CIK细胞体内外抗宫颈癌HeLa细胞活性的研究

目的：探讨细胞因子诱导的杀伤（cytokine-induced killer,CIK）细胞的体内外抗宫颈癌HeLa细胞活性。方法：收集8名健康献血者和8名宫颈癌患者的新鲜外周血,分别通过常规方法分离外周血单核细胞（peripheral blood mononuclear cells,PBMC）,应用相应细胞因子体外诱导分化出CIK细胞,动态观察CIK细胞的体外增殖活性、细胞表型和对HeLa细胞的杀伤活性;在BALB/c裸鼠皮下接种效应细胞,观察宫颈癌患者CIK细胞对接种HeLa细胞的荷瘤鼠的抑瘤作用,同时设淋巴因子激活的杀伤细胞（lymphokine activated killer cells,LAK）和PBMC细胞作为对照。结果：源于健康人和宫颈癌患者的CIK细胞间的增殖活性无明显区别（P〉0.05）。表型分析结果表明,两种来源的CIK细胞中CD3~＋CD56~＋双阳性细胞均得到了大量扩增,宫颈癌患者CIK细胞中CD3~＋CD56~＋双阳性细胞在实验开始前约占0.13%,到实验后第28天上升到25.8%。体外实验表明,宫颈癌患者的CIK细胞杀伤宫颈癌HeLa细胞的细胞毒活性明显高于PBMC细胞。裸鼠体内实验表明,宫颈癌患者CIK细胞能够显著抑制肿瘤的生长,其抑瘤率可达80.6%,高于LAK细胞的59.1%和PBMC细胞的38.3%（P〈0.01）。CIK治疗后肿瘤体积明显比空白对照组缩小（P〈0.05）。结论：宫颈癌患者CIK细胞具有较强的体内外抗宫颈癌细胞活性,有可能用于临床上宫颈癌的过继性免疫治疗。     

细胞因子诱导的杀伤细胞治疗恶性淋巴瘤的研究进展

细胞因子诱导的杀伤细胞（CIK细胞）是一群体外诱导的以CD3＋ CD56＋T细胞为主的异质细胞群,具有效应CD8＋T细胞的T细胞受体特异性和非主要组织相容性复合体（MHC）限制性抗肿瘤活性的特点.国内外研究结果表明CIK细胞对恶性淋巴瘤有明显的抗肿瘤效应,且有很好的耐受性.提示CIK细胞治疗对于恶性淋巴瘤患者尤其是不能耐受放化疗的淋巴瘤患者具有重要价值.     

CIK细胞-肿瘤过继免疫治疗的新希望

1 概述CIK细胞（cytokine－induced killer）是将人外周血单个核细胞在体外用多种细胞因子（如抗CD3McAb、IL－2、IFN－γ、IL－1α等）共同培养一段时间后获得的一群异质细胞。由于该种细胞同时表达CD3和CD56两种膜蛋白分子1,故又被称为NK细胞样T淋巴细胞,兼具有T淋巴细胞强大的抗瘤活性和NK细胞的非MHC限制性杀瘤优点。因此,应用CIK细胞被认为是新一代抗肿瘤过继细胞免疫治疗的首选方案。2 CIK细胞表型特点CIK细胞中的效应细胞CD3＋CD56＋细胞在正常人外周血中极其罕见,仅1％～5％,在体外经多因子培养28～30天,…     

CIK、DC-CIK细胞对神经母细胞瘤细胞杀伤作用的研究

目的：研究细胞因子诱导的杀伤细胞（ CIK）与树突状细胞（ DC）共培养后对神经母细胞瘤（ neuro-blastoma,NB）细胞株的杀伤作用。方法：取健康人和肿瘤患者外周血单个核细胞（ PBMC）,加入不同的细胞因子分别诱导出DC和CIK细胞,用流式细胞术测定诱导培养前后DC和CIK细胞的表型,MTT法测定不同组CIK细胞对NB细胞株的杀伤活性。结果：流式细胞仪检测健康人PBMC培养后CD3＋CD56＋淋巴细胞百分比以及对NB细胞株的杀伤活性均显著高于肿瘤患者（ P〈0.05）。此外,与单纯CIK细胞相比,DC-CIK细胞具有更强的杀伤NB细胞株的活性（ P〈0.05）。结论：DC-CIK细胞是一种细胞毒作用高于单纯CIK细胞的免疫活性细胞。健康人和肿瘤患者的PBMC经诱导培养获得的CIK细胞有显著差别,为临床进一步提高CIK细胞的治疗效果提供了实验依据。     

