gp96-肽复合物体外诱导脾淋巴细胞及其抗肿瘤效应

目的研究H22肿瘤细胞来源的热休克蛋白gp96-多肽复合物在体外诱导小鼠脾淋巴细胞的特异性的细胞毒性T细胞(CTL)的产生及其抗肿瘤效应。方法利用蛋白提取纯化技术、细胞培养技术、流式细胞术、CCK-8法、免疫荧光技术等。结果经鉴定获得纯化的热休克蛋白;流式细胞仪检测表明,经gp96-肽复合物诱导后的CD8T细胞比例达到近70%,远远高于对照组的35%、26%;该活化的CTL细胞在效靶比为50∶1时的肿瘤杀伤率达72%与对照组相比具有统计学意义;激光共聚焦显微镜观察证实实验诱导组的培养上清能诱导H22肿瘤细胞凋亡的形态学改变。结论肿瘤来源的热休克蛋白gp96-肽复合物能诱导小鼠脾淋巴细胞的CTL反应,该活化的CTL具有特异性抗H22肿瘤细胞的免疫保护作用并能分泌免疫活性物质诱导H22肿瘤细胞凋亡。     

gp96-肽复合物体外诱导CTL反应及其抗肿瘤效应研究

目的研究小鼠肝癌腹水瘤H22细胞来源的gp96-肽复合物体外诱导小鼠脾淋巴细胞特异性细胞毒T细胞的产生及其抗肿瘤效应.方法利用蛋白提取纯化技术、Western-blot法、细胞培养技术、流式细胞术、CCK-8法、RT-PCR法等研究gp96-肽复合物体外诱导扩增CTL及其抗肿瘤效应.结果流式细胞仪检测表明,经gp96-肽复合物诱导后的CD8 T细胞比例达到近70%,远远高于对照组的35%、26%.该活化的CTL细胞在效靶比为50:1时的肿瘤杀伤率达72%与对照组相比具有统计学意义;将活化的CTL细胞回输小鼠体内能产生对该肿瘤细胞良好的过继性免疫保护,延长荷瘤小鼠的生存时间;RT-PCR法检测该活化的脾细胞较正常脾淋巴细胞高表达IFN-γ mRNA.结论肿瘤来源的热休克蛋白gp96-肽复合物能诱导小鼠脾淋巴细胞的CTL反应,并增强脾细胞 IFN-γ mRNA的表达,该CTL具有特异性抗H22肿瘤细胞的免疫保护作用.     

利用gp96-肽复合物免疫小鼠脾淋巴细胞治疗H22肝癌的实验研究

目的探讨肿瘤细胞中提纯的热休克蛋白gp96-肽复合物的特异性肿瘤免疫作用对小鼠脾淋巴细胞的影响及其抗瘤作用和机制。方法将经gp96-肽复合物免疫过的小鼠的脾淋巴细胞作为效应细胞,用M TT法测定其体外细胞毒性;用gp96-肽复合物免疫小鼠的脾淋巴细胞进行特异性免疫腹腔回输,观察荷瘤鼠的生存时间;将上述效应细胞注射入直径约1 cm的小鼠皮下移植肝癌内,观察其抗瘤效果;用透射电镜观察瘤细胞的变化。结果gp96-肽复合物免疫小鼠的脾淋巴细胞较未经免疫小鼠脾淋巴细胞对靶细胞的杀伤率显著提高;gp96-肽复合物免疫小鼠脾淋巴细胞治疗组的小鼠瘤重最小,与各对照组相比差异有显著性;体内回输特异性免疫脾淋巴细胞显著提高了H 22荷瘤小鼠的生存时间。结论经gp96-肽复合物免疫激活的淋巴细胞,其毒性杀伤作用可能是引起肿瘤细胞凋亡的重要原因。     

