DC-CIK对急性髓细胞白血病干细胞的杀伤作用

目的:研究树突状细胞(dendritic cell,DC)联合同源细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对急性髓细胞白血病细胞株KG-1a中白血病干细胞(leukemic stem cell,LSC)的体外杀伤和诱导凋亡作用。方法:分离健康人外周血单个核细胞,贴壁细胞用GM-CSF和IL-4诱导培养DC,悬浮细胞用IL-2、IL-1、IFN-γ和CD3 mAb诱导培养CIK。将KG-1a细胞冻融物作为抗原负载DC(即Ag-DC),与CIK共培养作为实验组(Ag-DC-CIK),无抗原负载的DC与CIK共培养作为对照组(DC-CIK),单独CIK作为空白对照组,与KG-1a共育后流式细胞术检测各组细胞中CD34+CD38-CD123+白血病干细胞的比例。DC-CIK与KG-1a细胞共培养,流式细胞术检测各组细胞中KG-1a细胞与CD34+CD38-CD123+细胞的凋亡率。结果:外周血单个核细胞成功诱导DC。CIK组、DC-CIK组及Ag-DC-CIK组中CD3+CD56+细胞比例为(17.36±4.44)%、(28.22±3.66)%和(36.16±5.88)%,依次升高(P<0.05)。与对照组相比,Ag-DC-CIK组与DC-CIK组细胞中CD34+CD38-CD123+细胞比例显著降低[(8.78±0.62)%vs(3.95±0.53)%、(3.03±0.62)%,P<0.01〗。DC-CIK可诱导KG-1a细胞凋亡,凋亡率由(2.34±0.74)%上升至(12.27±1.01)%,但对其中CD34+CD38-CD123+细胞无明显的诱导凋亡作用。结论:DC联合CIK能杀伤急性髓细胞白血病干细胞,但无明显的诱导凋亡作用。     

PSMA/4-1BBL共转染DC疫苗联合CIK细胞体外抑制前列腺癌的实验研究

目的:本研究通过复制缺陷性腺病毒介导的人前列腺特异性膜抗原基因(PSMA)和肿瘤坏死因子配体超家族成员(4-1BBL)体外共同转染树突状细胞(DC)后,与细胞因子诱导的杀伤细胞(CIK)共培养,观察其体外抑制前列腺癌的生物学效应。方法:将Ad-PSMA、Ad-4-1BBL、Ad-GFP按MOI=200∶1转染DC作为刺激细胞,分为共转染组、PSMA转染组、4-1BBL转染组、阴性对照组(Ad-GFP-DC组)以及未转染DC组,Western blot法检测各组DC细胞中PSMA和4-1BBL蛋白的表达;流式细胞仪分析CD80、CD83、CD86等表型变化;取患者外周静脉血单个核细胞中淋巴细胞用于CIK细胞的诱导培养,按DC∶CIK=1∶10比例将上述不同转染组DC加入CIK中,共同孵育96h(设普通DC刺激组和单独CIK细胞组为对照),收获的细胞作为效应细胞,流式细胞仪测定不同DC刺激组CIK细胞CD3、CD56表达率,ELISA法检测不同DC-CIK组培养上清IFN-γ、IL-10水平;PE-7AAD凋亡试剂盒检测不同DC-CIK组对人前列腺癌细胞株LNCap、Du145的细胞毒作用。结果:Western blot结果证实PSMA、4-1BBL可在DC上成功表达;各转染组DC表面CD80、CD83、CD86、HLA-DR共刺激分子表达上调,与未转染组相比较差异显著(P<0.05)。培养14d,不同DC疫苗刺激组CIK以及单独CIK细胞组中CD3+、CD56+、CD3+/CD56+T细胞比例均高于培养7d的CIK,差异显著(P<0.05),其中经不同DC疫苗刺激的CIK组上升较单独培养14d-CIK组更明显(P<0.05)。DC-CIK培养上清中PSMA/4-1BBL-DC-CIK组IFN-γ分泌量最高,达1176.10±14.37pg/5×106cells,IL-10分泌量最低,为75.14±2.01 pg/5×106cells,与其他各组比较,差异显著(P<0.05)。不同组别DCCIK作用于LNCap、Du145细胞24h后,荧光显微镜下观察细胞凋亡和坏死明显。同一组别DC疫苗刺激下的CIK细胞对LNCap的杀伤作用强于DU145细胞,差异显著(P<0.05)。而在针对同一种肿瘤细胞时,PSMA/4-1BBL-DC-CIK组的杀伤作用最强,LNCap细胞的凋亡率可达(24.56±1.68)%,Du145细胞凋亡率可达(12.67±0.94)%,与PSMA-DC-CIK组、4-1BBL-DC-CIK组相比,差异显著(P<0.05),而PSMA-DC-CIK和4-1BBL-DC-CIK组的前列腺癌细胞的凋亡率则无显著差异(P>0.05),但与GFP-DC-CIK、普通DC-CIK、和单独CIK组相比差异显著(P<0.05)。结论:本实验中Ad-PSMA/4-1BBL高效转染的DC具有成熟DC的典型特征,与CIK共培养后提高了CD3+/CD56+T细胞比例,IFN-γ分泌量显著增高,并增加了对PSMA阳性靶细胞的特异性杀伤,比普通DC-CIK及单独CIK具有更强的杀伤活性。两种肿瘤相关抗原转染DC与CIK共培养后可以更有效地提高CIK细胞的杀伤活性。     

