表达EB病毒潜伏膜蛋白2A(LMP2A)的小鼠移植瘤模型的建立

目的：建立表达EB病毒LMP2A小鼠移植瘤模型，为树突状细胞治疗EB病毒相关肿瘤的动物试验奠定基础。方法：将EB病毒LMP2A基因克隆至逆转录质粒pLXSN中，重组质粒用脂质体Clonfectin转入BALB／c小鼠来源的骨髓瘤细胞SP2／0，经G418筛选获得阳性克隆，亚克隆得到EB病毒LMP2A表达株并经PCR、RT—PCR、IFA、流式细胞仪鉴定。腹股沟皮下接种该细胞入BALB／c小鼠得到移植瘤模型，两周后切除肿瘤组织并作苏木精-伊红(H—E)染色切片。结果：构建出含EB病毒LMP2A基因的重组逆转录质粒，获得表达EB病毒LMP2A的SP2／0细胞，该细胞在BALB／c小鼠可生长成肿块。结论：成功地构建表达EB病毒潜伏膜蛋白2A(LMP2A)的小鼠移植瘤模型。     

融合基因EBNA1-LMP1重组腺病毒的构建及其免疫学研究

目的 构建EB病毒(Epstein-Barr virus,EBV)EBNA1和LMP1融合基因的重组腺病毒,探究重组腺病毒的免疫学作用.方法 以质粒pCXWB-EBNA1和pMV261-LMP1为模板,通过PCR扩增EBNA1和LMP1基因,并通过linker以重叠延伸PCR的方式构建融合基因EBNA1-LMP1,将其...     

经癌胚抗原重组痘苗病毒转染的树突状细胞体外诱导特异性T细胞免疫

目的 研究人癌抗原重组痘苗病毒（rV－CEA）转染上周血树突状细胞（DC）后能否在体外诱导CEA特异性细胞性T淋巴细胞免疫。方法 将rV－CEA转染外周血单核细胞来源的DC后用于激发同源的T细胞，检测其对T细胞的增殖作用以及对CEA分泌性肿瘤细胞的杀伤活性，并与未经rV－CES转染的DC激发的T细胞进行比较。结果 经rV－CEA转染的DC激活的T细胞对CEA分泌性肿瘤细胞具有特异性杀伤作用。结论 rV－CEA转染的DC可以诱导CEA特异性T细胞活性。     

Antitumor effects of interleukin-18 gene-modified hepatocyte cell line on implanted liver carcinoma

Objective To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma. Methods Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines, BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line), tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2 ). Two weeks later, the serum levels of IL-18, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed, the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured, and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach. Results In the treatment group, the serum levels of IL-18, IFN-γ, TNF-α and NO increased significantly. The splenic CTL activity increased markedly (P<0.01) , accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice. Conclusions In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.     

EB病毒LMP2-LMP1Δ融合基因免疫效果的初步研究

目的构建EBV LMP2-LMP1Δ融合基因,初步观察融合基因体内诱导EBV特异性细胞免疫应答的效果。方法 （1）应用PCR方法构建包含EBV-LMP2全长和去除致癌基因的EBV-LMP1Δ融合基因,并将融合基因插入到pcDNA3.1（＋）-his真核表达载体中,构建重组表达质粒pcDNA-LMP2-LMP1Δ,Western blot和免疫荧光方法检测融合蛋白的表达。（2）使用pcDNA-LMP2-LMP1Δ及携带LMP1Δ和LMP2基因的重组腺病毒单独或联合免疫Balb/c小鼠,末次免疫1周后应用IFN-γELISPOT方法检测小鼠脾淋巴细胞中EBV特异性CTL水平。结果 （1）真核表达质粒pcDNA-LMP2-LMP1Δ能够在293细胞中表达LMP2-LMP1Δ融合蛋白,融合蛋白分子量大小正确,具有免疫原性。（2）重组质粒pcDNA-LMP2-LMP1Δ免疫小鼠能够诱导出EBV特异性的CTL,但诱导的CTL水平很低,1×106小鼠脾淋巴细胞中平均斑点数只有12个,远远低于LMP1Δ、LMP2重组腺病毒平均495个斑点数的免疫结果。但使用pcDNA-LMP2-LMP1Δ和重组腺病毒联合免疫,与只使用重组腺病毒相比,诱导的EBV特异性CTL水平能够显著提高,1×106小鼠脾淋巴细胞中平均斑点数可达1 001个（P〈0.01）。结论构建的EBV LMP2-LMP1Δ融合基因能够有效表达LMP2-LMP1Δ融合蛋白,并能够在小鼠体内诱导出EBV特异性的CTL反应,与重组腺病毒联合使用,可以提高特异性CTL应答水平。     

