巴细胞诱导的特异性细胞毒T淋巴细胞(CTL)抗肿瘤免疫的能力.方法:分离、纯化T、B混合淋巴细胞,并在CD40L、rmIL-4联合作用下培养.然后分离T、B淋巴细胞以备用.将凋亡的肝癌细胞及其冻融裂解物作为实验组,1640培养基作为空白对照组分别与B淋巴细胞共同培养,检测培养后各组B细胞表面抗原呈递细胞标记(CD40、CD80、CD86)及主要组织相容性抗原的表达情况.利用混合淋巴细胞增殖实验检测T细胞增殖情况.以负载肿瘤抗原的B淋巴细胞诱导的CTL作为效应细胞,肝癌细胞Hepal-6为靶细胞,LDH释放实验检测杀伤活性.结果:负载肿瘤抗原的B淋巴细胞具有刺激T细胞增殖的能力,实验组的B淋巴细胞,其组织相容性分子及其刺激分子表达明显高于空白对照组,其对靶细胞的杀伤率与空白对照组比较经统计学分析差异有显著性.结论:负载肝癌肿瘤抗原的B淋巴细胞作为抗原呈递细胞诱导的CTL可有效产生特异性抗肝癌免疫作用.     

白细胞介素2基因和白细胞介素3基因共转染白血病瘤苗的抗白血病作用的实验研究

目的：研究白细胞介素２（ＩＬ－２）和白细胞介素３（ＩＬ－３）基因共转染的白血病细胞瘤苗对白血病小鼠的治疗效果。方法：用ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒（Ａｄ－ＩＬ－３）转染红白血病细胞株ＦＢＬ－３，６０Ｃｏ照射后制备成瘤苗对实验性白血病小鼠进行治疗，观察肿瘤生长，小鼠生存期及治疗后小鼠腹腔巨噬细胞、ＮＫ细胞、诱导杀伤性Ｔ淋巴细胞（ＣＴＬ）杀伤活性，并与低剂量环磷酰胺（ＣＴＸ）合用，观察其抗肿瘤作用。结果：用ＩＬ－２或ＩＬ－３基因转染瘤苗治疗的白血病小鼠肿瘤生长明显减缓，生存期显著延长（Ｐ＜０．０５），用联合转染ＩＬ－２和ＩＬ－３基因的瘤苗治疗较单独转染ＩＬ－２或ＩＬ－３基因的瘤苗治疗效果更佳，与低剂量ＣＴＸ合用后抗肿瘤效果最佳。治疗后小鼠腹腔巨噬细胞、ＮＫ细胞、ＣＴＬ细胞杀伤活性显著增强。结论：ＩＬ－２、ＩＬ－３基因转染的瘤苗能够有效地通过诱导机体抗肿瘤免疫功能而发挥抗肿瘤作用，与低剂量ＣＴＸ合用后，抗肿瘤效果更佳。     

双顺反子人B7-1、IFN-γ共表达载体修饰的卵巢癌瘤苗免疫反应的研究

目的 :研究B7 1、IFN γ双基因修饰的卵巢癌瘤苗的免疫效果。方法 :构建了双顺反子人B7 1、IFN γ共表达逆转录病毒载体 ,用其修饰卵巢癌原代细胞 ,制备成肿瘤疫苗 ,体外刺激自体淋巴细胞 ,以MTT法进行杀伤抑制试验。对SCID小鼠进行人免疫重建 ,于皮下接种双基因修饰的卵巢上皮癌细胞系 3AO ,观察肿瘤的成瘤情况。结果 :体外试验证明人B7 1、IFN γ基因修饰的卵巢癌疫苗可诱导肿瘤特异性杀伤活性。体内试验观察到双基因修饰的卵巢癌细胞系成瘤性下降。结论 :B7 1、IFN γ双基因修饰的肿瘤细胞可诱导很强抗肿瘤免疫反应 ,为联合免疫基因修饰的卵巢癌瘤苗的制备提供了一定的借鉴。     