树突状细胞对自体CIK细胞体外杀伤肺腺癌细胞影响的研究

目的：研究人外周血树突细胞（dendriticcell,DC）对自体CIK细胞体外杀伤肺腺癌细胞的影响,以期获得具有抗原特异性杀伤功能的细胞毒活性细胞,并分别对CIK、LAK和CD3AK的杀伤效果进行比较.方法：采用某一肺腺癌肿瘤患者外周血单个核细胞（peripheral blood mononuclear cells,PBMNC）,经体外诱导分别扩增出CIK、LAK、CD3AK和DC细胞,再将靶细胞抗原孵育过的DC同三种细胞共同培养,通过镜下动态观察CIK联合DC对癌性胸腔积液中肿瘤细胞的杀伤活性,并利用MTT法检测CIK联合DC体外杀伤人肺腺癌细胞系（SPC-A1）的活性,同时比较CIK、LAK和CD3AK三种细胞的体外杀瘤活性.结果：CIK-ADC的杀伤活性最强为92.3%,明显高于单纯CIK的59.7%和DC-CIK的79.8%,（P值分别为0.025和0.042）,提示CIK A-DC细胞对肿瘤杀伤的特异性.而DC-CIK的杀伤活性为79.8%,也高于单纯CIK对照组59.7%,P=0.034,说明DC具有明显增强CIK细胞杀瘤活性的功能.同时,不论从单纯CIK、LAK、CD3AK细胞毒活性,或是从三种细胞联合DC的细胞毒活性比较,CIK细胞较后两种细胞都具有更强的杀伤活性,P值分别为0.038和0.022.联合DC的自体CIK细胞体外杀瘤活性显著增强,CIK细胞的杀伤活性显著高于LAK、CD3AK两种细胞.结论：DC可明显提高自体CIK细胞的体外杀瘤活性.     

树突状细胞与热休克处理抗原联合诱导抗肿瘤免疫的研究

目的建立体外培养与扩增脐血树突状细胞(DC)的方法,并利用DC与热休克处理的肿瘤抗原联合诱导产生一种高效特异的抗肿瘤免疫.方法用GM-CSF和IL-4联合刺激诱导脐血单个核细胞分化为DC,再用加热处理的肿瘤抗原负载,与同源的淋巴因子活化的杀伤(LAK)细胞共培养.用电镜观察DC形态,CD1a和HLA-DR试剂盒检测DC细胞表面CD1a和HLA-DR的表达情况,MTT法测定细胞杀伤活性.结果在体外,DC-LAK对同源的SPC-A-1细胞具有相对特异的杀伤作用,LAK和DC-L的作用不具有明显特异性,DC-LAK的细胞毒作用最强;加热抗原组作用明显优于未加热处理组.结论将DC与热休克处理的肿瘤抗原联合,可以诱导产生一种高效并具有相对特异性的抗肿瘤免疫活性细胞.     