In vitro inducing effect of dendritic ceils cotransfected with survivin and granulocyte-macrophage colony-stimulating factor on cytotoxic T cell to kill leukemic cells

Background Survivin is a rather specific gene in tumor tissue.We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells.Methods After derived from the peripheral,DCs was assayed by mixed leukocyte reaction (MLR) tests.Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL.Results Expression of survivin in transfected DCs was confirmed by Western blotting analysis.GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA).In MLR assay,DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5,1:10,1:50 and 1:100.DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only,Ad-GM-CSF only,or control DCs.Levels of interleukin-12 (IL-12) and interferon gamma (IFN-y) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group.Conclusion DCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor.Therefore,adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.     

四种不同尤文肉瘤树突状细胞免疫疫苗的体内外抗肿瘤免疫应答研究

目的:制备4种尤文肉瘤树突状细胞(dendritic cell,DC)疫苗,并比较和分析其在体内外抗肿瘤免疫反应中的效果.方法:从健康供者体中分离和培养外周血单核细胞,利用粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor, GM-CSF)和白细胞介素(interleukin-4,IL-4)将单核细胞诱生为DC,并对其表型进行流式细胞仪分析,分别制备4种DC疫苗:(1)融合瘤,将DC与A673细胞进行电融合,并通过流式细胞仪检测荧光双染色融合瘤,以确定其融合率;(2)负载肿瘤裂解物DC,将DC与A673细胞反复冻融物共培养;(3)EWS/FLI1修饰DC,用编码EWS/FLI1的腺病毒转染DC;(4)未作处理的成熟的DC.将各种DC疫苗与组织相容性白细胞抗原(histocompatibility leukocyte antigen,HLA)相配的CD8+T细胞共培养,用ELISA试剂盒检测刺激增殖细胞毒性T淋巴细胞(cytotoxicity T lymphocyte,CTL)分泌干扰素γ(interferon γ,IFN-γ)的量,用51Cr细胞杀伤试剂盒检测CTL在不同比例下对A673细胞的杀伤作用.通过腹腔注射人外周血淋巴细胞(peripheral blood lymphocyte,PBL),皮下接种A673细胞,构建重建人免疫系统的SCID鼠尤文肉瘤模型,应用ELISA试剂盒检测小鼠外周血中IgG的量,以确定免疫重建效果,并接种不同的DC疫苗,检测其对小鼠成瘤的影响.结果:流式细胞术检测出从外周血单核细胞诱生出的CD83、CD80、CD86及HLA-DR高表达的成熟DC.DCs/A673融合细胞的电融合效率达到16.32%.各种DC疫苗都可以诱导出有效的抗肿瘤免疫反应,IFN-γ分泌检测显示,融合瘤组较其他DC疫苗组产生CTL水平高,其后依次为肿瘤裂解物负载组、EWS/FLI1修饰组和未作处理组.而在体外杀伤A673实验中,融合瘤组杀伤效率最高,肿瘤裂解物负载组次之,EWS/FLI1修饰组与未作处理组最低,且后两组间差异无统计学意义.在抑制肿瘤生长的体内实验中,融合瘤组小鼠肿瘤最小,EWS/FLI1修饰组、肿瘤裂解物负载组和未作处理组之间差别不大.结论:尤文肉瘤DC疫苗可以诱导有效的抗肿瘤免疫反应,将各种尤文肉瘤DC疫苗进行体内外综合比较时发现,融合瘤是最佳的尤文肉瘤免疫疫苗.     

小鼠肠癌RNA转染mIL-12修饰的树突细胞诱发特异性细胞毒性T淋巴细胞的研究

目的：研究小鼠结肠癌细胞CT-26RNA体外转染mIL-12基因修饰的树突细胞（DC），观察其诱导特异性细胞毒性T淋巴细胞（CTL）能力。方法：小鼠骨髓细胞体外经rmGM-CSF、rmIL-4诱导培养获取DC，流式细胞仪检测其纯度；293细胞扩增携带mIL-12基因的重组腺病毒，体外转染DC；Trizol法提取CT-26细胞总RNA，应用TransMessenger体外转染mIL-12基因修饰的DC；ELISA法检测细胞上清及小鼠血液中mIL-12，LDH释放法检测小鼠体内特异性CTL活性。结果：小鼠骨髓细胞经诱导培养后，获得大量高纯度的DC，流式细胞仪检测CD11c^＋的DC〉90％；携带mIL-12基因重组腺病毒转染的DC细胞高表达mIL-12；CT-26细胞总RNA体外转染mIL-12基因修饰的DC后，回输小鼠，能明显提高小鼠体内mIL-12的水平，并可诱导体内生成较高水平的CTL活性，而以该RNA转染Ad-LacZ修饰DC后的对照组及RNA转染DC的对照组，可诱导机体生成中等水平的特异性CTL活性，DC、PBS对照组则均无此作用。结论：小鼠肠癌CT-26细胞总RNA转染mIL-12基因修饰的DC后，免疫接种小鼠，可提高小鼠体内mIL-12的水平，并能有效诱导机体产生较高水平的特异性CTL活性。     

LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer

ObjectiveTo evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patents with nasopharyngeal carcinoma (NPC).MethodsDCs were derived from peripheral blood monocytes of patents with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patents were immunized with 2 x 10 LMP2-DCs by intradermal injection at week 0 and after the second and fourth weeks. Specific responses to LMP2 were detected by enzyme-linked immunospot (ELISPOT) assay at week 0 and at the fifth and eighth weeks. Local clinicians performed the follow-up and tracking of patients.ResultsWe demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders.ConclusionIn this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.     

Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo

Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05) , and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance.     

In vitro induction of specific anti-tumoral immunity against laryngeal carcinoma by using human interleukin-12 gene-transfected dendritic cells

Background  Objective evaluation of the antitumor effect of interleukin-12 (IL-12) gene-transfected dendritic cell (DC) vaccine on laryngeal carcinoma requires in vivo and in vitro tests. The aim of this study was to investigate the function of IL-12 gene transfected DC at initiating specific immune response to laryngeal carcinoma in vitro．

Methods  Recombinant adenovirus with IL-12 gene was constructed. DCs were isolated from the peripheral blood of patients with laryngeal carcinoma, pulsed with tumor lysate of laryngeal carcinoma cells (DC⁺Ag), and transfected with IL-12 (DC-IL-12⁺Ag). The cells pheotypes including CD83, CD86 and HLA-DR on surface of DCs were assayed by flow cytometry (FCM). The concentration of IL-12 in culture supernatant of DCs and interferon γ (IFN-γ) in culture supernatant of T cells cocultured with DCs were quantified by ELISA. Methyl thiazolys tetrazolium (MTT) was used to evaluate proliferation of autologous T lymphocytes and activation of cytotoxic T lymphocytes (CTL) stimulated by IL-12-transfected DCs pulsed with tumor lysate against laryngeal carcinoma cells.

Results  The recombinant adenovirus expressing IL-12 gene was constructed successfully. Gene-transfected DC plused with tumor lysate with IL-12 (DC-IL-12⁺Ag) expressed higher level of CD83, CD86 and produced higher level of IL-12 than untransfected DCs (DC⁺Ag) (CD83: (60.2±1.8)% vs. (50.7±1.2)%, P <0.05; CD86: (88.9±2.1)% vs. (78.2±3.9)%, P <0.05; IL-12: (262.5±3.0) ng/L vs. (103.8±5.1) ng/L, P <0.05). The proliferation of autologous T lymphocytes and production of IFN-γ stimulated by DC transfected with IL-12 were more obviously than untransfected DCs. Cytotoxicity of CTL stimulated by IL-12-transfected DC pulsed with tumor lysate against laryngeal carcinoma cells were significantly stronger than stimulated by untransfected DC.

Conclusion  It is a promising approach for IL-12-transfected DC pulsed with tumor lysate to increase the antitumoral effect.

     

Recombinant Vaccinia Virus is an Effective and Non—perturbing Vector for Human Dendritic Cells Transfected with Epstein—Barr Virus Latent Membrane Protein 2A

Objective To study the effects of dendritic cells(DC) transfected with recombinant vaccinia virus encoding Epstein-Barr virus(EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic uaccines against EBV-associated malignancies.Methods Mature DC were transfected with EVB-LMP2A recombinant vaccinia virus(rVV-LMP2A).Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA-DR was measured by fluorescence activated cell sorter(FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions(MLR).Results LMP2A protein was highly expressed (66.1%) in DC after the transfection of rVV-LMP2A.No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection.Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells.Conclusion Recombinant vaccinia virus was an effective and non-perturbing vector to mediate the transfection of LMP2A into DC.The functions of mature DC were not affected significantly by the transfection of Vac-LMP2A.This study could provide evidence for the further immunotherapy of EBV-associated malignancies,e.g.nasopharyngeal carcinoma(NPC).     

