树突状细胞与细胞因子诱导的杀伤细胞共培养对白血病耐药细胞杀伤作用的实验研究

目的探讨负载P-糖蛋白(P-gp)高表达的多药耐药(MDR)白血病K562/A02细胞冻融抗原的树突状细胞(DC)与同源细胞因子诱导的杀伤细胞(CIK)共培养对MDRK562/A02杀伤作用的影响。方法提取健康人骨髓单个核细胞,常规诱导出DC及CIK,将K562/A02细胞冻融物作为抗原冲击的DC,与CIK共培养作为实验组,抗原不冲击的DC与CIK共培养作为对照组,以CIK及DC单独培养分别作为空白对照组1和空白对照组2。光镜下观察细胞形态,流式细胞术分析细胞表型,MTT法检测杀伤活性。结果实验组、对照组细胞增殖活性均大于CIK组(P<0.05)。实验组对K562/A02、K562的杀伤活性在效靶比5∶1、10∶1、20∶1时分别为(42.90±0.67)%、(49.85±0.28)%、(63.36±0.46)%和(23.56±0.43)%、(26.11±0.34)%、(34.46±0.35)%,均高于对照组及空白对照组1(P<0.05);实验组对K562/A02的杀伤活性高于K562和MCF7(P<0.05)。结论DC与CIK共培养物是一种增殖活性和细胞毒活性高于CIK的免疫活性细胞,而经冻融抗原冲击的DC与CIK共培养能明显提高对MDRK562/A02的杀伤活性。     

IL-24对细胞因子诱导的杀伤细胞的作用

目的:寻求增强CIK细胞的细胞毒活性的有效方法.方法:从健康人外周血中提取出单个核细胞,第1天加入IFN-γ,第2天加入IL-1,CD3 mAb,IL-2诱导CIK细胞,另一组与IL-1.CD3 mAb,IL-2同时加入IL-24.杀伤分为4组:未加IL-24培养组、单独IL-24杀伤组、加IL-24培养组、未加IL-24的CIK细胞培养组在杀伤时加入IL-24.细胞计数法测定细胞的增殖、MTT法测定细胞杀伤活性和流式细胞术分析细胞表型.扫描电镜和透射电镜观察CIK细胞对肿瘤细胞的杀伤和肿瘤细胞的改变.结果:未加IL-24培养组CIK细胞增殖高于加IL-24培养组,两者比较有明显差异(126.34±2.14 vs 108.87±1.29,P<0.05).加IL-24培养组细胞各个效靶比杀伤活性均达到90%以上,明显高于其他各组(效靶比为10:1时95.58%±2.21% vs 27.31%±2.69%,8.74%±2.41%,38.65%±21.30%,P<0.05;效靶比为20:1时91.97%±4.21% vs 34.27%±0.85%,11.54%±2.78%,48.32%±11.72%,P<0.05;效靶比为40:1时91.84%±9.28% vs 50.67%±1.30%,23.73%±11.07%,52.89%±12.26%,P<0.05).不同时间加IL-24培养组各个细胞表型与未加IL-24培养组相比没有差别.透射电镜下观察加IL-24培养组凋亡和坏死肿瘤细胞比未加IL-24培养组明显增多.结论:CIK细胞诱导过程中加入IL-24能明显增强其杀伤活性.     

骨髓瘤独特型抗原负载树突细胞介导的抗肿瘤效应研究

目的:研究骨髓瘤独特型抗原(Idiotype,Id)负载树突细胞(DC)对同源细胞因子诱导的杀伤细胞(CIK)体外抗瘤活性的影响。方法:采集健康供者外周血单个核细胞(PBMNC)用常规方法诱导DC和CIK细胞,将骨髓瘤OPM-2细胞培养上清提取的Id冲击或未冲击的DC与CIK细胞共培养(CIK、DC加CIK、Id-DC加CIK),用流式细胞术分析细胞表型,MTT法检测体外效应细胞杀伤活性。结果:在(5～20):1效靶比范围内, CIK细胞对OPM-2和K562细胞的杀伤率分别为(24．47±3．00)％～(40．64±1．62)％和(23．36±1．51)％～(42．52±2．06)％。DC加CIK及Id—DC加CIK对OPM-2和K562细胞的杀伤活性均高于CIK组,差异有统计学意义(P<0．05);而在相同效靶比之下,Id-DC加CIK对OPM-2细胞的杀伤活性最强,差异有统计学意义(P <0．05)。结论:CIK细胞对骨髓瘤细胞有强的杀伤活性,经Id负载的DC与CIK细胞共培养能进一步增强其特异性杀伤活性,对骨髓瘤可能有免疫治疗作用。     

