异种细胞疫苗的制备及其应用

采用异种疫苗可以打破机体对肿瘤细胞及其血管的免疫耐受，诱导针对肿瘤及其新生血管的自身免疫反应，抑制肿瘤生长，因而具有潜在的开发应用价值。现综述该新型疫苗制备方法及其优点。     

树突状细胞肿瘤疫苗研究进展

树突状细胞（DC）因为具备强大的专职递呈抗原诱导免疫应答的能力而用于肿瘤疫苗的制备和应用。一方面，免疫学及分子生物学等领域理论与技术的进步丰富了DC肿瘤疫苗所负载的内容和形式；另一方面，对DC肿瘤疫苗产生免疫应答的深入研究也为更合理地制备肿瘤疫苗提供了依据。现主要从以上两方面对DC肿瘤疫苗研究进展作一综述。     

胃癌疫苗的研究现状与进展

近年来，人们逐渐认识到机体免疫系统与肿瘤组织之间失衡是肿瘤发生发展的重要因素，因而肿瘤疫苗逐渐成为研究热点，新的肿瘤抗原不断被发现，新的肿瘤疫苗制备方法也不断出现，许多疫苗已进入临床试验研究，现就胃癌疫苗的研究现状与前景作一综述。     

树突状细胞疫苗的制备及其临床应用研究

本文主要就近 2年来树突状细胞 (DC)疫苗的发展和临床应用等方面作一综述 ,着重讨论DC疫苗的制备方法及存在问题。     

树突状细胞肿瘤疫苗研究进展

树突状细胞(DC)因为具备强大的专职递呈抗原诱导免疫应答的能力而用于肿瘤疫苗的制备和应用。一方面,免疫学及分子生物学等领域理论与技术的进步丰富了DC肿瘤疫苗所负载的内容和形式;另一方面,对DC肿瘤疫苗产生免疫应答的深入研究也为更合理地制备肿瘤疫苗提供了依据。现主要从以上两方面对DC肿瘤疫苗研究进展作一综述。     

树突状细胞疫苗研究进展

近十年来,随着对细胞毒性Ｔ淋巴细胞（ＣＴＬ）形成机制、作用及其和ＭＨＣⅠ、Ⅱ类分子关系的揭示,ＣＤ８＋ＣＴＬ细胞在抗肿瘤免疫反应中的核心地位已明确奠定。因此,树突状细胞（ｄｅｎｄｒｉｔｉｃｃｅｌｌ,ＤＣ）疫苗在肿瘤免疫治疗中的研究正引起人们的广泛关注。ＤＣ是体内最重要的抗原递呈细胞［１］,其在激发免疫反应中起核心作用。ＤＣ是骨髓来源的白细胞,也可由血液中的单核细胞、中性粒细胞及组织巨噬细胞衍生而来。与巨噬细胞相比,ＤＣ膜表面的ＭＨＣ分子数量多近５０倍,这样有可能形成较多的与Ｔ细胞受体结合的肽／Ｍ…     

胃癌疫苗的研究现状与进展

近年来,人们逐渐认识到机体免疫系统与肿瘤组织之间失衡是肿瘤发生发展的重要因素,因而肿瘤疫苗逐渐成为研究热点,新的肿瘤抗原不断被发现,新的肿瘤疫苗制备方法也不断出现,许多疫苗已进入临床试验研究,现就胃癌疫苗的研究现状与前景作一综述。     

肿瘤疫苗研究进展

肿瘤疫苗是通过激活患者自身免疫系统以清除或控制肿瘤的治疗方法.以肿瘤细胞为基础的疫苗取得了较满意的临床疗效.近几年随着免疫学、分子生物学等的发展,以及肿瘤相关抗原(TAA)的鉴定,出现了以TAA为基础的肿瘤疫苗,产生了明显的免疫应答.随着对肿瘤免疫逃避机制认识的深入和更多TAA的鉴定,肿瘤疫苗将成为肿瘤治疗的有效手段.     

