WT1致敏树突状细胞激活CTLs对HL60细胞作用的研究

目的研究分别负载WT1多肽抗原及HL60细胞冻融抗原的树突状细胞(DCs)产生的特异性细胞毒性T淋巴细胞(CTLs)对HL60细胞的杀伤效应。方法联合应用rhGM-CSF、rhIL-4、rhTNF-α及rh-sCD40L等细胞因子,诱导扩增自健康人外周血获取的单核细胞,培养出DCs,分别用WT1多肽抗原及冻融抗原冲击致敏,激活淋巴细胞。实验分4组WT1多肽致敏DCs为实验组A,HL60细胞冻融抗原致敏DCs为实验组B,未致敏DCs组为对照组A,单核细胞组为对照组B,LDH释放法检测CTLs对HL60细胞的杀伤作用。结果培养出具有典型特征的DCs,表达CD40达96%,CD80达77%,CD1α达69%,CD86达97%,体外能诱导强烈的同种异体混合淋巴结胞增殖反应。在效靶比为20∶1时,WT1多肽致敏DCs组时HL60细胞杀伤率为89.1%,冻融抗原负载DCs组为90.6%,未致敏DCs组为32.8%,单核细胞组为26.1%。以下多肽或冻融抗原负载DCs与对照组相比显示更强的杀伤效应,P<0.01。结论WT1多肽抗原及HL60细胞冻融抗原冲击致敏DCs均能有效诱导T细胞抗白血病作用,为临床研制DCs疫苗提供实验依据。     

H22细胞全细胞抗原负载的树突状细胞激活肿瘤浸润性淋巴细胞抗小鼠肝癌的实验

目的探讨H22小鼠肝癌细胞（H22细胞）全细胞抗原致敏的树突状细胞激活肿瘤浸润淋巴细胞抗小鼠肝癌细胞活性。方法取得小鼠骨髓细胞并诱导生成树突状细胞,由冻融法制备的H22细胞全细胞抗原致敏,然后用已致敏的树突状细胞激活肿瘤浸润性淋巴细胞,测定致敏前后的DC表面抗原CD11c、CD80、CD86、CD40、MHCⅡ,并评估激活前后的TIL对H22细胞的杀伤活性,同时脾淋巴细胞作为杀伤对照。结果CD11c阳性细胞中CD80、CD86、CD40、MHCⅡ阳性细胞所占比例在致敏后的DC表现为明显上调。经致敏后成熟DC激活的TIL对H22细胞杀伤活性明显高于未激活的TIL,并高于激活或未激活的小鼠脾脏淋巴细胞。结论在H22细胞全抗原致敏后,小鼠成熟DC中CD80、CD86、CD40、MHCⅡ的表达率明显高于未成熟DC。经H22细胞全细胞抗原致敏的DC能诱导活化TIL,明显提高其在体外对H22细胞的杀伤活性。     

递呈乳腺癌细胞MCF-7肿瘤抗原的树突状细胞对T细胞的激活

目的：在体外利用细胞因子的诱导获取树突状细胞（DC），并观察递呈肿瘤抗原后的成熟DC对T细胞的激活作用。方法：健康人外周血单个核细胞（PBMC）在体外用细胞因子诱导获取DC，并经乳腺癌细胞MCF-7肿瘤细胞裂解物体外冲击，再以1：5的比例将致敏DC与T细胞共同孵育5d并共同作为效应细胞，通过FACS分析T细胞激活前后的表型变化以及MTT试验观察体外特异性肿瘤杀伤效果。结果：DC在细胞因子的诱导下7d后，形态学表现为伸出许多伪足样突起，在摄取抗原后可见内吞样小泡。FACS检测呈现成熟DC的标志，CD40、CD83、CD80、CD86和HL-DR等高表达。另外，未经成熟DC激活的T淋巴细胞以辅助性T细胞（Th）为主，CD4^＋ 52．1％，CD8^＋ 仅22．1％；而经成熟DC激活的T淋巴细胞以杀伤性T细胞（Tc）为主，CD4^＋ 32．6％，CD8^＋ 64．2％。同时，MTT试验证实经递呈MCF-7肿瘤抗原的DC激活了细胞毒性T淋巴细胞（CTL），并对MCF-7具有特异性肿瘤杀伤作用。结论：体外递呈肿瘤抗原MCF-7的成熟DC对T细胞具有激活作用，并产生肿瘤特异性CTL。     