CIK细胞联合树突状细胞抗肿瘤作用的研究进展

树突状细胞（dendritic cell，DC）是当今肿瘤免疫治疗领域备受关注的热点之一。由DC激活的细胞免疫在机体抵御恶性肿瘤中发挥着十分重要的作用。细胞因子诱导的杀伤细胞（cytokine—induced killer cells，CIK）是一种高效的肿瘤杀伤细胞。研究表明树突状细胞联合CIK细胞可更有效地杀伤肿瘤细胞。现就树突状细胞联合CIK细胞抗肿瘤作用的基础和临床研究进展做一综述。     

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。     

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.     

mRNA转染树突细胞协同CIK细胞体内抗肿瘤作用的研究

目的:研究人肺腺癌细胞系SPC-Al mRNA基因转染的树突状细胞(dendritic cells, DCs)和细胞因子诱导活化杀伤细胞(cytokine induced killer, CIK)体内协同抗瘤活性.方法:按常规方法分离健康人外周血单个核细胞,并分别诱导培养为DC和CIK细胞.将人肺腺癌细胞mRNA转染DC细胞,并将转染后成熟DC与CIK细胞混合培养,流式细胞仪检测混合培养前后的CIK细胞表型变化,并通过建立人肺腺癌细胞移植模型研究其体内的协同抗肿瘤活性.结果:混合培养后的CIK细胞表面CD3 CD8 及CD3 CD56 表达显著升高,P<0.01;共孵育后的mRNA-DC-CIK细胞组与其余各组相比,体内肿瘤抑制效率明显增大.结论:利用该方法来负载DC瘤苗具有可行性,为临床上解决DC瘤苗因肿瘤抗原的来源困难问题及提高CIK细胞的特异性抗肿瘤活性提供实验依据,为肺癌的治疗开辟一种新的有效的免疫治疗策略.     

中晚期肺癌患者外周血CIK细胞水平及其临床意义

肿瘤免疫是目前肿瘤研究热点之一，其中免疫活性细胞及其在肿瘤免疫中作用备受关注。CIK（cytokine-induced killer cells）细胞是目前对体内肿瘤细胞产生非MHC限制性杀伤活性最强的免疫效应细胞之一。本研究采用流式细胞术检测25例中晚期肺癌患者外周血CIK细胞水平并分析其临床意义。     

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。     

Usefulness of Lymphokine-activated Killer Cells Generated from Bone Marrow Mononuclear Cells for the Purging of Residual Tumor Cells in Peripheral Blood Stem Cell Graft

Lymphokine-activated killer (LAK) cells were generated from bone marrow mononuclear cells (BM), and the usefulness of the BM-LAK for purging of residual tumor cells in autologous peripheral blood stem cell (PBSC) graft was determined. The BM and peripheral blood lymphocytes (PBL) were obtained from the same bone marrow donors. The BM-LAK and PBL-LAK were generated by incubation with interleukin-2 for 7 days. The BM-LAK demonstrated higher killer activity against a lymphoma cell line Raji than the PBL-LAK. The BM-LAK also had a higher percentage of CD4^-CD8^-CD16⁺ cells than the PBL-LAK, which suggests that their high killer activity is related to these cells. The BM-LAK did not show any killer activity against the PBSC graft. However, they killed tumor cells which contaminated the PBSC graft, and in particular, killed chimeric ber/abI messenger RNA-positive residual leukemic cells. These results suggest that the BM-LAK may be applicable for purging. As the BM-LAK possess higher killer activity than the PBL-LAK, they may be more useful than the PBL-LAK.     