高迁移率族蛋白B1对人T淋巴细胞免疫功能影响的体外研究

目的观察高迁移率族蛋白B1（HMGB1）对健康人T淋巴细胞增殖的影响，并对其机制进行初步探讨。方法分离健康人外周血单个核细胞（PBMCs），调整细胞浓度后接种于细胞培养板并加入重组人高迁移率族蛋白B1（rhHMGB1）进行刺激。以四甲基偶氮唑盐微量Ⅱ酶反应比色法（MTT）检测细胞数量和细胞活性，观察HMGB1对T淋巴细胞增殖活性的影响。采用四色流式细胞术（FCM）分析CD3^＋淋巴细胞CD4表达。细胞中自细胞介素-2（IL-2）、IL-2α受体（IL-2Rα）基因表达水平采用逆转录一聚合酶链反应（RT—PCR）分析。结果①500～1000μg／L rhHMGB1作用48h后T淋巴细胞增殖反应显著抑制，低于这一剂量对其增殖活性影响不显著。②不同rhHMGB刺激时间和作用剂量对CD4^＋T淋巴细胞未造成明显改变，但rhHMGB1能时间-剂量依赖性增加Th2亚群比例，并因此降低Th1／Th2比值，刺激后T淋巴细胞免疫功能出现Th1优势向Th2优势偏移。③经植物血凝素激活后12hT淋巴细胞IL-2和IL-2Ra基因表达达到峰值；rhHMGB1与T淋巴细胞共同培养12h后，10～100μg／L剂量可明显上调IL-2和IL-2Ra基因表达；而较高剂量rhHMGB1（100～1000μg／L）刺激持续48h上述效应衰竭，并表现出相反的变化趋势。结论HMGB1对T淋巴细胞包括增殖、分化和细胞因子分泌等免疫功能具有直接调节效应。剂量蓄积和持续刺激可诱导T淋巴细胞功能亚群从促炎优势向抗炎优势转化。     

Regulation of B cell lymphomagenesis by a malignant Qal+ inducer T cell clone

A series of Thy-1.2+ Ly-1+ Qa-1+ malignant T cell clones have been isolated from murine sarcoma virus-murine leukemia-Moloney (MSV-MuLV-M)- induced B cell lymphomas or from MSV-MuLV-M-infected B6 mice. These T cell clones enhance both antigen-independent and -dependent lymphocyte differentiation and function. They also induce the differentiation of granulocytes and erythrocytes in the stem cell compartment, a function that parallels the immunopathology of the disease in vivo. The malignant T cell appears to sustain B lymphoma growth in vivo by releasing a factor (BCGF) that promotes B cell proliferation.     

An antigen shared by a human T cell subset and B cell chronic lymphocytic leukemic cells. Distribution on normal and malignant lymphoid cells

We obtained a monoclonal antibody, A50, after immunizing Biozzi's high responder strain of mice with T cell chronic lymphocytic leukemia (T- CLL) cells. A50 recognized an antigen present on the surface of B cell chronic lymphocytic leukemia cells from many patients and from cells of T lineage from any subject we tested. We could not find this antigen either on the surface of normal B cell or on other non-T cell malignancies. On T cells, this antigen was present on a subpopulation of thymus cells, and on most peripheral T cells. The antigen was present on the surface of cells from T-CLL, Sezary's disease, and a subset o T cell lymphoma. The antigen seemed to belong to a complex set of antigenic determinants that we had defined with rabbit antisera.     

Integrase-defective lentiviral-vector-based vaccine: a new vector for induction of T cell immunity

INTRODUCTION: The development of new strategies for the induction of potent and broad immune responses is of high priority in the vaccine field. In this setting, integrase-defective lentiviral vectors (IDLV) represent a new and promising delivery system for immunization purposes. AREAS COVERED: In this review we describe the development and application of IDLV for vaccination. IDLV are turning out to be a new class of vectors endowed with peculiar characteristics, setting them apart from the parental integration-competent lentiviral vectors. Recent data suggest that IDLV are able to induce strong antigen-specific immune responses in terms of quantity, persistence and quality of CD8(+) T cell response following a single immunization in mice. EXPERT OPINION: IDLV are a recent acquisition in the field of genetic immunization, thus allowing for the opportunity of further upgrading, including increasing antigen expression and potency of immune response. Based on recent reports showing the potential of IDLV for immunization in mouse models, further development and validation of IDLV, including comparison with other vaccine protocols and use in non-human primate models, are warranted.     

Experimental study of specific immunotherapy induced by H22 autologous tumor as whole tumor cell vaccine

This study explores the novel H22 whole-cell vaccine of active specific immunotherapy in the treatment of hepatocellular carcinoma. H22 hepatoma tumor vaccine modified by human interleukin-2 (hIL-2) and mouse granulocyte-monocyte colony-stimulating factor (mGM-CSF) fusion gene was prepared to study its specific anti-tumor immunity. Mice were inoculated by these vaccines. Then tumor cells were injected into mouse models. The ⁵¹Cr release assay was used to examine the cytotoxicities of the splenocytes to H22 hepatoma cells in immunized mice, tumor-bearing mice and control mice. The blood was needed to test the level of IL-10 and interferon (IFN)-γ in serum. Survival time of mice was calculated. Specific cytotoxicity rate of splenocytes from the immunized mice to H22 cancer cell was 38%, significantly higher than 13.6% in the tumor-bearing group, 7.5% in the control group, and 9.1% in S180 cells (p < 0.05). Serum IFN-γ in the immunized group was significantly increased compared with other groups (p < 0.01), and serum IL-10 in the immunized group was significantly decreased compared with other groups(p < 0.01). The survival time of the transgenic vaccinated group was significantly longer.     

Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line

The association between T3 and the T cell antigen receptor was examined using the T3 bearing T cell leukemic line Jurkat. A monoclonal antibody, C305, was produced, which reacted with idiotypic-like determinants expressed on Jurkat. The molecule with which this antibody reacted was a disulfide-linked heterodimer of 90 kD, composed of polypeptides of 42 and 54 kD. Thus, C305 reacted with a molecule with characteristics of the putative T cell antigen receptor described by others. A series of mutants of Jurkat, induced with ethyl methane sulfonate or radiation, was selected for T3 or antigen receptor negativity. In every instance, there was a concomitant loss of both T3 and the antigen receptor as assessed by quantitative absorption, indirect immunofluorescence, and antibody plus complement-mediated cytotoxicity. The absence of antigen receptor molecules was confirmed on diagonal gels, excluding the possibility that conformational changes of the antigen receptor on such T3-negative mutants were responsible for the failure of such mutants to react with C305. Moreover, in a mutant that expressed a marked decrease in the level of T3 expression, there was a comparable decrease in the expression of antigen receptor determinants. These results suggest that there is an obligate requirement for the coexpression of T3 and the T cell antigen receptor. Furthermore, attempts to activate such mutants with the lectin phytohemagglutinin suggested that the expression of T3 and/or the antigen receptor was required for activation of these cells.     

L6细胞胰岛素抵抗的骨骼肌细胞模型

背景:在胰岛素抵抗的细胞模型中,以游离脂肪酸诱导3T3-L1脂肪细胞和高胰岛素诱导的HepG2肝癌细胞最为常见,对分布最为广泛靶组织骨骼肌的胰岛素抵抗模型报道较少.目的:拟使用L6细胞株建立胰岛素抵抗的骨骼肌细胞模型.设计、时间及地点:细胞学体外观察,于2007-06/2008-05在辽宁医学院生物化学与分子生物学教研室完成.材料:L6细胞株由中国协和医科大学细胞中心提供.α-MEM培养基为美国Gibco公司产品.方法:复苏后的L6细胞置于α-MEM培养基中诱导培养14 d,以考马斯亮蓝染色观察肌管出现来确定L6成肌细胞株分化完成.将分化后的细胞建立胰岛素抵抗模型,按1 × 108/孔接种于24孔板内,用含体积分数为0.02胎牛血清的α-MEM培养,以L6细胞贴满24孔板的板底为宜.换液后,向各孔内分别加入含体积分数为0.02牛血清白蛋白的α-MEM无血清培养基饥饿培养5 h.设立2组,实验组的板孔内加入含1×107mol/L胰岛素的HEPES缓冲液(136mmol/LNaCl,4.7mmol/LKCl,1.25 mmol/L MgSO4,1.2 mmol/L CaCl2,0.2%牛血清白蛋白,20 mmol/L HEPES,pH 7.4)孵育1 h;对照组的板孔内加入不含胰岛素的HEPES缓冲液孵育1 h.弃上清后用HBS缓冲液冲洗,每孔内分别加入含0.1 mol/L葡萄糖的HBS缓冲液100 μL,孵育10 min后取出置于冰板上快速回收上清.主要观察指标:考马斯亮蓝染色观察L6细胞出现肌管结构情况,葡萄糖氧化酶法检测上清中葡萄糖浓度.结果:诱导前L6细胞未见肌性结构出现,诱导分化后L6细胞内出现肌管结构.使用α-MEM无血清培养基饥饿培养1,3 h,两组上清中葡萄糖浓度基本相似(P>0.05);使用α-MEM无血清培养基饥饿培养5 h后,实验组上清中葡萄糖浓度明显高于对照组(P<0.05).结论:诱导分化成肌后的L6骨骼肌细胞经α-MEM无血清培养基饥饿培养5 h,在胰岛素浓度为1×10-7mol/L的刺激下可形成胰岛素抵抗的骨骼肌细胞模型.