HIFU活化的CTL免疫治疗后荷瘤鼠免疫功能变化

[目的]探讨过继免疫高强度聚焦超声(HIFU)治疗后活化的杀伤性T淋巴细胞(CTL)引起的荷瘤鼠肿瘤免疫功能变化.[方法] C57BL/ 6J近交系荷瘤鼠在H22肝癌移植后7d分别接受HIFU治疗和HIFU假照治疗,治疗后14d分离两组荷瘤鼠和正常小鼠外周血CTL.选30只H22荷瘤鼠随机分为治疗组、假照组和对照组,分别尾静脉注射之前分离的3组CTL.于过继免疫治疗后2周测定各组小鼠外周血T淋巴细胞亚群的变化,采用MTT法测定脾淋巴细胞的体外杀伤活性,ELISA法检测脾淋巴细胞与肿瘤细胞共培养24h后上清液IFN-γ、TNF-α含量.[结果]CTL过继免疫治疗后2周,与假照组和对照组比较,治疗组小鼠肿瘤体积较小,外周血CD4+水平明显增加,CD8+水平明显降低,CD4+/CD8+的比值明显升高,脾脏淋巴细胞对H22肿瘤细胞毒活性明显增加,与H22肿瘤细胞共培养上清液INF-γ和TNF-α浓度明显增加,差异有显著统计学意义.[结论] HIFU活化的CTL过继免疫治疗H22移植性肝癌,宿主细胞免疫功能增强,同时机体产生抗肿瘤的免疫作用.     

热休克蛋白72-甲胎蛋白抗原表位肽复合物抗小鼠肝癌Hepal-6细胞免疫的实验研究

目的:以热休克蛋白72(HSP72)-甲胎蛋白(AFP)抗原表位肽复合物免疫小鼠,研究该复合物针对AFP肿瘤是否具有特异性抗肿瘤免疫.方法:以HSP72-AFP多肽复合物皮下注射免疫昆明小鼠,并分别以AFP多肽和HSP72单独免疫小鼠为对照组.ELISA法检测免疫后小鼠血清IFN-γ水平、MTT法检测各免疫组小鼠淋巴细胞对肝癌Hepal-6细胞的杀伤作用、以小鼠体内瘤负荷实验评价蛋白复合物的免疫效应.结果:HSP72-AFP多肽复合物组小鼠血清IFN-γ水平、淋巴细胞对Hepal-6细胞的杀伤作用均显著高于AFP多肽和HSP72组(P＜0.01).HSP72-AFP多肽复合物组瘤体积也明显小于AFP多肽和HSP72免疫组(P＜0.01).结论:HSP72-AFP多肽复合物疫苗可诱导荷瘤小鼠产生针对AFP肿瘤的特异性细胞免疫,其对瘤细胞的杀伤效应明显优于两者单一的多肽纯化疫苗.表明HSP72-AFP多肽复合物可诱导小鼠产生有效的抗肿瘤免疫.     

肺癌gp96-多肽复合物/DC疫苗体外诱导CTL反应的实验研究

目的 研究热休克蛋白gp96－多肽复合物负载树突状细胞（dendritic cell,DC）后，能否诱导出gp96－多肽复合物特异性的细胞毒性T细胞（cytotoxic T lymphocyte,CTL）反应。方法 从一例肺癌患者肿瘤组织中提取gp96-多肽复合物和自体肿瘤细胞溶解物，分别负载从该患者骨髓血中培养的DC。以不同形式的抗原／DC疫苗分别刺由患者外周血中分离的淋巴细胞。采用ELISA法检测淋巴细胞所释放的IFN－γ量作为CTL反应的指标；以Cr^51释放实验分析致敏后的淋巴细胞对不同靶细胞的裂解和杀伤作用。结果 所有肿瘤抗原致敏淋巴细胞后均可以诱导产生CTL反应，其中以gp96－多肽复合物／DC疫苗诱导释放的IFN－γ量最高。肿瘤抗原致敏淋巴细胞后对原代培养的肿瘤细胞的杀伤作用高于PG细胞和K562细胞。结论 自体肿瘤组织中提取的gp96－多肽复合物能诱导出肿瘤特异性CTL反应，而负载DC后能激发起更强的CTL。     