细胞因子联合人参皂甙及三氧化二砷诱导HL-60细胞向树突状细胞分化的实验研究

目的研究HL-60细胞在体外经细胞因子联合人参皂甙及三氧化二砷(AS2O3)诱导向树突状细胞(DC)分化的情况。方法选用HL-60细胞与重组人粒单核细胞集落刺激因子(rh-GM-CSF)、白细胞介素4(IL-4)、γ干扰素(INF-γ)、TSPG及AS2O3共培养。根据细胞因子、TSPG及AS2O3不同组合分为12组。在培养后7天及14天以光学和电子显微镜观察细胞的形态学特征，用HLA-DR、CD1a、CD86等五种单克隆抗体标记流式细胞术检测细胞表型，用ELISA法测定HL-60-DC培养上清液中的IL-12及INF-γ的量，培养14天后的细胞再继续以1：10的比例与HL-60细胞共培养24h，观察HL-60-DC对白血病细胞的杀伤情况，用流式细胞仪检测凋亡率。结果HL-60细胞在组合细胞因子并分别加入TSPG及AS2O3后，均可诱生出不同比例的DC。光镜及电镜下均有典型的树突状细胞的形态学特征，细胞表达DC的表面分化抗原，诱生的DC培养上清中可测出不同量的IL-12及INF-γ。细胞凋亡率均明显高于对照组，联合中药各组的凋亡率均高于GM-CSF与IL-4组。结论①GM—CSF、IL-4与HL-60细胞共培养可诱导出HL-60-DC，在此基础上加入适当浓度的TSPG、AS2O3可使树突状细胞的特异性抗原表达加强。②在DC诱生过程中，GM—CSF联合IL-4组DC特异性抗原表达率高于IL-4联合INF组，证明GM—CSF在DC诱生过程中起重要作用。③单独应用TSPG，也可出现部分DC特异性抗原表达，但表达率明显低于联合用药组。④诱生后的HL-60-DC具有自分泌IL—12及INF-γ的功能。⑤诱生后的DC具有抗白血病细胞作用，与HL-60细胞共培养后可使白血病细胞凋亡明显增强。     

自体肿瘤抗原负载DC-CIK细胞治疗胃癌的疗效分析

目的:探讨自体肿瘤抗原负载树突状细胞联合细胞因子诱导的杀伤细胞(DC-CIK)治疗胃癌的疗效及安全性.方法:回顾性分析2005年1月至2010年1月在福建省肿瘤医院行胃癌切除术患者270例,根据是否进行DC-CIK治疗,分为对照组(150例)和DC-CIK治疗组(120例).应用流式细胞术分析DC-CIK细胞表型,比较两组患者的总生存期(OS),观察DC-CIK治疗的不良反应,Cox回归法分析胃癌预后影响因素.结果:成熟DC中HLA-DR+、CD8+、CD83+和CD86+的细胞比例分别为(96.16±1.51)％、(96.58±2.66)％、(96.44±2.20)％和(98.74±0.76)％.CIK中CD3+、CD3+ CD4+、CD3+CD8+和CD3+ CD16+/CD56+的细胞比例分别为(91.98 ±-5.9)％、(25.19±10.5)％、(71.15±7.8)％和(18.73±7.4)％.DC-CIK治疗组中位OS为77个月,与对照组的51个月相比差异明显(x2=4.431,P=0.035).DC-CIK治疗、辅助化疗和TNM分期是胃癌预后的独立影响因素.DC-CIK治疗的常见不良反应为发热、寒战和乏力.结论:胃癌术后应用自体肿瘤抗原负载DC-CIK治疗可延长胃癌患者的OS,且不良反应轻,安全有效.     