抗原致敏树突状细胞诱导CIK对胃癌细胞杀伤作用的研究

[目的]探讨抗原致敏的树突状细胞（DC）诱导CIK（cytokine induced killer）的杀伤作用。[方法]联合应用粒/巨细胞集落刺激因子（GM-CSF）及白介素-4（IL-4）从外周血单个核细胞中培养DC，用人胃癌细胞SGC提取肿瘤抗原致敏DC，流式细胞仪检测致敏前后DC表型的变化，用人胃癌细胞SGC提取肿瘤抗原致敏DC诱导CIK，用MTT法检测淋巴细胞的增殖及原致敏DC诱导CIK对SGC的杀伤效应。[结果]①利用GM-CSF及IL-4可从外周血单个核细胞中获取DC，肿瘤抗原可促进DC的成熟，肿瘤抗原致敏可促进DC成熟，细胞表面分子HLA-DR、CD86、CD86升高，CD14下降。②混合淋巴细胞反应提示成熟的DC可促进CIK细胞增殖。③SGC肿瘤抗原致敏DC诱导CIK对胃癌细胞SGC有特异性的杀伤作用，随着效靶比的升高，杀伤效应随之增强。[结论]抗原致敏的DC可通过诱导特异性CIK细胞及促进CIK细胞增殖两方面显著提高CIK细胞的杀瘤效应。     

树突状细胞对肿瘤浸润性淋巴细胞抗结肠癌活性影响的研究

目的:探讨树突状细胞(DC)激活的肿瘤浸润性淋巴细胞(TIL)体外对结肠癌细胞的杀伤活性。方法:从结肠癌患者外周血获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)和肿瘤抗原激活DC,然后用DC激活TIL,观察TIL在体外对自体结肠癌细胞和VoLo细胞的杀伤活性。结果:DC激活的TIL对自体结肠癌细胞具有很强的杀伤活性,杀伤率为87.62%±3.01%,明显高于未经DC激活的TIL、DC激活的T淋巴细胞和未经DC激活的T淋巴细胞对自体结肠癌细胞的杀伤率分别为53.72%±1.50%、52.23%±1.46%和3.55%±0.25%,而它们对VoLo细胞的杀伤活性则相对较低。结论:结肠癌患者外周血DC能诱导TIL产生高效而特异的抗结肠癌免疫活性。     

抗原致敏DC诱导CIK细胞对肺腺癌细胞的杀伤作用

[目的]研究肿瘤抗原致敏的树突状细胞（DC）诱导淋巴因子激活的杀伤细胞（LAK）和细胞因子诱导的杀伤细胞（CIK）对肺腺癌原代细胞的杀伤作用，并与单独LAK、CIK细胞的杀伤效果进行比较。[方法]取健康人外周血单个核细胞（PBMNC），常规诱导出DC、CIK、LAK细胞；用肺癌A549细胞提取的肿瘤抗原冲击DC，倒置显微镜下观察DC形态，流式细胞仪检测DC经抗原冲击和未经抗原冲击后其表型变化；把CIK细胞、DC-CIK细胞、LAK细胞和DC-LAK细胞作为效应细胞，肺腺癌原代细胞作为靶细胞，共分为4组，在10：1、20：1、50：1的效靶比时，进行杀伤试验，使用LDH释放法测定杀伤活性。[结果]DC经肿瘤抗原冲击后在镜下呈典型成熟形态；流式细胞仪检测DC经肿瘤抗原冲击和未经肿瘤抗原冲击其表面分子CD40、CD80、CD86和HLA-DR的表达，前者明显高于后者，两者有显著性差异（P〈0．01）；DC—CIK细胞对肺腺癌原代细胞的杀伤活性高于DC—LAK细胞、CIK细胞和LAK细胞（P〈0．05），随着效靶比的升高，DC-CIK细胞对肺癌细胞的杀伤效应随之增强（P〈0．05）。[结论]肿瘤抗原致敏的DC可诱导特异性CIK细胞，DC-CIK细胞对肺腺癌原代细胞的杀伤作用明显高于DC—LAK、CIK、LAK细胞。     