树突状细胞增强细胞因子诱导的杀伤细胞抗神经胶质瘤细胞作用及机制研究

目的探讨细胞因子诱导的杀伤细胞（CIK）与树突状细胞（DC）共培养后DC-CIK混合细胞抗神经胶质瘤细胞的免疫作用。方法分离健康人外周血单个核细胞,分别于体外诱导DC和CIK,然后共培养成DC-CIK细胞。实验分DC组、DC-CIK组、DC-T组和CIK组。Elisa试剂盒检测各组培养上清中IL-12和IFN．1的含量,流式细胞仪检测细胞表型,CCK一8法体外检测对神经胶质瘤细胞的杀伤活性。结果DC-CIK组培养上清中IL-12和IFN-1的含量分别为（110．24±2．22）mg／L和INF·Y／（913．46±20．64）mg／L,明显高于其它三组（P〈0．05）。DC—CIK组cDi细胞（61．34-1．31）％、CD3／CD56细胞（29．4±1．03）％也明显增加（P〈0．05）。对神经胶质瘤细胞的杀伤活性,DC—CIK组为（54．67±2．62）％,与DC组（19．44±1．07）％、DC—T组（21．27±1．85）％和CIK组（36．52±2．06）％比较,差异有统计学意义（P〈0．05）。结论DC—CIK细胞能诱导明显的神经胶质瘤细胞杀伤活性,为颅内肿瘤的免疫治疗提供了依据。     

CIK细胞对结肠癌SW620和LOVO细胞株的杀伤作用观察

目的观察细胞因子诱导的杀伤细胞（C IK细胞）对结肠癌SW 620和LOVO细胞株的杀伤作用。方法无菌采集健康人和结肠癌患者外周血,诱导制备C IK细胞。采用流式细胞术检测两者细胞表型。将健康人、结肠癌患者来源的C IK细胞分别以20∶1、10∶1的效靶比作用于结肠癌SW 620、LOVO细胞株,测算杀伤活性。结果用健康人来源的C IK细胞处理SW 620细胞,效靶比为20∶1时的杀伤活性为80.86%±6.08%,10∶1时为78.00%±7.63%;处理LOVO细胞,效靶比为20∶1时的杀伤活性为86.13%±6.97%,10∶1时为82.15%±6.60%。用结肠癌患者来源的C IK细胞处理SW 620细胞,效靶比为20∶1时的杀伤活性为63.36%±5.26%,10∶1时为65.35%±6.28%;处理LOVO细胞,效靶比为20∶1时的杀伤活性为60.33%±4.09%,10∶1时为55.16%±5.82%。相同靶细胞和相同效靶比下健康人来源的C IK细胞的杀伤活性明显高于结肠癌来源的C IK细胞（P均〈0.05）。结论健康人、结肠癌患者来源的C IK细胞对结肠癌SW 620、LOVO细胞均有杀伤作用。健康人来源的C IK细胞对结肠癌SW 620、LOVO细胞株的杀伤作用更强。     