树突状细胞与细胞因子诱导的杀伤细胞共培养对多药耐药肿瘤细胞系的杀伤活性

目的探讨在细胞因子诱导的杀伤细胞(CIK)表达特异性抗原的肿瘤细胞过程中,是否存在抗原特异性杀伤。方法分离健康人骨髓获得单个核细胞,分别诱导为树突状细胞(DC)和CIK细胞,将人类乳腺癌耐药细胞株MCF-7/ADR细胞的冻融物抗原冲击或未冲击DC与CIK细胞共培养(pulsed-DC CIK、DC CIK),CIK细胞单独培养作对照。用流式细胞仪分析细胞表型,用酶联免疫吸附法(ELISA)检测IL-12、和IFN-γ分泌水平,用二苯基溴化四氮唑蓝(MTT)法测定细胞毒效应。结果DC与CIK共育后,两组DC成熟表型较共育前明显提高(P=0.003、P=0.001);pulsed- DC CIK组与DC CIK组、CIK组比较,细胞表型(CD3、CD8、CD56)明显提高(P=0.003、P= 0.011),CD3 CD56 细胞明显增多(P=0.001,P<0.001),CD3 CD8 细胞亦明显增多(P=0.002, P=0.002);CD45RA表型则明显降低(P<0.001,P=0.004)。IL-12和IFN-γ水平在pulsed-DC CIK组表达最高,分别为(254±14.5)pg/ml和(3100±286)pg/ml。对有耐药抗原表达的MCF-7/ADR细胞,pulsed-DC CIK组杀伤效应最强,pulsed-DC CIK组、DC CIK组和CIK组比较,差异均有统计学意义(pulsed-DC CIK组与DC CIK组比较,P=0.039;pulsed-DC CIK组与CIK组比较,P= 0.002;DC CIK组与CIK组比较,P=0.049);而对于无P-gp抗原表达的MCF-7细胞的杀伤效应,pulsed- DC CIK组和DC CIK组之间无明显差异,但均高于CIK组,差异有统计学意义(pulsed-DC CIK组与CIK组比较,P=0.007;DC CIK组与CIK组比较,P=0.048)。结论从人骨髓培养得到DC和CIK细胞共培养后,能促进各自特征性表面标志的表达上调,并分泌大量相关细胞因子。细胞杀伤效应的明显提高及可能的特异性细胞杀伤效应,为多药耐药肿瘤的临床生物免疫治疗提供实验基础。     

脐血来源树突状细胞体外诱导抗卵巢癌免疫特异性

[目的]研究脐血来源树突状细胞(DC)体外诱导特异性抗卵巢癌细胞的免疫效应.[方法]①从脐血中分离单个核细胞(MNCs)后,获得单核细胞(Mo).粒单集落刺激因子(GM-CSF)和白介素4(IL-4)诱导分化,培养7天后应用流式细胞仪进行细胞表型分析.②诱导单核细胞分化的第3天加入人卵巢癌细胞株3AO的冻融抗原,共培养4天后获得负载肿瘤抗原的成熟DC;将致敏DC与从脐血中分离的同种异体T淋巴细胞共培养3天,获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝(MTT)法检测CTL及上清对人卵巢癌细胞株3AO、人胚肾细胞株293T(对照细胞)、人肝癌细胞株HCCC-9810的细胞毒作用.[结果]①脐血来源单核细胞(Mo)在GM-CSF和IL-4作用下,7天后可分化生成成熟的DC,高表达DC特异性抗原CDla、CD80(B7-1)、CD86(B7-2)、HLA-DR、CD83.②DC可负载并递呈肿瘤抗原,激活同种异体T淋巴细胞,诱导肿瘤特异性CTL产生.不同浓度CTL及上清对卵巢癌细胞3AO有特异性杀伤、抑制作用(P＜0.05).[结论]脐血中单核细胞可体外分化扩增为成熟的功能性DC,并诱导出特异性杀伤卵巢癌细胞的免疫效应.     