树突细胞疫苗用于多发性骨髓瘤的免疫疗法

多发性骨髓瘤（MM）是一种浆细胞恶性增生性疾病,其独特型是一种肿瘤特异性抗原,该病的发病率呈上升趋势,而传统治疗对其效果不佳,借助树突细胞（DC）强大的抗原递呈功能结合独特制备疫苗,可在体内外活化CD4＋,CD8＋T细胞,从而激发特异性抗肿瘤效应,为多发性骨髓瘤的免疫治疗提供了新方法。     

树突状细胞融合瘤苗的制备及其在肿瘤免疫治疗中的应用

树突状细胞（DC）融合瘤苗的制备主要包括DC体外诱导扩增及肿瘤细胞体外培养、诱导融合、瘤苗纯化等流程。在黑色素瘤、胃癌、肾癌等肿瘤的动物实验及临床前期试验中融合瘤苗表现出良好的抗肿瘤免疫效果。     

Humoral and cellular immunity induced by tumor cell vaccine based on the chicken xenogeneic homologous matrix metalloproteinase-2

Matrix metalloproteinase-2 (MMP-2) has been used as a target for cancer immunotherapy. The activation of immunization by breaking immune tolerance to self-MMP-2 may be one of the promising approaches for the treatment of MMP-2-positive tumors. In this study, we constructed the xenogeneic tumor cell vaccine c-MMP-2 by transfecting CT26 and LLC cells with chicken MMP-2 cDNA constructs. MMP-2-specific autoantibodies in sera and tumor cells were found in mice immunized with c-MMP-2. Protection against tumor growth was evaluated in respect of the relative contributions of autoantibodies, CD4+, and CD8+ T cells. Treatment with this vaccine (c-MMP-2) also prolonged the survival time of mice bearing cancer. The specific cytotoxic T-cell responses suggested that the treatment increased CD8+ T-cell activity. The antitumor activity of c-MMP-2 was abrogated by in vivo depletion of CD4+ and CD8+ T-lymphocytes and improved by adoptive transfer of CD4+ and CD8+ T-lymphocytes from the mice treated with c-MMP-2. An alternative DNA vaccination strategy for cancer therapy was identified in this study by eliciting humoral and cellular immunoresponse with a crossreacting transfectant.     

Combination of low-dose cisplatin and recombinant xenogeneic endoglin as a vaccine induces synergistic antitumor activities

Angiogenesis is critical to the growth and metastasis of solid tumors, and acquired drug resistance is one of the major hindrances to chemotherapy. Thus, we sought a rational strategy using the combination of antiangiogenic biotherapy and chemotherapy for cancer therapy. We explored the efficacy of a strategy combining low-dose cisplatin and a recombinant xenogeneic endoglin as a protein vaccine, which we previously demonstrated to have effective antiangiogenic effects in several mouse models. We found that both low-dosage cisplatin and xenogeneic endoglin vaccine individually resulted in effective suppression of tumor growth in 2 tumor models via inhibition of tumor angiogenesis. Remarkably, the combination therapy resulted in not only significant antiangiogenic effects but also additional promotion of tumor cell apoptosis and inhibition of tumor cell proliferation, without any ensuing increase in host toxicity during the course of treatment, which lasted for 6 months. In addition, the combination demonstrated a synergistic relationship, which was shown in all of the synergistic indexes, i.e., tumor volume, angiogenesis, apoptosis and proliferation. Both antibodies and antibody-producing B cells against mouse self-endoglin were observed in all mice immunized by the xenogeneic endoglin vaccine (alone and combination), which suggested that low-dose cisplatin did not suppress the host immune response but potentiated the antitumor activity of the xenogeneic endoglin vaccine. These observations may provide the basis for an effective alternative strategy for cancer therapy in the near future.     

血性胸腔积液细胞块制作方法研究及其应用

目的:探讨一种适合血性胸腔积液的细胞块制作方法.方法:收集细胞学涂片中见到肿瘤细胞的血性胸水48例,分别用直接离心沉淀法、去红细胞离心沉淀法和富集有核细胞层沉淀法制作细胞块,对三种方法获得的细胞块的HE染色效果、免疫组化效果及提取DNA的合格率进行比较.结果:直接离心沉淀法、去红细胞离心沉淀法、富集有核细胞层沉淀法制作细胞块的提取DNA合格率分别为72.9%(35/48)、85.4%(41/48)和93.8%(45/48),富集有核细胞层沉淀法提取DNA的合格率明显高于直接离心沉淀法,两者合格率差异有统计学意义(P<0.01).富集有核细胞层沉淀法提取DNA的合格率高于去红细胞离心沉淀法,但两者合格率差异无统计学意义(P>0.05).HE、免疫组化效果富集有核细胞层沉淀法最好,去红细胞离心沉淀法次之,直接离心沉淀法最差.结论:血性胸水细胞块推荐的方法依次是富集单个核细胞层沉淀法、去红细胞离心沉淀法、直接离心沉淀法.     

