树突状细胞疫苗对人肺腺癌GLC-82细胞体外杀伤作用的实验观察

目的 通过建立负载人肺腺癌细胞株GLC-82可溶性抗原的树突状细胞(DC)疫苗,探讨应用DC疫苗的致敏特异性杀伤性T细胞(CTL)体外杀瘤细胞的可行性和实验条件,为后期l临床应用提供实验依据.方法 通过用定量摩尔氯化钾提取法获得人肺腺癌细胞GLC-82的可溶性抗原多肽(TSA),从人外周血单核细胞(PBMC)中用GM-CSF、白细胞介素-4和肿瘤坏死因子-α体外诱导扩增并鉴定获取DC,构建DC疫苗;利用DC疫苗刺激同种异体外周血T淋巴细胞活化增殖,诱导产生具有识别肺癌细胞抗原的特异性CTL的可行性及MTT法检测该CTL对GLC-82、肺癌CALU-6和人红白血病K 562细胞的体外杀伤效应.结果 人PBMC体外经7d诱导出的DC,经形态学、免疫组化证实具有典型的树突状细胞特性;负载GLC-82抗原的DC疫苗能有效诱导同种异体T淋巴细胞活化增殖产生CTL,最适浓度为1:10;诱导活化的CTL对靶细胞的杀伤活性明显高于未经肿瘤抗原致敏的组.结论 诱导培养人外周血PBMC中的Mo可获取大量DC,诱导出的DC功能较强,适宜临床应用;DC疫苗能强烈刺激初始型同种异体T淋巴细胞增殖产生CD 8+表达增加的CTL;激活的CTL对肺癌靶细胞发挥高效而特异的细胞毒效应,对非肺组织瘤靶细胞也具有非特异性杀伤效应.     

热休克蛋白抗原肽致敏的脐血树突状细胞治疗肺癌的实验研究

目的研究热休克蛋白抗原肽致敏的脐血树突状细胞体外对肺癌细胞的杀伤作用。方法用体外构建的热休克蛋白-抗原肽复合物刺激经组合细胞因子诱导的脐血树突状细胞,MTT法检测其对肿瘤细胞的杀伤作用。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70。热休克蛋白-抗原肽负载可以促进脐血树突状细胞对T细胞的激发作用,使其对靶细胞有了更强的生长抑制作用。各组T细胞杀伤活性分别为:负载抗原组(85.77±1.03)%(正常T细胞)、(45.01±1.66)%(肺癌患者T细胞);未负载抗原组(41.92±1.38)%(正常T细胞)、(13.99±3.07)%(肺癌患者T细胞)。组间比较差异有统计学意义(P<0.01)。结论热休克蛋白抗原肽负载脐血树突状细胞能有效激发外周血T淋巴细胞,使其对靶细胞有了更强的生长抑制作用。本研究为解决树突状细胞的来源及开发特异性CTL的治疗开创了条件。     

负载冻融抗原的DC诱导特异性CTL杀伤胃癌细胞的实验研究

目的 研究负载肿瘤抗原的树突状细胞(DC)活化的特异性细胞毒性T淋巴细胞(CTLs)对胃癌细胞的体外杀伤作用.方法 冻融法获取胃癌细胞抗原,联合应用粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)诱导培养外周血BC并负载肿瘤抗原,激活自体T淋巴细胞,制备特异性CTLs,MTT法检测对胃癌细胞的体外杀伤作用.ELISA法检测γ干扰素(IFNγ)分泌情况.结果 负载胃癌抗原的DC激活的CTLs表现出对胃癌细胞的特异性杀伤作用,产生高水平的IFNY(P<0.01);而对B16黑色素瘤细胞没有杀伤作用不产生高水平的IFNγ.未负载胃癌抗原DC刺激的CTLs对胃癌细胞无杀伤作用.结论 应用细胞因子从人外周血中诱导的DC负载胃癌抗原后,激活的CTLs在体外对胃癌细胞能产生高效而特异性的杀伤作用.     