肝癌热休克蛋白—抗原肽复合物的构建及诱导CTL反应的初步研究

目的：研究体外构建热休克蛋白-抗原肽复合的方法,观察其在体外的抗肿瘤作用。方法：用经43℃热处理1小时的人肝细胞悬液,经过裂解液裂解,60%-80%饱和硫酸铵沉淀,用SephadexG-100柱制备,取分子量为70KD组份,Westen blot进行性质鉴定。应用多肽解离液处理该组份,SephadexG-25柱过滤获得未结合多肽的蛋白分子,使其在体外与肝癌抗原肽SLIVHLNEV结合,构建成热休克蛋白-抗原肽复合物。应用此复合物树突状细胞,激活同源外周血T淋巴细胞产生肿瘤特异性杀伤T淋巴细胞（CTL）,应用MTT法检测其对T2细胞及肿瘤细胞的杀伤活性。结果：所得蛋白经电泳及Western blot进行蛋白分子量及性质鉴定为热休克蛋白70。SephadexG-25柱双分离法证实应用上述方法成功构建了肝癌热休克蛋白70-抗原肽复合物,用该复合物负荷树突状细胞在体外可以诱导出较强的肝癌抗原肽物特异性CTL,可以杀伤负荷有该肽的T2细胞及递呈该肽的肿瘤细胞系。结论：体外构建的热休克蛋白-抗原肽复合物可以增强抗原肽诱导CTL反应能力,热休克蛋白是良好的T细胞免疫佐剂,有可能在肿瘤疫苗治疗中发挥重要作用。     

肿瘤基因工程纳米疫苗NL(MH)抗肿瘤复发的实验研究

目的：在小鼠体内观察纳米疫苗NL（MH）抗肿瘤复发作用。方法：以PBS、空白脂质体、MH、MH／NL、NL（MH）免疫C57BL／6小鼠3次后，IFN-γ ELISPOT和LDH杀伤实验检测纳米疫苗激活小鼠特异性细胞免疫反应的情况；以肿瘤攻击实验和肿瘤切除后复发实验来评价纳米疫苗预防肿瘤复发作用。结果：与对照组相比，NL（MH）组小鼠脾淋巴细胞中分泌IFN-γ的T细胞数量明显增多（P〈0．05），CTL对B16-MAGE3细胞具有显著的特异性杀伤作用；在肿瘤攻击实验中，NL（MH）组B16-MAGE3肿瘤成瘤时间长、成瘤率低；肿瘤切除后，NL（MH）组B16-MAGE3肿瘤复发时间延迟，复发率明显降低。结论：NL（MH）能够刺激机体产生强烈的MAGE3特异性的细胞免疫反应，对表达MAGE3的肿瘤细胞具有显著的杀伤作用，能有效预防B16-MAGE3切除后复发。     

Hsp70-肿瘤抗原肽复合物防治小鼠黑色素瘤B16肺转移的作用

目的:探讨热休克蛋白70-肿瘤抗原肽复合物(Hsp70-antigen peptide complexes)对小鼠黑色素瘤B16转移的防治作用.方法:分别从小鼠腿部接种的B16实体瘤及小鼠肺B16转移灶提取混合抗原肽,体外与Hsp70结合制得复合物,此复合物免疫小鼠后用于预防或治疗经尾静脉接种转移至肺的B16黑色素瘤,观察其对肿瘤转移的防治作用.结果:Hsp70-肿瘤抗原肽复合物免疫后肺转移灶节结数显著减少(P＜0.01),体外脾细胞表现出对B16较高的杀伤率(P＜0.01);并对肺转移灶有显著的治疗作用(P＜0.01),而从B16实体瘤提取的混合抗原肽制得的复合物比从肺转移灶提取的混合抗原肽制得复合物有更好的治疗效果(P＜0.01),表现出体外脾细胞对B16更高的杀伤率(0.01＜P＜0.05).结论:Hsp70-肿瘤抗原肽复合物对肿瘤的转移有明显的防治作用,而从实体瘤提取的混合抗原肽比从转移灶提取的混合抗原肽更为有效.     