MGBA负载脐带血DC诱导特异性CTL对乳腺癌细胞的杀伤作用

目的：观察乳腺珠蛋白（mammaglobin A, MGBA）负载脐带血来源树突状细胞（dendritic cell, DC）诱导产生的细胞毒性T细胞（cytotoxic T lymphocyte, CTL）对乳腺癌细胞的体外杀伤效果。方法：采集健康剖宫产女性志愿者的脐带血,分离脐带血单个核细胞并诱导DC生成,用MGBA致敏DC与自体淋巴细胞共培养诱导CTL。采用流式细胞术测定DC的表型(CD83、CD86、HLADR)变化,ELISA法测定IL-10、IL-12分泌水平,CCK-8法测定CTL对乳腺癌细胞的杀伤活性。结果：成功培养出形态典型、功能成熟的DC,MGBA负载DC诱导的MGBA特异性CTL对乳腺癌细胞MDA-MB-415 产生显著的杀伤效果（P<0.05）;加入HLA-I 抗体可显著减弱杀伤效果,加入HLA-II 抗体细胞毒活性无显著变化,加入HLA-I 抗体或HLA-II 抗体对正常乳腺细胞的杀伤性均无影响。结论：MGBA 能明显加强脐带血DC 诱导的CTL 对乳腺癌细胞的杀伤活性,该杀伤活性具有MHC限制性。     

人脐血树突状细胞抗神经胶质瘤的体外免疫效应研究

目的　研究肿瘤抗原致敏的树突状细胞 (DC)对神经胶质瘤细胞的免疫杀伤效应。方法　应用免疫磁珠分选脐血 CD34细胞 ,经 SCF FL3 GM- CSF IL- 4 TNF- α的联合诱导培养 DC,采用相差显微镜观察树突状细胞的形态 ;流式细胞仪作 DC的表面标志检测 ;MTT比色法测定同种异型的混合淋巴细胞反应能力和诱导CTL 毒性的检测。结果　 (1)脐血 CD34细胞在体外经细胞因子联合刺激后呈典型的 DC形态 ;(2 )流式检测 CD4 0、CD80和 CD86等成熟 DC特异性表面标志呈高表达 ,分别与诱导前比较 ,差异均有显著性 ;(3) MTT法测得脐血来源的 DC较外周血 DC刺激同种异体 T淋巴细胞增殖能力弱 ;经肿瘤抗原负载的 DC体外诱导出较强 CTL 毒性。结论　脐血 CD34细胞经体外扩增诱导的 DC具有典型的 CTL 毒性 ,为临床应用脐血来源 DC抗神经胶质瘤的生物治疗提供了理论依据     

肺癌可溶性抗原联合超抗原诱导的DC疫苗对肺癌细胞杀伤作用的研究

背景与目的 通过经肺癌肿瘤可溶性抗原(TSA)和超抗原金黄色葡萄球菌肠毒素A(SEA)联合修饰致敏树突状细胞(DC)体外诱导对肺癌细胞杀伤作用的研究,为以DC为基础的肺癌免疫瘤苗的临床应用提供一定的实验依据.方法 3M KCI法提取人肺癌可溶性抗原;从人外周血单个核细胞(PBMC)中诱导扩增DC并鉴定;肺癌TSA和不同质量浓度的SEA联合修饰致敏DC;以联合抗原修饰后的DC、单纯肺癌抗原致敏的DC和未经抗原修饰的DC分别与同种异体T淋巴细胞共同孵育,MTT法测定混合淋巴细胞反应中DC的免疫刺激活性,诱导产生具有识别肺癌抗原的特异性CTL(作为效应细胞分别称为TSA-SEA-DCL、TSA-DCL、DCL);用流式细胞仪FCS及免疫染色法分析鉴定DC和效应细胞表型;M1T法检测各效应细胞对靶细胞体外杀伤效应.结果 诱导出表达CD1a,CD80,HLA-DR分子的DC;经肺癌TSA和SEA联合修饰致敏后,上述分子表达上调;联合修饰后的DC具有较强的免疫刺激活性,DC/T比例1:10可能为最适比例;TSA-SEA-DCL中CD3+CD8+细胞的比例大幅度增加;TSA-SEA-DCL对靶细胞GLC-82的杀伤率明显高于TSA-DCL及DCL,亦明显高于对肺癌CALU-6和人红白血病K562细胞的杀伤率.结论 经肺癌TsA和sE^联合修饰致敏的DC可诱导同种异体T淋巴细胞活化增殖产生CD8+表达增加的CTL;联合抗原诱导的DC疫苗对肺癌细胞有高效特异性的杀伤作用,肺癌TSA和超抗原SEA联合修饰的DC的活性明显强于单用肺癌TSA修饰.     