Lymphokine-activated killer cell activity of peripheral blood,spleen, regional lymph node,and tumor infiltrating lymphocytes in gastric cancer patients

Lymphokine-activated killer (LAK) cell activity of peripheral blood mono-nuclear cells (PBM), spleen cells (SPC), regional lymph node cells (LNC), and tumor-infiltrating lymphocytes (TIL), induced by activation with in-terleukin 2 (IL 2) for 4 days, was evaluated in patients with gastric carcinoma. TIL exhibited the lowest LAK activity and the cytotoxicity of LNC was significantly lower than that of either PBM or SPC. There was no difference between PBM and SPC. Then, there were significant correlations of LAK activity among PBM, SPC, and LNC, whereas poor correlations were observed in the cytotoxicity between TIL and PBM, SPC, or LNC. Phenotypic analysis of each cell population was performed before and after activation with IL 2. Before culture, the cells mediating natural killer (NK) activity such as CD16⁺, CD56⁺, and CD57⁺ cells were few in LNC and TIL. However, CD56⁺ and CD57⁺ cells in TIL were increased after culture. Then, CD4⁺ Leu8⁺ and CD8⁺ CD11⁺ cells, which identify suppressor cell function, were not elevated in LNC or TIL, as compared to that in PBM or SPC. Further, the proportions of OKIa1⁺ and CD25⁺ cells expressing T-cell activation and IL 2 receptor were uniformly increased in all cell populations after culture. These results indicate the differential reactivity of each lymphocyte population to IL 2 and fundamental dysfunction of LNC and, especially TIL, suggesting the specific influence of the local tumor environment on the lymphocyte function in the area in patients with gastric carcinoma. © Wiley-Liss, Inc.     

双特异抗体介导的CD_3AK对人小细胞肺癌细胞株的细胞毒作用

目的 :研究在双特异性抗体的介导下CD3AK细胞 (CD3单克隆性激活的杀伤细胞 )对人小细胞肺癌细胞株LTEP sm1的细胞毒作用。方法 :用51Cr Na2 CrO4 释放试验检测单抗 (2D6、UCHT1)和双特异性抗体 (WST H7)与CD3AK共同对人小细胞肺癌细胞株的杀伤活性。结果 :双特异性抗体体外能明显增强CD3AK细胞对人小细胞肺细胞株LTEP sm1的杀伤活性。结论 :与单纯CD3AK相比 ,加入双特异性抗体后的CD3AK细胞的细胞毒活性显著增强     

康赛迪胶囊对食管癌放疗患者细胞免疫功能的影响

目的　观察中药复方制剂康赛迪胶囊对食管癌患者放疗前后细胞免疫功能的影响。方法　采用FACSCalibur流式细胞仪 ,SimulSETv3 1分析软件 ,分别检测 2 0例食管癌患者在放疗 6 0Gy前后 (对照组 )和口服康赛迪同时放疗 6 0Gy前后 (实验组 )患者外周血NK细胞活性、T淋巴细胞亚群 (CD3+、CD4 +、CD8+)、CD4 +/CD8+、B淋巴细胞 (CD19+)、NK淋巴细胞 (CD16 +,CD5 6 +)水平。结果　二组患者放疗前外周血NK细胞活性、CD3+、CD4 +、B淋巴细胞百分数、CD4 +/CD8+的平均值均低于健康人参考值 ,其中CD4 +细胞和B淋巴细胞明显下降 ,CD8+细胞明显升高。对照组患者放疗 6 0Gy后 ,除CD8+细胞和NK淋巴细胞百分数稍有升高外 ,其他细胞均略有下降 (P >0 0 5 )。实验组患者在治疗后CD3+、CD4 +、NK淋巴细胞百分数、CD4 +/CD8+明显升高 ,并明显高于对照组 (P <0 0 5或P <0 0 1)。结论　食管癌患者在放疗前后细胞免疫功能持续低下。放疗 6 0Gy后患者细胞免疫功能受到轻度抑制 ,康赛迪可以明显改善食管癌患者放疗后的细胞免疫功能状态。提示食管癌患者在放疗的同时应加强免疫治疗 ,以改善患者的免疫抑制状态。     