B-NHL患者NK细胞中CD16ζ表达及利妥昔单抗与LAK细胞的联合抗瘤作用

背景与目的:自然杀伤细胞(nature killer cell,NK)是抗体依赖细胞介导的细胞毒作用的主要效应细胞,肿瘤患者普遍存在NK细胞活化功能的缺陷可能会影响单克隆抗体的治疗效果.因此如能逆转NK细胞的CD16ζ链信号转导的功能缺陷,并与单克隆抗体联合免疫治疗,可能会产生协同抗肿瘤作用.本研究的目的是了解B细胞非霍奇金淋巴瘤(B-cell non-Hodgkin's lymphoma,B-NHL)患者是否存在NK细胞的活化障碍,体外白细胞介素-2(interleukin-2,IL-2)是否能完全逆转其活化障碍,并观察利妥昔单抗与LAK细胞联合对肿瘤细胞的杀伤作用.方法:使用密度梯度离心方法分别分离69例B-NHL患者和30例健康志愿者外周血单个核细胞(peripheral blood mononuclear cell,PBMC),将两种PBMC在体外与1 000 U/ml IL-12共同培养制备LAK细胞,流式细胞仪检测PBMC和LAK细胞中CD16ζ链的阳性率和平均荧光强度.流式细胞仪检测Raji细胞表面CD20的表达;Annexin V/PI方法检测利妥昔单抗单药对Raji细胞的促凋亡作用,乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验进行杀伤活性的检测.结果:在B-NHL组和健康对照组,CD56 细胞表达CD16ζ链的阳性率为(63.3±16.4)%、(97.8±3.1)%(P＜0.001),CD16ζ链MFI值分别为1.3±1.3和3.6±1.7(P＜0.001).在体外1 000 U/ml的IL-2共培养的LAK细胞中,两组CD16ζ链的阳性率分别为(99.3±4.1)%和(99.7 3.9)%,其MFI值分别为29.2±12.5和31.4±13.8,均无显著性差异(P=0.15和P=0.44).40 μg/ml利妥昔单抗可以完全结合细胞表面CD20抗原,在24 h时才开始出现对Raji细胞的明显的凋亡作用.利妥昔单抗与LAK细胞联合对Raji细胞的杀伤率在不同的浓度组均明显高于不加利妥昔单抗组(P＜0.05).LAK细胞与Herceptin(40 μg/ml)联合的杀伤率与不加Herceptin组相比,在各效靶比浓度梯度均无明显提高(P＞0.05).LAK细胞与利妥昔单抗联合对Jurket细胞的杀伤率在各效靶比浓度梯度均与不加利妥昔单抗组无显著性差异(P＞0.05).结论:B-NHL患者普遍存在NK细胞CD16ζ链的表达下调,高剂量的IL-2可以显著增强CD16ζ链的表达,利妥昔单抗与LAK细胞联合可增强对Raji细胞的抗肿瘤作用.     

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。     

Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and gammadelta T cells

Tumor vaccines using dendritic cells (DCs) have been shown to induce antitumor CTL responses. The choice of the tumor antigen preparation used for DC loading is still an unresolved issue. We compared DCs pulsed with cell lysates, whole apoptotic tumor cells or their supernatants of the HLA-A2(+) human pancreatic carcinoma cell line Panc-1 for their capacity to activate T cells. Monocyte-derived DCs from HLA-A2(+) donors were pulsed with tumor antigen, matured subsequently, and cocultured with autologeous peripheral blood mononuclear cells. After three weekly restimulations with DCs, T-cell activation was assessed by intracellular IFN-gamma staining and cytotoxicity assays. Compared with lysate, pulsing DCs with the supernatant of apoptotic tumor cells induced a higher frequency of activated CTLs and T-helper cells, as well as an enhanced MHC class I-restricted tumor cell lysis. No activation of natural killer (NK) or gammadelta T cells was detected. Pulsing DCs with whole apoptotic tumor cells induced an even more pronounced lytic effect. However, in this case, MHC class-I blocking was only partially effective, and unrelated cell lines were also killed. IFN-gamma staining revealed activation of CTLs and T-helper cells, as well as NK and gammadelta T cells. Trans-well cultures of NK cells, apoptotic tumor cells, and DCs showed that NK cell activation was dependent on direct cell-to-cell contact with tumor cells and the presence of interleukin-12 produced by DCs. These results indicate that the choice of antigen preparation is a critical determinant in the induction of antitumor immunity. Tumor vaccines consisting of DCs and apoptotic tumor cells may be able to activate CTLs, as well as effector cells of the innate immune system.     

DC与CIK共培养对骨肉瘤细胞的杀伤作用

目的 探讨树突状细胞(dendritic cells,DC)与细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK)共培养对人骨肉瘤细胞的杀伤效应.方法 从健康志愿者中分离外周血单个核细胞,制备DC和CIK,并以5:1比例混合培养.采用CCK-8法和流式细胞术检测DC-CIK共培养...     