热休克蛋白gp96-肽复合物体外对人肝癌细胞的抗肿瘤效应

目的研究热休克蛋白gp96-肽复合物修饰树突状细胞(DC)后对人肝癌细胞的体外特异抗肿瘤效应。方法从人原发性肝癌组织中提取gp96-肽复合物,用其修饰DC细胞后与T淋巴细胞共同培养,以MTT法检测该复合物介导的细胞毒性T细胞(CTL)对不同来源人肝癌细胞的抗肿瘤效应,并与粗提抗原修饰DC细胞组相比较。结果 gp96-肽复合物诱导的CTL对原代肝癌细胞杀伤效率为74.3%,明显高于粗提抗原组(42.5%)和未加抗原组(14.4%)(P<0.01),粗提抗原组对肝癌细胞杀伤效率高于未加抗原组(P<0.01)。且该复合物的抗肿瘤效应具有一定的组织特异性。结论 gp96-肽复合物修饰的DC细胞,在体外能刺激产生特异的抗肿瘤细胞毒性T细胞,因而热休克蛋白gp96在肿瘤免疫治疗中具有重要的实用价值。     

胎儿来源的树突状细胞诱导抗膀胱癌效应的研究

目的：研究胎儿来源的树突状细胞（DC）体外诱导抗膀胱癌的特异性细胞免疫的效果.方法：从胎儿骨髓获得单个核细胞,经粒细胞-单核细胞集落刺激因子（GM-CSF）、IL-4和TNF-α诱导产生DC.利用50%～70%硫酸铵饱和沉淀法获取膀胱癌细胞系EJ含热休克蛋白（HSP）成分的细胞溶解物,以该抗原负载DC,激活胎脾细胞产生肿瘤特异性的细胞杀伤性T淋巴细胞（CTL）.利用IL-2刺激胎脾细胞产生LAK细胞.应用MTF法分别检测CTL和LAK细胞对EJ细胞的杀伤效应.结果：胎儿骨髓可诱导出功能成熟的DC,高表达CD1a、CD86、HLA-DR和CD83.负载EJ抗原的DC可诱导产生CD8＋CTL.其对EJ细胞的杀伤作用明显强于LAK细胞.结论：含HSP成分的肿瘤细胞溶解物负载胎儿来源的DC,体外可诱导出更强的特异性抗肿瘤免疫应答.     

奥沙利铂联合Exosome诱导的效应性T细胞抑制HepG2细胞增殖的研究

目的观察奥沙利铂(OXA)联合负载HepG2细胞裂解物的Exosome诱导的效应性T细胞对肝癌HepG2细胞系增殖的抑制作用。方法自健康人外周血中提取单个核细胞和T淋巴细胞,并以GM-CSF+IL-4的培养方案获得树突状细胞(DC)。将反复冻融后产生的HepG2细胞裂解物体外致敏DC,收集培养上清后以超高速离心法分离得到Exosome,电镜下观察其形态特征。用致敏DC及其分泌的Exosome刺激T淋巴细胞的增殖和活化。以ELISA试验检测不同刺激环境下T细胞分泌IL-1β、IL-2、IL-4、IL-6、IL-10、IFN-γ和TNF-α的能力。以细胞杀伤实验(MTT法)检测不同因素刺激后的T细胞、OXA(2.5、5.0、10.0mg.L-1)以及两者联合应用对HepG2细胞生长的抑制效应。结果与未致敏DC及其分泌的Exosome相比,HepG2细胞冻融裂解物致敏的DC及其分泌的Exosome能显著促进T细胞的增殖,并使其活化和分泌大量的IL-1β、IL-2、IL-6、IFN-γ和TFN-α等Th1型细胞因子。活化后的T细胞、OXA以及两者联合应用对HepG2细胞体外增殖均有抑制作用,而活化T细胞联合10mg.L-1OXA对HepG2细胞的杀伤率最高,但与联合5 mg.L-1OXA的效应相比,差异无统计学意义(P0.05)。结论负载肿瘤细胞抗原的DC所分泌的Exosome可在体外刺激T细胞,使其活化为具有抗肿瘤效应的细胞毒性T细胞,该细胞联合低浓度OXA能显著抑制肝癌细胞的体外增殖,并降低OXA的用量。     