DC-CIK过继性细胞免疫治疗对卵巢癌患者的预后及免疫功能的影响

目的:探讨自体DC-CIK细胞疗法改善卵巢癌患者的预后及免疫功能的能力。方法:回顾性分析2014年1月至2015年12月在中国医科大学附属盛京医院均接受了规范的初始治疗、达到完全缓解（CR）的卵巢癌患者90例。分析比较其基本条件、免疫指标、无瘤生存时间及不良反应。结果:经DC-CIK生物免疫治疗的患者外周血CD3+、CD4+细胞百分率,CD3+、CD8+细胞百分率及CD4+/CD8+比值均上升,NK细胞即CD16+、CD56+细胞增多,同时随着疗程数的增多,升高的更加明显（P<0.05）。一线治疗后,单纯观察组平均无瘤生存时间为15.88±2.7个月明显少于DC-CIK生物免疫治疗组的22.5±4.13个月（P=0.039）。其中使用DC-CIK生物免疫治疗疗程≥5程的平均无瘤生存时间为27.25±4.53个月明显高于DC-CIK生物免疫治疗疗程≤4程的19.69±2.81个月,两者相比差异无明显统计学意义（P=0.412）。所有患者DC-CIK细胞治疗后血常规及肝肾功能检测与治疗前及单纯观察组患者相比无明显变化,也未见明显不良反应。结论:DC-CIK免疫治疗提高卵巢癌患者的免疫反应,改善卵巢癌患者预后,未见明显不良反应。     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

应用晚期乳腺癌患者外周血单个核细胞体外增殖诱导新型CIK细胞的实验研究

目的 应用晚期乳腺癌患者外周血来源的单个核细胞体外培养诱导产生新型(Cytokine induced killer cells,CIK)细胞的可能性,为乳腺癌患者应用自体免疫细胞治疗提供理论基础。方法 取8例晚期乳腺癌患者外周血提取单个核细胞,经体外培养诱导增殖,并应用细胞计数法和流式细胞仪检测增殖细胞表面特异性标志CD3、CD16和CD56,应用51Cr release assay及MTT方法测定其对MCF7及BT20乳腺癌细胞株的杀伤能力。应用ELISA试验方法对诱导得到的新型CIK细胞的培养上清液进行解析。结果 经过体外18天培养,平均得到8.2×10⁸个以上纯度为95.2%~98.1%的CD16⁺、CD56⁺和CD16⁺CD56⁺阳性高纯度NK细胞的新型CIK细胞,且对乳腺癌细胞株MCF7及BT20具有明显的抑制作用。结论 成功应用晚期乳腺癌外周血单个核细胞选择性扩增诱导高纯度NK细胞的新型CIK细胞,且证明其对MCF7及BT20乳腺癌细胞具有明显抑制作用,为应用自体高纯度NK细胞的新型CIK细胞为基础的过继性免疫细胞治疗乳腺癌提供理论研究基础。     

Clinical stage-depending decrease of NK cell activity in multiple myeloma patients

Natural killer cells, as an important subpopulation of cells of the innate immune system have an essential role in defense of the rise and spread of malignancy. These cells have a CD3-CD16 + CD56+ phenotype and they are functionally defined by their ability to lyses tumor cells. We here show that decrease of NK cell activity was significantly associated with advanced clinical stage, increased lactate dehydrogenase (LDH), percentage infiltration of bone marrow with plasma cells, and β-2 microglobulin. The patients with higher NK cell activity at presentation after receiving VAD protocol have better cumulative survival in comparison with those with low NK cell activity.     

Effect of Cytotoxic Cells on Minimal Residual Leukemia

The presence of minimal residual disease is indicated by the high frequency of relapses after twin bone marrow transplants and after allogeneic bone marrow transplants without graft versus host disease (up to 75% and 45% of cases, respectively). The graft versus leukemia effect may be mediated by IL-2 activation of natural killer cells (CD16 +, CD56 +, CD3-, CD8+/-) or cytotoxic T cells (CD3 +, CD56+/-). These activated killer cells can bind to targets and cause their lysis, and then recirculate to kill other targets. Killing can be blocked by anti-perforin antibodies and enhanced by protein kinase C-activation of effectors

There are several studies indicating that a high percentage of leukemic cells can be killed by LAK-cells. However even in the most sensitive cases, lysis of all the cells cannot be achieved. The finding that leukemic clonogenic cells are generally sensitive to both NK and LAK cytotoxicity provides a more hopeful possibility

The lack of tumor specific antigents, leukemic cell-immunoheterogeneity and maturation asynchrony explains why antigen dependent T-cell mediated cytotoxicity is only partly effective in eradicating residual leukemic cells. Future work should therefore include more studies of the mechanism of resistance to LAK cells, possibilities of further enchancing cytotoxicity and the mechanism of graft-versus-leukemia effect