多肽负载DC联合CIK治疗激素难治性前列腺癌的疗效

目的:研究多肽负载树突状细胞(dendritic cell,DC)联合细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对激素难治性前列腺癌(hormone refractory metastatic prostate cancer,HRPC)患者免疫治疗的效果。方法:选择无锡市第四人民医院中西医结合科收治的HLA-A2+HRPC患者26例,分离外周血单个核细胞,其中贴壁细胞经GM-CSF、IL-4联合诱导培养为成熟DC,负载前列腺癌特异性抗原(prostate specific antigen,PSA)、前列腺酸性磷酸酶(prostatic acid phosphatase,PAP)、前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)三个多肽,制备成DC疫苗,经患者腹股沟皮内注射;未贴壁细胞经IFN-γ、IL-2、抗CD3单抗、IL-1体外诱导培养成CIK,经静脉回输给患者。在治疗后1周进行迟发型超敏反应(delayed type hypersensitivity,DTH)检测,在患者治疗前后进行血清中细胞因子和PSA检测,治疗结束后4周进行短期疗效评价。结果:26例HRPC患者对DC联合CIK治疗的耐受良好。治疗后患者血清中IL-2、IL-12、IFN-γ水平较治疗前显著升高(上升幅度分别为65.07%、67.69%和125.38%,P<0.05或P<0.01),TNF-α和IL-10水平变化不大;DTH的阳性率为43.5%(10/23);7例患者的CD8+IFN-γ+T细胞比例较治疗前显著提高[(8.95±2.74)%vs(0.39±0.15)%,P<0.01];8/26例患者的PSA下降,降幅为13%～66%。26例患者短期疗效评价,3例PR、4例PD、19例SD,所有患者治疗中未出现明显不良反应。结论:多肽负载DC联合CIK治疗HRPC能激发患者的免疫应答、诱导Th1型细胞因子的分泌,近期疗效良好,是一种安全的治疗方法。     

共培养的树突细胞和CIK细胞对肺癌的体内外抑癌作用

背景与目的:细胞因子诱导的杀伤(cytokine-inducedkiller,CIK)细胞是高效的肿瘤杀伤细胞。树突细胞(dendriticcells,DCs)是体内最强的抗原递呈细胞,并且能够提高效应细胞的抗瘤活性。本实验将DCs和CIK细胞共培养观察DCs对CIK细胞的细胞表型、增殖活性及体内外的抗肺癌作用的影响。方法:从健康人外周血单个核细胞中常规诱导出DCs、CIK细胞后,将DCs和CIK细胞按1∶10比例共培养5天获得DC-CIK细胞。流式细胞仪测DC-CIK细胞表型变化,3H-TdR掺入法测定其体外的细胞毒活性,并用肺腺癌细胞株A549建立裸鼠模型观察DC-CIK体内的抗肿瘤效果。结果:在培养第14天,DC-CIK细胞与单独CIK细胞培养组相比,增殖速率提高[(17.0±1.8)倍vs.(10.9±2.0)倍,P<0.05],CD3 CD56 表达水平明显上调[(36.0±4.2)%vs.(25.7±2.9)%,P<0.05],同时对A549细胞的细胞毒活性明显增强(P<0.05)。裸鼠体内实验表明,接种肺癌细胞51天后DC-CIK组、CIK组的抑瘤率分别为62.9%、41.5%,与对照组相比DC-CIK组及CIK组均抑制裸鼠皮下移植瘤的生长(P<0.01),且DC-CIK组与CIK组抑瘤效应差异有统计学意义(P<0.05)。结论:DCs与CIK细胞共培养可使CIK细胞获得更高的增殖活性和更强的抑癌作用。     

腺病毒介导的HBsAg基因修饰树突状细胞的体外生物学特性

目的： 〖HT5"SS〗探讨腺病毒介导乙型肝炎病毒表面抗原（AdVHBsAg）基因修饰树突状细胞（dendritic cell,DC）瘤苗体外生物学活性。〖HT5W〗方法〖HT5"SS〗：将腺病毒表达载体AdVHBsAg转染人单个核细胞来源的DC,构建AdVHBsAgDC肝癌瘤苗,采用Western blotting法鉴定转染基因表达,FACS检测表面分子和内吞功能,3HTdR法检测T细胞增殖反应的能力,MTT法检测CTL活性。〖HT5W〗结果〖HT5"SS〗：HBsAg基因转染后,Western blotting法检测结果示HBsAg基因表达于转染的DC,表明腺病毒介导的HBsAg基因转染的有效性。AdVHBsAgDC可高表达CD1a、CD11c、 CD83、CD86和HLADR,但内吞功能较DC组降低(P<0.05)。AdVHBsAg DC刺激自体T细胞增殖功能均明显高于DC对照组和AdVLacZDC组(P<0.05)。AdVHBsAg DC体外诱导CTL对HepG2215肿瘤细胞的杀伤作用具有特异性。〖HT5W〗结论〖HT5"SS〗：肝癌相关基因HBsAg可作为乙型肝炎病毒相关性肝癌的切入点,该研究为HBV相关肝癌DC体内免疫治疗提供了实验依据。     