CpGODN结合CD28/CD80单抗活化T细胞对结肠癌细胞的杀伤作用

目的研究联合应用CpGODN和CD28/CD80单抗共刺激健康人外周血T淋巴细胞(PBLs)在体外对结肠癌细胞株HT-29的杀伤作用,为结直肠癌的过继免疫治疗可能性提供参考。方法PBLs的分离与体外培养;CD28/CD80共刺激活化PBLs;用含共刺激活化PBMC或/和CpGODN的100ml/L胎牛血清的RPMI1640培养基培养细胞,测生长曲线。MTT法检测活化细胞的体外淋巴细胞毒作用,并用电镜观察效应细胞杀伤的结肠癌细胞超微结构及用流式细胞仪检测结肠癌细胞的相关凋亡情况。结果CpGODN本身对HT-29细胞有一定的杀伤作用(P<0.05),CD28/CD80共刺激活化PBLs在体外对结肠癌细胞株HT-29杀伤作用明显(P<0.01),CpGODN与CD28/CD80共刺激活化PBLs合用,对HT-29的杀伤作用显著大于CpGODN单独对HT-29细胞杀伤作用(抑制率92.31%vs68.00%,P<0.01),亦大于单独应用CD28/CD80共刺激活化PBMC对HT-29细胞的杀伤作用(P<0.05),CpGODN增加了HT-29细胞对共刺激活化PBMC的敏感性。电镜结果显示,效应细胞作用24h肿瘤细胞就部分发生坏死,部分肿瘤细胞可见凋亡。流式细胞仪检测效应细胞HT-29细胞72h明显凋亡。结论联合应用CpGODN可以提高单抗协同诱导的效应细胞对结肠癌细胞株HT-29杀伤作用,而坏死细胞进一步增多说明效应细胞是通过诱导肿瘤细胞坏死及细胞凋亡两条途径来实现抑制杀伤作用从而说明活化的淋巴细胞杀伤结肠癌细胞是通过坏死及凋亡实现的。     

hKDR融合基因致敏树突状细胞对小鼠肝癌细胞杀伤活性研究

目的:探讨hKDR融合基因致敏树突状细胞(DC)激发的细胞毒性T淋巴细胞(CTL)对小鼠肝癌细胞的杀伤活性.方法:自小鼠骨髓中获取DC,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)和hKDR融合基因的mRNA致敏DC;用致敏的DC免疫小鼠取其脾脏获得CTL,即效应细胞,用脂质体将pCDNA hKDR转染至小鼠肝癌Hepal-6细胞株中,作为靶细胞;将效应细胞与靶细胞按不同比例混合,用乳酸脱氢酶(LDH)释放法检测细胞毒作用.结果:未经修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为19.2%、12.3%、6.9%;经hKDR修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为71.6%、55.8%、22.7%,将两组结果在相同的比例下两两比较,均有显著性差异(P＜0.05).结论:hKDR融合基因致敏DC激活的CTL对小鼠肝癌细胞具有较强的杀伤作用,且杀伤能力随效应细胞与靶细胞比例增高而增强.     