树突状细胞对肿瘤浸润淋巴细胞抗小鼠肝癌活性影响的研究

目的探讨H22细胞全细胞性抗原致敏的DC激活的TIL体外抗小鼠肝癌活性,并将H22-DC-TIL过继免疫荷瘤小鼠,研究其对荷瘤小鼠免疫功能的影响及抑瘤作用。方法从小鼠四肢长骨骨髓中获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤全细胞性抗原致敏DC,然后用DC激活TIL,观察TIL在体外对H22细胞、Hepal-6细胞和B16细胞的杀伤活性;检测应用H22-DC-TIL后荷瘤小鼠的脾淋巴细胞的NK、LAK、CTL活性、血清TNF活性、抑瘤作用以及瘤体病理改变,并与对照组相比较。结果①H22-DC-TIL具有很强的对H22细胞杀伤活性[杀伤率为(71.31±3.11)%],明显高于其对Hepal-6和B16细胞的杀伤活性[杀伤率分别为(50.11±3.03)%,(30.31±2.89)%],也明显高于未经DC激活的TIL、H22-DC-脾淋巴细胞和未经DC激活的脾淋巴细胞对H22细胞杀伤活性[杀伤率分别为(49.80±3.21)%,(48.76±3.60)%和(19.23±2.71)%]和对Hepal-6细胞杀伤活性[杀伤率分别为(39.40±3.21)%,(38.62±2.87)%和(18.73±2.40)%]以及对B16细胞杀伤活性[杀伤率分别为(26.38±2.51)%,(25.82±2.70)%和(18.34±3.01)%],同时B16-DC-TIL(TIL来源于H22瘤体)也可诱导相对较低的对B16细胞的特异性细胞杀伤活性。②H22-DC-TIL可明显诱导提高荷瘤脾淋巴细胞NK、LAK和CTL活性[活性为(30.43±1.35)%、(31.40±1.80)%、(35.30±1.20)%],并可检测到血清TNF水平明显上升[血清TNF为(40.41±1.85)U/ml],它们均达正常对照组水平,与未经DC激活的TIL组、H22-DC-脾淋巴细胞组、未经DC激活的脾淋巴细胞组、生理盐水组分别对应比较,差异均有显著性(P<0.01)。该组瘤体内淋巴细胞浸润程度也高于对照组,其瘤体生长明显受到抑制。结论①H22-DC-TIL可产生很强的体外针对H22细胞的特异性杀伤活性。②H22-DC-TIL具有很强的特异性抗小鼠肝癌作用。     

Anti-Tumor Effect of Heat Shock Protein 70-Peptide Complexes on A-549 Cells

Objective: To investigate the anti-tumor immunity in vitro of heat shock protein 70-peptide complexes (HSP70-PC) from human lung cancer tissue. Methods: HSP70-PC was purified from lung tumor tissues and corresponding non-tumor lung samples with the methods of ADP-affinity chromatography, DEAE ion-exchange chromatography and Western-blot. The activation and proliferation of PBMC induced by different HSP70-PC and tumor cytotoxic reactivity to A549 cells in vitro were measured by the MTT cell proliferation assay. Results: The purified HSP70-PC had a very high purity found by SDS-PAGE and Western-blot. Human lymphocytes were sensitized efficiently by HSP70 preparation purified from lung cancer tissues and a definite cytotoxicity to A-549 cells was observed. There was significant difference with HSP70-PC purified from lung cancer, compared with the control group (P<0.001). Conclusion: High purity of HSP70-PC could be achieved from tumor tissues in this study. HSP70-PC purified from human tumor tissues can induce anti-tumor immunity in vitro mainly implemented by eliciting CTL immunity.     