异种EGFR口服DNA疫苗抑制小鼠Lewis肺癌的生长

目的：观察表达异种鸡EGFR(chicren EGFR, cEGFR)与 IgGγFc融合基因的口服减毒鼠伤寒沙门菌疫苗对高表达EGFR 的肺癌Lewis细胞小鼠移植瘤生长的抑制作用。方法：将pVAX1-cEGFR-γFc质粒转化减毒沙门菌SL7207,重组菌SL7207/pVAX1-cEGFR-γFc体外感染小鼠腹腔巨噬细胞,免疫荧光法检测cEGFR-γFc融合蛋白的表达。SL7207/pVAX1-cEGFR-γFc重组菌口服免疫小鼠3次后接种Lewis细胞,Western blotting检测小鼠体内融合蛋白的表达,ELISA法检测免疫小鼠血清抗EGFR抗体的水平。接种Lewis细胞14 d后处死小鼠,瘤体称质量,检测SL7207/pVAX1-cEGFR-γFc疫苗对Lewis肺癌生长的抑制作用,测定荷瘤小鼠的生存时间。结果：成功构建减毒沙门菌疫苗SL7207/pVAX1-cEGFR-γFc,SL7207/pVAX1-cEGFR-γFc感染后,在小鼠后体内外都能检测到cEGFR-γFc融合蛋白的表达;SL7207/pVAX1-cEGFR-γFc疫苗口服免疫后小鼠能够产生高水平的抗EGFR抗体,口服SL7207/pVAX1-cEGFR-γFc疫苗能够有效抑制小鼠Lewis移植瘤的生长,延长荷瘤小鼠的生存时间。结论：异种EGFR口服DNA疫苗能够有效地抑制高表达EGFR肺癌的生长,是EGFR分子靶向治疗的一条新途径。     

Production and characterization of xenogeneic antisera to a human renal cell carcinoma-associated antigen.

Antisera to human renal cell carcinomas were produced by the immunization of goats and rabbits with dissociated tumor cells and/or tumor homogenates from single donors. After absorptions with human red blood cells and homogenates of human liver, lung, spleen, and heart, all the immune sera reacted on immunofluorescence with the brush border of the proximal convoluted tubules of adult and fetal human kidneys and with the proximal convoluted tubular epithelia of rabbits, guinea pigs, rats, and mice. After further absorption with pooled normal human kidney homogenates, the immune sera on immunofluorescence showed cytoplasmic staining of smears and sections of 21 of the 22 human renal cell carcinomas tested. These sera did not show any staining of normal adult human tissues including normal kidney adjacent to the carcinomas, perirenal fibroblasts and peripheral blood leukocytes from the patients, human fetal kidneys, transplanted renal adenocarcinoma of BALB/c mice, and several human tumors tested, i.e., transitional cell carcinoma of the bladder, adenocarcinomas of the breast and colon, squamous cell carcinoma of the lungs, malignant lymphoma, and melanoma. Autoradiography of tissue sections with 131I-labeled antitumor globulins revealed greater localization of radioactivity in tumors than in adjacent normal kidney. Membrane immunofluorescence with the immune sera rendered tumor-specific after appropriate absorptions revealed tumor-associated antigens on the surfaces of all 5 human renal carcinoma cell lines tested.     

Results of xenogeneic I-RNA therapy in patients with metastatic renal cell carcinoma

Six patients with metastatic renal cell carcinoma were treated with five intravenous infusions (every other day) of autologous lymphocytes incubated in vitro with I-RNA extracted from the lymphoid tissue of guinea pigs immunized with the patient's own tumor. No toxicity was evident. One patient showed regression of multiple pulmonary metastases beginning three months after therapy with complete remission by six months. She remained without evidence of disease until 18 months after therapy. Two other patients had more than 50% regression of measurable metastases lasting eight and ten months after therapy. Two patients showed stabilization of previously growing renal cell carcinoma pulmonary metastases. A single patient with renal cell carcinoma metastatic to brain had progressive tumor growth after a single I-RNA treatment. Serial peripheral blood lymphocyte samples obtained from each of the patients during I-RNA therapy demonstrated progressive increase in in vitro cytolysis of allogeneic renal cell carcinoma targets. Boosts in cytolytic effect were shown in all patients during I-RNA treatment regardless of their subsequent clinical course. These results seem to justify a randomized, prospective trial of xenogeneic I-RNA therapy in renal cell carcinoma patients with lesser tumor burden.     