hKDR融合基因致敏树突状细胞对小鼠肝癌细胞杀伤活性研究

目的:探讨hKDR融合基因致敏树突状细胞(DC)激发的细胞毒性T淋巴细胞(CTL)对小鼠肝癌细胞的杀伤活性.方法:自小鼠骨髓中获取DC,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)和hKDR融合基因的mRNA致敏DC;用致敏的DC免疫小鼠取其脾脏获得CTL,即效应细胞,用脂质体将pCDNA hKDR转染至小鼠肝癌Hepal-6细胞株中,作为靶细胞;将效应细胞与靶细胞按不同比例混合,用乳酸脱氢酶(LDH)释放法检测细胞毒作用.结果:未经修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为19.2%、12.3%、6.9%;经hKDR修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为71.6%、55.8%、22.7%,将两组结果在相同的比例下两两比较,均有显著性差异(P＜0.05).结论:hKDR融合基因致敏DC激活的CTL对小鼠肝癌细胞具有较强的杀伤作用,且杀伤能力随效应细胞与靶细胞比例增高而增强.     

CD80联合CD28/CpG ODN活化的PBMC对胃癌细胞杀伤作用的实验研究

目的研究CD80和CD28/CpGODN(含CPG的核脱氧核苷酸)活化单个核细胞(PBMC)在体外对胃癌细胞株MKN45杀伤作用机理,为胃癌的过继免疫治疗可能性提供参考。方法外周血PBMC的分离与体外培养,共刺激活化外周血PBMC,用含共刺激活化PBMC或/和CD800.4mg/L的100ml/L胎牛血清的RPMI1640培养基培养细胞,测生长曲线;MTT法检测活化细胞的体外淋巴细胞毒作用,并用电镜观察效应细胞杀伤的胃癌细胞超微结构及用流式细胞仪检测结肠癌细胞的凋亡指数。结果CD80本身对胃癌细胞株MKN45有一定的杀伤作用(P<0.05),CD28/CpGODN共刺激活化PBMC在体外对胃癌细胞株MKN45杀伤作用明显(P<0.01),CD80与CD28/CpGODN共刺激活化PBMC合用,对胃癌细胞株MKN45的杀伤作用显著大于CD80本身对胃癌细胞株MKN45杀伤作用(P<0.01),亦大于单独应用CD28/CpGODN共刺激活化PBMC对胃癌细胞株MKN45的杀伤作用(P<0.05),CD80增加了胃癌细胞株MKN45对共刺激活化PBMC的敏感性。电镜结果显示,效应细胞作用24h肿瘤细胞就部分发生坏死,部分肿瘤细胞可见凋亡。流式细胞仪检测效应细胞胃癌细胞株MKN4572h明显凋亡。结论CD80与共刺激活化PBMC合用,提高了CD28/CpGODN活化PBMC对胃癌细胞株MKN45的靶向性杀伤作用。单抗协同诱导的效应细胞对胃癌细胞株MKN45杀伤效果较好,以上二者合用较单用任何一种处理对胃癌细胞株MKN45杀伤作用更强,而坏死细胞进一步增多说明效应细胞是通过诱导肿瘤细胞坏死及细胞凋亡两条途径来实现杀伤作用,从而说明活化的淋巴细胞杀伤胃癌细胞是通过坏死及凋亡实现的。     

食管癌可溶性抗原和超抗原构建肿瘤疫苗的免疫活性成分分析

目的对食管癌可溶性抗原和超抗原SEC构建的肿瘤疫苗中的免疫活性成分进行分析,从而为探讨其抗癌机制打下理论基础。方法提取食管癌抗原,和超抗原SEC构建成肿瘤疫苗;分离人外周血单个核细胞（peripheral blood mononuclear cells,PBMC）,经肿瘤疫苗联合作用,进行体外培养,观察其增殖活性;流式细胞术FCM和细胞毒试验测定效应细胞表型和杀伤活性;提取食管癌细胞中的细胞膜抗原（mAg）、热休克蛋白70（HSP70）、DNA、RNA和细胞内多肽（Peptides）,用于诱导PBMC增殖,培养6d,用3H-Tdr掺入法测定细胞增值,确定其有效成分。结果经肿瘤疫苗刺激的PBMC组增殖活性最强,于72h达到高峰,并且特异性刺激CD8＋T细胞群增值。肿瘤疫苗刺激的PBMC组诱导的CTLs对靶细胞杀伤活性显著高于单纯PBMC组（P〈0.01）。食管癌细胞中的膜抗原和细胞内多肽能显著刺激PBMC增殖。结论食管癌抗原与超抗原构建的肿瘤疫苗能诱导效应细胞明显增殖、活化、并产生高效、特异的抗肿瘤效果,具有免疫活性的成分是细胞中的mAg和细胞内多肽。     