HSP70-Id复合物修饰的树突状细胞体外诱导特异性抗肿瘤作用

目的观察人慢性B淋巴细胞性白血病（B-CLL）细胞的独特型抗原Id-ScFv与热休克蛋白70（HSP70）形成复合物修饰的树突状细胞（DC）体外诱导特异性抗肿瘤作用,并初步探讨其机制。方法将HSP70与Id-ScFv体外结合形成复合物HSP70-Id,修饰自人外周血单核细胞获取的DC。倒置相差显微镜观察DC的形态特征;流式细胞仪检测修饰前后DC的表型变化,酶联免疫吸咐试验（ELISA）检测DC分泌的白细胞介素12（IL-12）和肿瘤坏死因子-α（TNF-α）,四甲基偶氮唑蓝（MTT）法检测修饰的DC对自身淋巴细胞的激活和增殖作用,流式细胞仪检测激活的自身淋巴细胞T细胞亚群的变化,台盼蓝染色法检测其对Daudi、K562和HepG2等细胞的杀伤作用。结果DC体外诱导培养成功,HSP70-Id复合物可使DC成熟,镜下可见典型的DC形态,其CD1a表达率为20％-30％,CD83表达率〉72％,CD86和HLA-DR表达显著增加（P〈0．05）,上清中IL-12、TNF-α亦显著高于DC对照组（P〈0．01）。HSP70-Id复合物修饰的DC激活自身淋巴细胞,对Daudi细胞的杀伤率为71．24％,而对K562细胞杀伤作用较弱,对HepG2细胞无明显作用。其淋巴细胞亚群中,CD4^＋T细胞、CD8^＋T细胞的比例均显著增加,分别为56．51％和70．21％,CD4^＋T细胞／CD8^＋T细胞比值由空白对照组的1．49倒置为0．81。结论HSP70-Id复合物修饰的DC生物学活性增强,经其刺激后,传代培养的淋巴细胞可产生高效而特异性的抗肿瘤免疫效应,可能是CD4^＋T细胞、CD8^＋T细胞及DC协同作用的结果。     

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。     

Intratumoral injection of immature dendritic cells enhances antitumor effect of hyperthermia using magnetic nanoparticles

Dendritic cells (DCs) are potent antigen-presenting cells that play a pivotal role in regulating immune responses in cancer and have recently been shown to be activated by heat shock proteins (HSPs). We previously reported that HSP70 expression after hyperthermia induces antitumor immunity. Our hyperthermia system using magnetite cationic liposomes (MCLs) induced necrotic cell death that was correlated with HSP70 release. In the present study, we investigated the therapeutic effects of DC therapy combined with MCL-induced hyperthermia on mouse melanoma. In an in vitro study, when immature DCs were pulsed with mouse B16 melanoma cells heated at 43 degrees C, major histocompatibility complex (MHC) class I/II, costimulatory molecules CD80/CD86 and CCR7 in the DCs were upregulated, thus resulting in DC maturation. C57BL/6 mice bearing a melanoma nodule were subjected to combination therapy using hyperthermia and DC immunotherapy in vivo by means of tumor-specific hyperthermia using MCLs and directly injected immature DCs. Mice were divided into 4 groups: group I (control), group II (hyperthermia), group III (DC therapy) and group IV (hyperthermia + DC therapy). Complete regression of tumors was observed in 60% of mice in group IV, while no tumor regression was seen among mice in the other groups. Increased cytotoxic T lymphocyte (CTL) and natural killer (NK) activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of B16 melanoma cells. This study has important implications for the application of MCL-induced hyperthermia plus DC therapy in patients with advanced malignancies as a novel cancer therapy.     

MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor

Summary Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n=23), untransduced DCs (n=17), or sterile saline (n=22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challengein vivo.     