树突状细胞诱导抗人肝癌细胞系的肿瘤免疫

 目的 以树突状细胞(DC)在体外诱导抗肝癌免疫.方法 自肝癌患者外周血中分离DC;以粒/巨噬细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)联合刺激DC;以人肝癌细胞系HepG2细胞和BEL-7402细胞的肿瘤相关抗原(TAA)激活DC;DC诱导自体T淋巴细胞增殖、分化为细胞毒素性T细胞(CTL);检测CTL对HepG2细胞、BEL-7402细胞、SGC-7901细胞、LOVO细胞及HOS-8603细胞的细胞毒作用.结果 肝癌患者外周血DC能够诱导自体T淋巴细胞增殖分化为CTL,该CTL对HepG2细胞和BEL-7402细胞有强大的杀伤力(杀伤率分别为90%±10%,86%±11%),对SGC-7901细胞、LOVO细胞及HOS-8603细胞则无明显的细胞毒作用(杀伤率分别为11%±6%,8%±4%,6%±4%).结论 肝癌患者外周血DC体外能够诱导高效而特异抗肝癌免疫.提示DC可能在治疗肝癌及预防肝癌术后复发和转移中发挥重要作用.     

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

负载肝癌抗原的树突状细胞瘤苗诱导的抗肿瘤作用

目的 探讨原发性肝癌患者外周血树突状细胞（DC）体外经自体肝癌细胞抗原致敏后诱导的抗肿瘤作用。方法肝癌患者外周血经梯度密度离心法分离，获得DC前体细胞，用重组人粒细胞-巨噬细胞集落刺激因子（rhGM—CSP）和重组人白细胞介素-4（rhIL-4）联合培养，诱导扩增DC。制备自体肝癌细胞抗原，体外脉冲DC，检测DC诱导自体T细胞增殖能力及细胞毒性T细胞（CTL）在体外对自体肝癌细胞的杀伤活性，并检测肿瘤抗原致敏DC分泌的IL-12水平。结果经自体肝癌细胞抗原致敏的DC能分泌IL-12和诱导较强的自体T细胞增殖，且能诱导特异性CTL，该CTL对自体肝癌细胞具有很强的杀伤活性，杀伤率明显高于DC、未经肝癌细胞抗原致敏的DC激活的CTL及T淋巴细胞的杀伤率，而对3LLLEWIS肺癌细胞、H22肝癌细胞则无明显的．杀伤作用。结论肝癌患者外周血DC经自体肝癌细胞抗原致敏后能诱导高效而特异的抗肝癌免疫，其机制可能与增强T细胞应答和诱导机体产生肿瘤特异CTL而发挥特异性的抗肿瘤作用有关。     

树突状细胞负载的exosome疫苗抗胶质瘤的实验研究

目的：旨在分离胶质瘤细胞释放的exosomes,致敏外周血树突状细胞（dendritic cell,DCs）,观察其对细胞毒性T淋巴细胞（cytotoxic T lymphocytes,CTLs）的激活效应。方法：离心超滤和蔗糖密度梯度离心法分离胶质瘤细胞释放的exosomes,固相免疫电镜法（SPIEM）制备exosomes的MAGE-1及ICAM-1免疫电镜标本。常规方法从外周血单个核细胞诱导DCs并分离T细胞,将胶质瘤细胞来源的exosomes冲击或未冲击的DCs与T细胞共培养。MTT比色法检测体外细胞毒活性。结果：胶质瘤细胞分泌的exosomes为直径50-100nm的膜性微囊,经抗MAGE-1、ICAM-1抗体-胶体金免疫电镜标记,囊外膜可见点状？颗粒状电子致密物沉积。培养后DCs的表面标志物CD1a、HLA-DR、CD83、CD86的表达率较培养前明显升高,差异有显著性（P〈0.05）。exosomes致敏的外周血DCs激活CTLs的能力显著高于肿瘤冻融抗原致敏的DCs组,在效靶比为50：1时,两组CTLs对胶质瘤细胞的杀伤率为70.4%±4.1%vs30.1%±2.8%,（P〈0.05）。结论：胶质瘤细胞分泌的exosomes负载外周血DCs后活化CTLs,发挥抗肿瘤活性,可以作为一种有效的抗胶质瘤免疫治疗策略和方法。     