树突状细胞联合细胞因子诱导的杀伤细胞体外抗肺癌的实验研究

目的探讨树突状细胞（DC）联合细胞因子诱导的杀伤细胞（CIK）在体内外能否有效的抗肺癌。方法正常人外周血单个核细胞经不同细胞因子作用后培养成DC和CIK细胞,单个核细胞经贴附塑料平皿获得单核细胞,加入GM—CSF,IL-4及TNF—a诱导获得DC,悬浮细胞加入IFN—g,24h后IL-1a,IL-2及CD3McAb诱导获得CIK,将A549制备成肿瘤细胞冻融物．作为肿瘤抗原刺激DC,并将DC与CIK细胞联合培养（DC＋CIK,负载抗原的DC＋CIK）,以CIK细胞单独培养作为对照。CytotoxicityTOX96体外杀伤实验测定体外细胞毒活性。结果在培养的第15d．与负载抗原的DC共同培养的CIK与CIK细胞单独培养细胞相比,增殖速率明显提高[（23．4±2．3）倍vs（16．7±2．7）倍,P〈0．05],CD3＋CD56＋细胞表达水平也明显提高,[（64．3±3．6）％vs（43．9±2．1）％,P〈0．05],同时负载抗原的DC的CIK对A549细胞的体外下细胞毒活性增强。结论DC与CIK细胞共培养后可提高CIK细胞的增殖速率,提高CIK细胞表型的表达水平,增强CIK抗肺癌的活性．将来可作为一种临床有效的过继免疫治疗策略。     

Inhibitory effect of apoptosis in human astrocytes CCF-STTG1 cells by lemon oil

The effects of lemon pure essential oils on the heat shock-induced apoptosis in human astrocytes cell line CCF-STTG1 were examined. In previous studies, heat shock has been reported to induce the apoptosis or programmed cell death through the activation of caspase-3. Treatment of heat shock on CCF-STTG1 cells markedly induced apoptotic cell death as determined by flow cytometry. Interestingly, pre-treatment with lemon pure essential oils on CCF-STTG1 cells inhibited the heat shock-induced apoptosis. Lemon oil also inhibited the heat shock-induced apoptosis in primary cultured rat astrocytes. To determine whether lemon oil inhibits the heat shock-induced activation of the apoptotic proteases, activation of caspase-3 was assessed by Western blotting. DNA fragmentation, giemsa staining, and heat shock-induced activation of caspase-3 were blocked by lemon pure essential oil, which is consistent with flow cytometry. Poly-ADP-ribose polymerase (PARP), the cysteine protease substrate, was fragmented as a consequence of apoptosis by heat shock. Lemon oil inhibited the PARP fragmentation. These results suggest that lemon pure essential oils may modulate the apoptosis through the activation of the interleukin-1 beta -converting enzyme-like caspases.     