MART-1 Adenovirus-Transduced Dendritic Cell Immunization in a Murine Model of Metastatic Central Nervous System Tumor

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially.Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n = 23), untransduced DCs (n = 17), or sterile saline (n = 22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival.Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challenge in vivo.     

体外构建的HTA-HSP70BCG冲激的树突状细胞疫苗的抗肿瘤作用

 目的 观察体外构建的榄香烯复合瘤苗抗原-卡介苗热休克蛋白70复合物（H_TA—HSP70_BCG）诱导的树突状细胞疫苗的抗肿瘤效应。方法 来源于小鼠的肝癌Hca-F榄香烯复合疫苗的抗原（HTA）与卡介苗来源的HSPT0（HSP70_BCG）在体外构建成HTA—HSP70_BCG复合物，用GM-CSF和IL-4诱导树突状细胞（DCs），分别用HTA—HSP70_BCG、HTA和HSP70麟对其冲激。用舯法检测该DCs刺激的全脾细胞的增殖活性及被刺激的脾细胞的细胞毒活性。用流式细胞仪检测DCs表面（瑚6和CD40的表达。结果 体外构建HTA—HSP70_BCG可以诱导DCs成熟，表现为DCs表达CD86和CD40上调，该DCs可以刺激全脾细胞增殖并使其产生特异性杀瘤活性，其强度明显大于HTA。结论 体外构建HTA—HSP70麟复合物可以诱导DCs成熟，该DCs可以激活脾细胞产生较强的特异性抗瘤效应。     

Enhanced T-cell immunity induced by dendritic cells with phagocytosis of heat shock protein 70 gene-transfected tumor cells in early phase of apoptosis

The dual role of heat shock protein 70 (HSP70), as antigenic peptide chaperone and danger signal, makes it especially important in dendritic cell (DC)-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T-cell stimulation and vaccine efficacy. We found that DCs with phagocytosis of MCA/HSP in early phase of apoptosis expressed more pMHC I complexes, stimulated stronger cytotoxic T lymphocyte (CTL) responses (40% specific killing at an E:T cell ratio of 50) and induced immune protection in 90% of mice against MCA tumor cell challenge, compared with 25% specific CTL killing activity and 60% immune protection seen in mice immunized with DC with phagocytosis of MCA/HSP in late phase of apoptosis (P<0.05). Similar results were confirmed in another EG7 tumor model also expressing HSP70. Taken together, our data demonstrate that HSP70 on apoptotic tumor cells stimulate DC maturation, and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DCs with phagocytosis of apoptotic tumor cells in late phase of apoptosis. These results may have an important impact in designing DC-based antitumor vaccines.     

rhHSP70联合冻融抗原修饰树突状细胞诱导的抗乳腺癌作用

  目的   利用rhHSP70联合树突状细胞递呈肿瘤抗原的特性提高细胞毒T淋巴细胞（CTLs）对乳腺癌细胞的杀伤活性。   方法   外周血单个核细胞体外经GM-CSF和IL-4诱导产生树突状细胞,负载冻融抗原肽的同时加入新型热休克蛋白（rhHSP70）,不同分组分别诱导自体CTLs产生。ELISA测定CTLs杀伤活性和细胞因子的分泌。   结果   冻融抗原肽致敏的DCs促进CTLs增殖,上调CTLs中CD3+和CD8+T细胞群及Th1型细胞因子的分泌;体外实验中具有对人乳腺癌细胞MCF-7的杀伤活性,在加入rhHSP70后效果更加明显,并能显著增强CTLs对肿瘤细胞的杀伤率。   结论   hHSP70联合肝癌冻融抗原修饰DCs,能够促进DCs的成熟,增强DCs刺激淋巴细胞增殖的能力,诱导的CTLs在体外对乳腺癌细胞能产生高效杀伤力。rhHSP70增强DCs抗肿瘤能力的机制可能与其促进DCs成熟有关。