异种EGFR胞外区重组DNA疫苗与蛋白质疫苗抗小鼠Lewis肺癌的作用研究

目的:构建异种表皮生长因子受体(epidermal growth factor receptor,EGFR)胞外区重组DNA和蛋白质疫苗,观察其对小鼠Lewis肺癌的抗肿瘤作用.方法:克隆鸡源性EGFR胞外Ⅱ-Ⅲ区与IgG-Fc融合基因,构建重组DNA疫苗质粒pVAX1-cEGFR-Fc和原核表达质粒pET28a(+)-S-cEGFR-Fc,后者转化大肠杆菌,将诱导表达的重组蛋白作为蛋白质疫苗.免疫小鼠4次后接种Lewis肺癌细胞,19 d后处死小鼠,剥离肿瘤称重并计算抑瘤率,检测小鼠血清中抗EGFR抗体效价,并检测特异性细胞毒性T淋巴细胞( cytotoxicity T lymphocyte,CTL)活性.结果:成功构建异种EGFR胞外区重组DNA和蛋白质疫苗.DNA疫苗组、蛋白质疫苗组和联合免疫组的抑瘤率分别为37%、49%和63%.3组小鼠均检测出抗EGFR抗体和CTL反应.结论:重组疫苗能打破机体对自身EGFR分子的免疫耐受,诱导特异性免疫反应,对小鼠Lewis肺癌有明显的抑制作用.2种疫苗的联合应用可进一步加强抑瘤作用.     

Preparation of murine B7.1-glycosylphosphatidylinositol and transmembrane-anchored staphylococcal enterotoxin. A dual-anchored tumor cell vaccine and its antitumor effect

BACKGROUND: The authors have previously reported a tumor cell vaccine modified with superantigen staphylococcal enterotoxin A (SEA) and its antitumor effect. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger immune responses against established tumors than single-modified vaccines. METHODS: The authors explored the effectiveness of a tumor cell vaccine transduced with immune activators, dual-modified using the protein transfer technique. First, a glycosylphosphatidylinositol (GPI)-anchored murine B7.1 (mB7.1-GPI) and a transmembrane-anchored SEA (TM-SEA) were genetically generated. Then, the murine lymphoma EL4 cells were dual modified with the incorporation of mB7.1-GPI and TM-SEA onto the cell surface. Flow cytometry and laser confocal microscopy showed that the incorporation of B7.1 and SEA molecules onto EL4 cells was quite stable. RESULTS: The dual-modified tumor cell vaccine EL4/mB7.1-GPI + TM-SEA elicited significantly stronger antitumor immune responses both in vitro and in vivo when compared with the single-modified tumor cell vaccines EL4/mB7.1-GPI and EL4/TM-SEA. CONCLUSIONS: The results of the current study validated the novel approach for preparing tumor cell vaccines modified with dual immune active molecules using the protein transfer technique, and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine.     

体外诱导尤文肉瘤DC疫苗的特异性抗肿瘤免疫鉴定及初步临床应用报告

目的：探讨尤文肉瘤细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力及临床初步应用的效果。方法：分离尤文肉瘤患者外周血单核细胞体外诱导为DC细胞。Trizol法提取患者尤文肉瘤细胞总RNA,总RNA转染DC并于体外诱导特异性CTL克隆扩增。RT-PCR方法检测肿瘤总RNA加载的DC细胞内EWS-FLI1mRNA的表达。MTT法检测淋巴细胞的增殖和CTL的杀伤活性。在试验成功基础上,将成熟DC疫苗接种患者并测定免疫学OKT值。结果：RT-PCR测定显示尤文肉瘤细胞总RNA转染的DC细胞可以提呈肿瘤特异性抗原,并特异性地表达其特有序列EWS-FLI1。经总RNA转染的DC特异性表面标志及功能相关分子表达均上调,转染后的DC可显著刺激自体T淋巴细胞增殖。诱导的特异性CTL对携带EWS-FLI1抗原的靶细胞的杀伤率显著高于LAK细胞和未经转染的DC。经疫苗免疫的患者血清CD8明显升高（P〈0.05）。结论：尤文肉瘤细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫,并在临床初步证实可提高尤文肉瘤患者的特异性抗肿瘤免疫力。