腺病毒介导人IL-2基因治疗的抗肿瘤作用及机理分析

目的　观察重组人白介素 2腺病毒 (advh IL- 2 )在小鼠肝癌 H2 2中的转染效率、表达时程、体内抗肿瘤作用及其免疫机理。方法　小鼠皮下接种肝癌 H2 2 ,将肿瘤生长至体积为 10 0～ 2 0 0 mm3的荷瘤小鼠随机分组 ,经肿瘤局部注射给药。用 X- gal染色法检测重组腺病毒载体 adv L ac Z的转染效率 ;用 RT- PCR检测人 IL- 2基因的持续表达时间 ;以 H2 2瘤体生长及荷瘤小鼠存活时间评价 advh IL - 2抗肿瘤作用 ;以 51 Cr 4小时释放法测定治疗小鼠脾细胞的 L AK与 CTL 杀伤活性 ,免疫荧光法分析肿瘤组织内 CD4 +与 CD8+ T细胞浸润情况。结果　肿瘤局部单次注射 1× 10 9pfu重组腺病毒 ,可在肿瘤组织中进行有效的转染及表达 ,人 IL- 2基因的表达时间长于 12 d。AdvhIL- 2的抗肿瘤作用具有剂量依赖性 ,与 PBS组比较 ,2× 10 9pfu advh IL- 2可非常明显地抑制 H2 2皮下瘤的生长、延长荷瘤小鼠的生存时间 (P<0 .0 1) ,同时明显增强脾细胞 L AK与 CTL杀伤活性 (P<0 .0 1) ,增加肿瘤组织内CD4 + 与 CD8+ T细胞的浸润。结论　 Advh IL - 2通过介导人 IL - 2基因在小鼠 H 2 2肿瘤组织中的持续表达 ,产生明显的体内抗肿瘤作用 ,其机理与激活荷瘤小鼠抗肿瘤免疫反应有关     

Induction of T-cell immunity against leukemia by dendritic cells pulsed with total RNA isolated from leukemia cells

Objectives To assess the feasibility and efficacy of eliciting leukemia-specific T-cell responses in syngeneic mice in vitro and in vivo using dendritic cells (DCs) pulsed with total RNA from leukemia cells.Methods DCs generated from bone marrow culture in vitro in the presence of combined cytokines were pulsed with cellular total RNA isolated from cultured L615 cells by cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP). T-cell responses were evaluated by in vitro proliferation, and cytotoxicity assay. And in vivo immune protection and proghosis of mice with leukemia were studied.Results DCs pulsed with total RNA isolated from cultured L615 cells (DCs/RNA) were remarkably effective in stimulating L615-specific T-cell response in vitro, but did not cross-react with other leukemia cells from syngeneic mice. Vaccination of naive mice with viable DCs/RNA vaccine was able to partly protect from challenge with a lethal dose of live L615 cells, leading to low leukemia incidence and overall survival prolongation. Statistically significant survival was also observed in a low lethal dose of L615-bearing mice that received treatment using viable DCs∕RNA vaccine alone, suggesting that systemic administration of IL-2 could enhance the anti-tumor efficacy of leukemia RNA/DCs vaccine.Conclusions These data support the use of DCs/RNA vaccine as a feasible and effective route to elicit leukemia immunity against unidentified leukemia-associated antigens for treatment of leukemia-bearing animals.     

PADRE/MUC4 重组腺病毒转染树突状细胞诱导特异性CTL体外杀伤作用研究

目的:观察PADRE/MUC4重组腺病毒转染树突状细胞(DC)诱导特异性细胞毒性T细胞(CTL)及体外特异性杀伤作用.方法:pAd-CMV-PADRE/MUC4转染HLA-A2健康志愿者外周血单个核细胞(PBMC)来源的未成熟DC,TNF-α诱导成熟后与自体PBMC混合培养刺激3周,Cr51和Elispot实验检测CTL体外杀伤活性.结果:Cr51和Elispot检测结果显示PADRE/MUC4转染DC可以诱导产生特异性CTL,而pAd-CMV-GFP组和空白对照组不能产生有效特异性CTL.结论:腺病毒载体介导多表位嵌合基因(PADRE/MUC4)转染未成熟DC,在体外可以诱导产生特异性CTL,对表达MUC4肿瘤细胞具有杀伤效应.     