Dendritic cells transduced with tumor-associated antigen gene elicit potent therapeutic antitumor immunity: comparison with immunodominant peptide-pulsed DCs

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-gamma by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.     

抗原冲击树突状细胞介导细胞毒性T淋巴细胞特异性抗白血病作用的体外研究

 目的　研究白血病来源树突状细胞体外诱导的有效方法;观察不同抗原诱导的特异性细胞毒性T淋巴细胞（CTL）的抗白血病效应。方法　分离白血病患者骨髓单个核细胞,经钙离子载体A23187诱导分化,将弱酸洗脱抗原、低渗抗原分别冲击树突状细胞,96 h后树突状细胞与T细胞共同培养,采用MTT法比较CTL细胞对白血病细胞的杀伤活性。结果　骨髓来源的单个核细胞经过100 ng／ml的GM-CSF、500 ng／ml钙离子载体A23187成功诱导为树突状细胞,倒置显微镜下具有树突状细胞的典型形态,流式细胞术测定其CD1a、CD83表达较诱导前明显升高,差异有统计学意义（P＜0.01）。弱酸洗脱法获得抗原冲击树突状细胞致敏T细胞后杀伤白血病的能力最高,未负载抗原的树突状细胞致敏T细胞杀伤白血病细胞的能力最低,两者差异具有统计学意义（P＜0.01）。结论　白血病来源的骨髓单个核细胞经GM-CSF、钙离子载体A23187能够成功诱导为树突状细胞;弱酸洗脱后获得的抗原冲击树突状细胞致敏T细胞能够获得更强的杀伤白血病细胞的能力。     

Hyperthermia improves cellular immune response to human hepatocellular carcinoma subsequent to co-culture with tumor lysate pulsed dendritic cells

Dendritic cells play a major role in cellular immunity. The crucial steps of antigen presentation and processing by DCs may be limiting factors for adoptive cellular immunotherapy. Here, we investigated whether hyperthermia of human hepatocellular carcinoma (HCC) cells induces enhanced cytotoxic cellular immune response. Peripheral blood mononuclear cell (PBMC)-derived DCs were pulsed with tumor cell lysate of the human HCC cell line HepG2, which had been heat shocked prior to incubation for 5 h. Subsequent to TNFalpha-induced maturation DCs were co-cultured with autologous CD4+ and/or CD8+ cells, and T cell mediated cytolysis of HepG2 cells was assessed. We observed enhanced CD4+/8+ cellular cytotoxicity against HepG2 cells subsequent to co-culture with the heat shocked tumor lysate pulsed DCs as compared to pulsing DCs with lysate of non-heat shocked tumor cells. The improved cellular immune response can be related to enhanced expression of HSP 70 and 90 in HepG2 cells upon hyperthermia.     

DC活化淋巴细胞对自体乳腺癌细胞的杀伤作用

 目的探讨负载自体抗原DC诱导的CTLs对自体乳腺癌细胞的杀伤作用。方法以负载自体癌细胞抗原的DCs体外诱导CTLs,用ELISA法检测IFN-γ和IL-12的表达水平,用LDH法检测CTL对自体癌细胞的杀伤作用。结果负载自体抗原DC组IFN-γ和IL-12的浓度高于未致敏DC组、抗原组及单核细胞对照组(P<0.05),且负载抗原DC组所刺激的CTLs的杀伤作用也强于未致敏DC组、抗原组及单核细胞组(P<0.01)及对照的MCF-7组和HT-29组(P<0.01)。结论肿瘤裂解物致敏DC可持续刺激特异性CTLs从而可在体外杀伤乳腺癌患者的自体肿瘤细胞,说明肿瘤抗原致敏的DC疫苗对于治疗残留的和(或)对化疗耐药乳腺癌可能是适合的,因而有可能成为标准的补救措施。     