5-Fluorouracil enteric-coated nanoparticles for improved apoptotic activity and therapeutic index in treating colorectal cancer

5-Fluorouracil (5-FU) is one among the anti-cancer agents in FOLFORINOX treatment along with oxaliplatin and irinotecan for the treatment of colorectal cancer. Despite its potential activity on the tumor cells, it lacks site specificity partly attributed by its biodistribution to healthy cells resulting in toxic effects to healthy cells. Therefore, we have formulated 5-fluorouracil enteric-coated nanoparticles (5-FUEC) to localize the drug in the colon area that enables its prolonged presence in target area in a sustained manner. The current work emphasizes on enhanced anti-cancer activity of 5-FUEC sequencing its apoptotic activity on HCT 116 colorectal cancer cell lines in vitro. MTT assay exhibited 5.5-fold decrease in IC50 value of nanoparticles comparable to 5-FU. Nuclear fragmentation with irregular edges in nucleus of cells justified its improved activity. Furthermore, flow cytometric analysis confirms the majority of cells gated in early apoptotic (39.75%) and late apoptotic phase (36.25%). Acridine orange/ethidium bromide staining (AO/EB) exhibited cells with red fluorescence (indicating apoptosis) comparable to the control and 5-FU. γ-Scintigraphic studies determined the applicability and feasibility of the enteric coating with mean gastric emptying time, mean intestinal transit time and mean colon arrival time of 1.89?±?0.03, 2.15?±?0.05 and 4.03?±?0.27?h, respectively. Moreover, nanoparticulate approach was found significant in reducing tumor size and volume in xenograft tumor models in vivo along with sustained release. These superior anti-cancer activities exhibited by 5-FUEC indicated that it could be a potential alternative to chemotherapy for colorectal cancer.     

脐血浆培养的脐血树突状细胞对胃癌细胞的特异杀伤作用

目的尝试用脐血浆代替胎牛血清培养脐血树突状细胞（DCs）,并使之负载胃癌抗原,观察其对胃癌细胞的特异杀伤作用。方法分离脐血单个核细胞（CBMCs）并在含10％同源脐血浆的培养体系中诱导培养,部分细胞加入胃癌冻融抗原冲击。流式细胞仪检测细胞表面抗原CD1a和CD83表达,MTT法检测DCs体外刺激淋巴细胞增殖活性,LDH法检测DCs诱导的细胞毒T淋巴细胞（CTLs）对胃癌及肝癌细胞的细胞毒作用。结果CBMCs能分化为表型正常的未成熟DCs,并进而吞噬胃癌细胞冻融抗原成熟,刺激淋巴细胞增殖并诱导对胃癌细胞株特异的CTLs毒性。结论脐血浆培养的脐血DCs能有效捕获胃癌冻融抗原,激发针对胃癌细胞的特异杀伤效应。本实验采用的DCs制备方法具有方法简单、成本较低、瘤苗制备时间较短等优点。     

Apoptic renal carcinoma cells are better inducers of cross-presenting activity than their primary necrotic counterpart

Vaccination with tumor-loaded dendritic cells (DC) is a promising treatment strategy for patients with renal cell carcinoma (RCC). Cells undergoing cell death proved useful as a source of tumor antigen for DC loading. Both apoptotic and necrotic tumor cells have been shown to efficiently load RCC-tumor antigens on DC. However, no direct comparison of these two kinds of death has been attempted in the same RCC. We compared DC pulsed with apoptotic cells, whole cell lysates or their supernatants of the cell line K1, derived from a patient with clear cell RCC, to determine their ability to activate T cells. Monocyte-derived DCs were pulsed with the different sources of tumor antigen, matured and co-cultured with autologouos peripheral blood lymphocytes. After three weekly re-stimulations with DCs, generation of cytotoxic T lymphocytes CTL was assessed by IFN-gamma release in an ELISpot assay in the presence of the sensitizing target. By comparison with lysate, apoptotic tumor cells induced a higher frequency of MHC class I-restricted IFN-gamma releasing lymphocytes. A higher CTL response was induced by pulsing DCs with cell lysate supernatant compared with whole cell lysate. These results indicate that, although necrotic death has been regarded as highly permissive when compared to apoptotic death, the immunogenicity of the death treatment may vary from one tumor to another.     

Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL

Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation. We compared electroloading of tumor cell lysates directly into mature DCs with the traditional method of lysate co-incubation with immature DCs. Electroloaded mature DCs were more potent in vitro, as judged by their ability to elicit significantly (p < 0.05) greater expansion of peptide antigen-specific CD8⁺ T cells, than either lysate-electroloaded immature DCs or lysate-co-incubated immature DCs, both of which must be subsequently matured. Expanded CD8⁺ T cells were functional as judged by their ability to produce IFN-γ upon antigen-specific re-stimulation. The electroloading technology used herein is an automated, scalable, functionally closed cGMP-compliant manufacturing technology supported by a Master File at CBER, FDA and represents an opportunity for translation of enhanced potency DC vaccines at clinical/commercial scale.     

Inhibitors of β-catenin affect the immuno-phenotype and functions of dendritic cells in an inhibitor-specific manner

Many tumors are characterized by mutation-induced constitutive activation of β-catenin which promotes tumor growth and survival. Consequently, the development of specific β-catenin inhibitors for tumor therapy has come into the focus of drug development. β-Catenin was also shown to contribute to the tolerance-promoting function of unstimulated dendritic cells (DCs). In response to activation, DCs acquire potent T cell stimulatory capacity and induce profound tumor antigen-specific immune responses. Here we asked for effects of pre-clinically established β-catenin inhibitors (CCT-031374, iCRT-5, PNU-75654) on mouse bone marrow-derived (BM)DCs. All three inhibitors moderately increased surface expression of MHCII, CD80, and CD86 on unstimulated DCs, but had no enhancing effect on their capacity to stimulate the proliferation of ovalbumin (OVA) specific CD4⁺ T cells. CCT-031374 interfered with upregulation of costimulators (CD40, CD86) and cytokines (IL-1β, TNF-α, IL-6, IL-10, IL-12) by LPS-stimulated DCs. Accordingly, this DC population displayed an impaired CD4⁺ T cell stimulatory activity. iCRT-5 and PNU-75654 had no detrimental effects on the immuno-phenotype of stimulated DCs. Hence, DCs treated with iCRT-5 in the course of stimulation exerted comparably strong T cell proliferation as did control DCs. In contrast, DCs stimulated in the presence of PNU-75654 induced less T cell proliferation than the control population despite enhanced uptake and processing of OVA. Our findings suggest that the differential effects of β-catenin inhibitors on stimulated DCs reflect off target effects. Concerning potential application of β-catenin inhibitors for tumor therapy, iCRT-5 may be most beneficial, since it did not exert detrimental effects on stimulated DCs.     

树突状细胞瘤苗诱导的抗神经胶质瘤作用体外实验研究

目的 :研究负载自体神经胶质瘤抗原的树突状细胞 (DCs)瘤苗在体外诱导的特异性细胞毒性淋巴细胞 (CTL)对神经胶质瘤细胞的杀伤效应。方法 :以组合酶消化法从新鲜神经胶质瘤手术标本中获取神经胶质瘤细胞 ,冻融制备神经胶质瘤抗原。GM -CSF、IL -4体外诱导外周血单个核细胞 (PBMC)获得DCs并负载神经胶质瘤抗原 ,继而以其刺激自体T淋巴细胞制备神经胶质瘤抗原特异性CTL ;用CytoTox96TM检测CTL对患自身神经胶质瘤细胞体外杀伤效应。结果 :负载神经胶质瘤抗原DCs诱导的特异性CTL对患者自身神经胶质瘤细胞的杀伤率达88 17 % ,显著高于LAK细胞的杀伤率 (P<0 05)。且其对同种不同分化类型的神经胶质瘤细胞株 (P<0 01)。结论 :负载神经胶质瘤抗原的DCs体外可诱导出高效而特异的抗神经胶质瘤效应 ,提示以DCs为中心的肿瘤生物治疗作用可望提高神经胶质瘤综合治疗水平