Immune Responses of Dendritic Cells Loaded with Antigens from Apoptotic Cholangiocarcinoma Cells Caused by γ-Irradation

Objective:To investigate the induction cytotoxic T cells(CTLs) with antitumor activity and therapeutic efficacy after dendritic cells(DCs) acquired antigen from apoptotic cholangiocarcinoma cells caused by γ-irradiation. Methods:DCs from peripheral blood mononuclear cells (PBMC) that maintain the antigen capturing and processing capacity charateristic of immature cells have been established in vitro, using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Then, in cholangiocarcinoma cells apoptosis was induced by γ-irradiation. The experimental groups were as follows:(1)coculture of DCs and apoptotic cancer cells and T cells;(2)coculture of DCs and necrotic cancer cells and T cells;(3)coculture of DCs, cultured cancer cell and T cells. They are cocultured for 7 days.DCs and T cells were riched, isolated and their antitumor response was tested. Results:The cells had typical dendritic morphology, expressed high levels of CDla and B7, acquired antigen from apoptotic cells caused by γ-irradiation and induced an increased T cell stimulatory capacity in mixed lymphocyte reactions (MLR). Conclusion:DCs obtained from PBMCs using GM-CSF and IL-4 can efficiently present antigen derived from apoptotic cells caused by γ-irradiation and efficiently induce T cells.This strategy, therefore, may present an effective approach to transduce DCs with antigen.     

Therapeutical doses of temozolomide do not impair the function of dendritic cells and CD8+ T cells

The median survival of patients with glioblastoma multiforme (GBM) remains poor. Innovative immunotherapies with dendritic cell (DC) vaccination might be combined with standard temozolomide (TMZ) treatment. Here, we evaluated the influence of TMZ on the phenotype and function of DCs and CD8+ T cells. DCs were generated from the peripheral blood of healthy volunteers (HVs) and GBM patients. DCs were analyzed by light microscopy and flow cytometry. Phagocytic activity was tested by FITC-dextran engulfment. Mixed lymphocyte peptide cultures were followed by enzyme-linked immunospot (ELISPOT) and flow cytometry assays. TMZ was added to DC and T cell cultures at concentrations up to 500 μM. Mature DCs were generated from HVs and GBM patients. Cells displayed a typical DC morphology and a mature DC phenotype. Expression of CD209 was even higher in DCs generated from patients under therapy than from HVs (75.2 vs. 51.1%). In contrast, CD40 (1.1 vs. 13.5%) and BDCA4 (26.5 vs. 52.9%) were lower expressed in GBM patients at time of diagnosis. Immature DCs showed high phagocytic activity. Addition of TMZ at concentrations up to 50 μM did neither impair the phenotype nor the function of DCs. In ELISPOT and flow cytometry assays, no impairment of CD8+ T cell responses to viral antigens could be observed. Taken together, TMZ does not impair the function of either DCs or the CD8+ T cells.     