淋巴细胞趋化因子基因修饰增强肿瘤抗原多肽致敏的树突状细胞的 …

目的 通过选择性增强树突状细胞（ＤＣ）与Ｔ细胞的体内相互作用。优化其体内抗原提呈的微环境,为进一步增强ＤＣ介导的肿瘤免疫治疗效果。方法 体外培养的小鼠骨髓树突太细胞体外经Ｌｔｎ重组腺病毒感染后（Ｌｔｎ－ＤＣ）,用３ＬＬＬｅｉｗｓ肺癌细胞株的Ｍｕｔｌ抗原肽冲击致敏,按不同剂量免疫正常同系小鼠体内,观察其体内诱导的细胞毒性Ｔ淋巴细胞（ＣＴＬ）活性保护性免疫反应。通过体内阻断试验探讨免疫细胞亚群及免疫分     

TNF-α gene-modified dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity in mice

Objective To investigate the antitumor immune efficiency of mouse dendritic cells (mDCs) by using adenovirus-mediated tumor necrosis factor-alpha (AdV-TNF-α) gene transfer.Methods MDCs infected with AdV-TNF-α and AdV-pLpA (no gene insert) at 100 multiplicity of infection (MOI) were analyzed by RNase protection assay for their cytokine secretion. Mixed lymphocyte reactions were also performed to analyze their capacity for alloantigen-presentation. C57BL/6 mice were challenged with R3LL tumor cells (Lewis lung carcinoma line) 10 days after vaccination with different engineered DCs and regular DCs as well.Results Compared to AdV-pLpA and mock-infected DCs, AdV-TNF-α-infected DCs displayed up-regulated expression of alpha tumor necrosis factor, interleukin-12 (IL-12), interleukin-18 (IL-18) and granulocyte macrophage colony stimulation factor (GM-CSF), and indicated stronger allogeneic T cell proliferative responses. Furthermore, vaccination of mice with dendritic cell tumor necrosis factor-alpha (DCTNF-α) pulsed with Mut1 peptide induced more efficient tumor-specific cytotoxic T lymphocyte (CTL) cytotoxicity against R3LL tumor cells in vitro and with efficient antitumor immunity in vivo. Conclusion This type of engineered DCs could be applied in clinical settings of DC-based cancer vaccines     

两种抗原致敏方式负载树突状细胞疫苗对结直肠癌细胞株的体外杀伤作用

目的 比较两种抗原负载方式对树突状细胞(DCs)疫苗的影响.方法 抽取HLA表型为A11的健康志愿者外周血,分离单核细胞,体外培养.通过反复冻融LoVo细胞,提取肿瘤细胞裂解物或者使用含CEA片断的重组腺相关病毒转染未成熟DCs,诱导特异性细胞毒性T细胞.检测体外培养的DCs和CTL活性,并使用MTT法检测两组CTL对LoVo细胞的杀伤作用.结果 两种方式均可培养的成熟DCs,诱导的CTL细胞分泌IFN-γ有所增加;转染后DCs诱导特异性CTL可有效识别并杀伤HLA-A11阳性的LoVo细胞,腺相关病毒提呈抗原制备的DCs疫苗对LoVo细胞的杀伤作用明显高于肿瘤细胞裂解物的抗原负载方式.结论 两种抗原提呈方式均可培养出成熟的DCs,不明显改变DCs表型和刺激淋巴细胞增殖、分化功能,并可诱导自体CTL增殖.使用腺相关病毒转染DCs的方式明显优于肿瘤细胞裂解物的抗原负载方式.     

双表达B7-1、B7-2基因肿瘤疫苗治疗小鼠肝癌优于单表达疫苗的研究

目的:研究B7-1、B7-2基因对肿瘤的免疫治疗作用。方法:应用转染有B7-1、B7-2基因的肝癌细胞株H22/B7-1、H22/B7-2,建立小鼠肝癌模型,观察小鼠成瘤期、荷瘤小鼠存活期及肿瘤结节大小。结果:各实验组动物都发生肿瘤,接种H22/B7-1 H22/B7-2组成瘤率低,对照组动物肿瘤呈进行性生长;组间成瘤潜伏期不同,与对照组相比,凡接种有H22/B7-2的小鼠肿瘤形成有迟发性;接种转B7基因细胞的小鼠肿瘤生长都较对照组慢;同时接种H22/B7-1和H22/B7-的小鼠能负载大于107的肿瘤细胞。转基因细胞在体外刺激淋巴细胞增殖和诱导CTLs的能力明显增强。结论:B7-1、B7-2都能增强肝癌细胞的免疫原性。B7-2在抗肿瘤早期发挥作用,B7-1随后起放大和调节作用。B7-1与B7-2联用效果优于单一应用。