K-ras 抗原致敏的DC诱导CTL 对胰腺癌体内杀伤活性的研究*

目的:研究K-ras多肽的致敏树突状细胞（DC）活化的特异性细胞毒性T 淋巴细胞（CTL）对胰腺癌的体内外杀伤作用。方法:联合应用粒细胞- 巨噬细胞集落刺激因子和白细胞介素-4 诱导培养外周血DC。表达K-ras突变体的胰腺癌细胞株全瘤、单纯K-ras突变体多肽和K-ras突变体表位肽阳离子纳米颗粒分别致敏DC。致敏DC刺激T 淋巴细胞得到肿瘤抗原特异的细胞毒性T 淋巴细胞（CTL）。 Patu 8988、SW1990细胞系制备荷瘤裸鼠模型评价CTL 体内抗肿瘤活性。结果:负载全瘤抗原的DC其诱导产生的CTL 对胰腺癌有较好的抑制,负载单纯K-ras（12-Val ）突变体多肽、K-ras（12-Val ）突变体表位肽阳离子纳米颗粒的DC其诱导产生的CTL 对表达K-ras（12-Val ）突变体阳性（Patu 8988）的胰腺癌有较特异的抑制作用,而对K-ras（12-Val ）突变体阴性（SW1990）的胰腺癌的抑制作用与对照组比较无显著性差异。结论:负载肿瘤抗原的DC诱导的CTL 可显著提高对荷瘤裸鼠的生存时间,抑制肿瘤的生长速度,并显示其可增加抗肿瘤特异性。      

Enhancement of DNA vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain of Fms-like tyrosine kinase 3-ligand

Recently, Flt3 (Fms-like tyrosine kinase 3)-ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). A recombinant chimera of the extracellular domain of Flt3-ligand (FL) linked to a model antigen may potentially target the antigen to DCs and their precursor cells. Using human papillomavirus-16 E7 as a model antigen, we evaluated the effect of linkage to FL on the potency of antigen-specific immunity generated by naked DNA vaccines administered intradermally via gene gun. We found that vaccines containing chimeric FL-E7 fusion genes significantly increased the frequency of E7-specific CD8+ T cells relative to vaccines containing the wild-type E7 gene. In vitro studies indicated that cells transfected with FL-E7 DNA presented E7 antigen through the MHC class I pathway more efficiently than wild-type E7 DNA. Furthermore, bone marrow-derived DCs pulsed with cell lysates containing FL-E7 fusion protein presented E7 antigen through the MHC class I pathway more efficiently than DCs pulsed with cell lysates containing wild-type E7 protein. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing metastatic tumors. The FL-E7 fusion vaccine mainly targeted CD8+ T cells, and antitumor effects were completely CD4 independent. These results indicate that fusion of a gene encoding the extracellular domain of FL to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways.     

抗原致敏树突状细胞诱导CIK对肺癌细胞杀伤作用的研究

[目的]研究肿瘤抗原致敏的树突状细胞（DC）诱导淋巴因子激活的杀伤细胞（LAK）和细胞因子诱导的杀伤细胞（CIK）对肺癌细胞株A549和肺腺癌原代细胞的杀伤作用。[方法]取健康人外周血单个核细胞,常规诱导出DC、CIK、LAK细胞。用肺癌A549细胞提取的肿瘤抗原冲击DC,倒置显微镜下观察DC形态。流式细胞仪检测DC经抗原冲击和未经抗原冲击后其表型变化。LDH释放法测定杀伤活性。[结果]DC经肿瘤抗原冲击后在镜下呈典型成熟形态,其表面分子CD40、CD80、CD86和HLA-DR的表达明显较未经肿瘤抗原冲击的DC高。DC＋CIK细胞对A549和肺腺癌原代细胞的杀伤活性高于CIK细胞、LAK细胞和DC＋LAK细胞（P〈0.05）,随着效靶比的升高,其杀伤效应随之增强（P〈0.05）。[结论]肿瘤抗原致敏的DC可诱导特异性CIK细胞,DC＋CIK细胞对A549和肺腺癌原代细胞的杀伤作用明显高于DC＋LAK、CIK、LAK细胞。