肿瘤射频消融裂解物负载的DC—CIK细胞体外抗肿瘤活性实验研究

目的研究负载射频消融肿瘤原位裂解物的树突状细胞（Dc）联合细胞因子诱导杀伤活性细胞（CIK）体外抗肿瘤活性。方法制备BALB／C小鼠脾脏来源的CIK细胞及骨髓来源的Dc。建立射频消融灭活小鼠皮下结肠癌的实验模型,将其原位裂解的肿瘤组织反复冻融后取上清,lowry蛋白定量法定量,以终浓度5μg／ml载培养第5天的Dc（即Ag-Dc）,2d后再与培养第7天的CIK细胞共培养48h,流式细胞术分析其表面共刺激分子的表达,细胞增殖与毒性检测试剂盒（CCK-8试剂盒）检测其体外杀伤活性。结果DC表面分子表达共刺激分子CD86＋ CD11c＋、MHCⅡ＋ CD11c＋、MHCⅡ＋ CD80＋双阳性细胞百分含量分别为9．50％、42．4％、53．4％;Ag-DC表面分子表达共刺激分子双阳性细胞百分含量明显提高,分别为19．2％、74．2％、61．1％。CIK细胞培养第1天,CD3＋ NK1．1＋双阳性的百分含量为1．45％,第7天CD3＋ NK1．1＋双阳性表达明显提高为36．9％。Ag—DC—CIK细胞对结肠癌细胞C26的杀伤活性明显高于DC-CIK、CIK细胞,且相同效靶比下前者的杀伤活性明显高于后者。效靶比为5：1时,Ag-DC-CIK细胞杀伤率为（74．9±3．5）％,DC-CIK细胞杀伤率为（71．2±2．1）％,CIK细胞杀伤率为（68．7±2．9）％,差异有统计学意义（F=7．007,P=0．007）;效靶比为10：1时,Ag—DC-CIK细胞杀伤率为（82．3±4．5）％,DC-CIK细胞杀伤率为（77．1±5．1）％,CIK细胞杀伤率为（72．7±2．8）％,差异有统计学意义（F=7．727,P=0．005）;效靶比为20：1时,Ag-DC-CIK细胞杀伤率为（83．2±1．9）％,DC-CIK细胞杀伤率为（77．2±4．2）％,CIK细胞杀伤率为（73．0±2．6）％,差异有统计学意义（F=16．594,P=0．000）。结论负载肿瘤射频消融原位裂解产物的DC联合CIK细胞可以提高体外细胞毒活性,为肿瘤综合治疗提供新策略。     

DC-CIK细胞的生物学活性及抗淋巴瘤细胞的作用

Objective: To investigate the proliferation capabilities, immunophenotype changes, level of secreted cytokines and activities against lymphoma cells under the condition that cytokine-induced killer (CIK) cells co-cultured with dendritic cells (DC) in vitro. Methods: DC and CIK cells were induced from peripheral blood mononuclear cells of healthy volunteers. They were co-cultured meanwhile CIK cells were cultured alone as controls. Increased number of cells were counted by tapan-blue staining, killing activities were detected by MTT assay, immunophenotype changes were analyzed by flow cytometry, the IL-12 and INF-γ levels of the cultured supernatants were detected by ELISA kits. Results: The proliferation capabilities of DC-CIK cells were significantly higher than that of CIK cells (P < 0.05). Under the same condition, the ratio of double positive cells such as CD3 CD8 , CD3 CD56 in CIK cells was significantly enhanced by co-cultured with DC cells (P < 0.05). The level of IL-12 and INF-γ secreted in supernatants was increased noticeably by co-cultured DC-CIK cells on day 3 compared to CIK cells which were cultured alone (P < 0.01 and P < 0.05). Within the effector-target ratio range between 5:1 to 40:1, the activi-ties against lymphoma cells of DC-CIK cells were much higher than that of CIK cells (P < 0.05), and this effect was showed a positive correlation with the effector-target ratio. Conclusion: The proliferation capabilities, the level of secreted cytokines and the activities against lymphoma cells of DC-CIK cells were significantly higher than those of CIK cells. The research might provides theoretical and experimental basis for clinical immunotherapy of DC-CIK cells.     

Preclinical evaluation of autologous dendritic cells transfected with mRNA or loaded with apoptotic cells for immunotherapy of high-risk neuroblastoma

Children with high-risk neuroblastoma (NB) have a poor clinical outcome. The purpose of the present study was to evaluate different strategies for immunotherapy of high-risk NB based on vaccination with antigen-loaded dendritic cells (DCs). DCs are professional antigen-presenting cells with the ability to induce antitumor T-cell responses. We have compared DCs either loaded with apoptotic tumor cells or transfected with mRNA from the NB cell line HTB11 SK-N-SH, for their capacity to induce T-cell responses in vitro. Monocyte-derived DCs from healthy donors were loaded with tumor-derived antigens in the form of apoptotic cells or mRNA, matured and used to prime autologous T cells in vitro. After 1 week, T-cell responses against antigen-loaded DCs were measured by ELISPOT assay. DCs loaded with apoptotic NB cells or transfected with NB-cell mRNA were both able to efficiently activate autologous T cells. Both T cells of the CD8+ and CD4+ subset were activated. T cells activated by NB mRNA transfected DCs extensively crossreacted with DCs loaded with apoptotic NB cells and vice versa. The results indicate that loading of DCs with apoptotic NB cells or transfection with tumor mRNA represent promising strategies for development of individualized cancer vaccines/cancer gene therapy in treatment of NB.