树突细胞-细胞因子诱导的杀伤细胞在抗肿瘤生长免疫治疗中的应用

目的研究树突细胞(DC)与细胞因子诱导的杀伤细胞(CIK)在抗肿瘤生长免疫治疗中的应用情况。方法采集26例恶性肿瘤患者外周血进行细胞培养与回输,随机分为观察组(PC、CIK回输)和对照组(CIK回输)。采用流式细胞仪检测细胞免疫表型、采用酶联免疫吸附试验(ELISA)双抗体夹心法检测干扰素(IFN)-γ、白细胞介素(IL)-12、肿瘤坏死因子(TNF)-α细胞因子水平,记录患者不良反应与疗效。结果观察组CD3~+/CD8~+细胞、CD3~+/CD56~+细胞双阳性细胞比例显著高于对照组(P<0.05);CIK细胞的IFN-γ、IL-12、TNF-α细胞因子均显著低于DC-CIK细胞(P<0.05);观察组不良反应率显著低于对照组、治疗效果显著优于对照组(P<0.05)。结论肿瘤患者免疫结构受损,DC-CIK免疫功能优于单纯CIK;DC-CIK生成IFN-γ、IL-12、TNF-α细胞因子能力强、杀伤肿瘤细胞效果优;DC-CIK免疫治疗可有效缓解治疗中发热、发量减少、白细胞数量降低等副作用。     

脐血DC-CIK共培养体外抗肿瘤效应及趋化性的实验研究

目的探讨细胞因子诱导的杀伤细胞（CIK）与同源树突状细胞（DC）共培养后对人白血病K562细胞、人淋巴瘤raji细胞、人乳腺癌MCF-7细胞的杀伤作用及CIK细胞的趋化性。方法采集健康产妇分娩的正常足月胎儿脐血,分离单个核细胞,诱导培养CIK、DC细胞。将成熟DC和CIK混合培养3d,用MTT法检测CIK、DC-CIK对K562、raji、MCF-7细胞的杀伤活性;趋化试验检测CIK细胞的趋化性。结果 CIK、DC-CIK细胞对K562、raji、MCF-7细胞均具有较强的杀伤作用,DC-CIK杀伤活性明显高于CIK。趋化试验显示,IL-8、MCP-1作用后穿过微孔滤膜的细胞数明显高于阴性对照。结论 DC-CIK共培养可明显提高CIK对K562、raji、MCF-7细胞的杀伤作用,IL-8、MCP-1对CIK细胞存在趋化性。     

树突状细胞联合细胞因子诱导的杀伤细胞对胃癌细胞杀伤作用

目的探讨树突状细胞联合细胞因子诱导的杀伤细胞对胃癌细胞的杀伤作用。方法采用胃癌患者自身血液中单个核细胞(peripheral blood mononuclear cells,PBMC),经体外诱导分别扩增出DC和CIK细胞,二者共同培养后,利用MTT法检测DC细胞联合CIK细胞体外杀伤人胃癌细胞株(MNK-45、MNK-28、SG-7901)的活性。结果DC与CIK细胞共培养后得到的细胞群高表达CD3 CD56 ,平均值达到(56.74±7.63)%。通过彼此相互作用诱导出的细胞群体对胃癌细胞株MNK-45、MNK-28、SG-7901有杀伤作用,且杀伤活性随着效靶比的增加而增强。结论DC与CIK细胞共培养后有很强的增殖能力,对胃癌细胞具有杀伤活性,且其杀伤作用与胃癌细胞类型无相关性。     

DC-CIK共培养在AL化疗后免疫治疗中的应用及其对NKG2D表达的影响

目的观察树突状细胞(DC)—细胞因子诱导的杀伤细胞(CIK)共培养用于急性白血病(AL)化疗后免疫治疗的疗效,及其对自然杀伤(NK)细胞表面活化受体NKG2D表达的影响。方法 31例AL患者经诱导治疗骨髓达完全缓解后,给予DC-CIK共培养治疗。治疗前后检测外周血中T细胞亚群表达、血清IL-2、IL-12、TNF-α、IFN-γ水平及外周血中NKG2D的表达。结果所有患者治疗结束后骨髓均处于完全缓解状态;DC-CIK共培养治疗后,CD3+、CD3+CD8+、CD3+CD5+6/CD1+6高于治疗前(P均<0.05);血清IL-2、IL-12、TNF-α、IFN-γ水平高于治疗前(P均<0.05);外周血单个核细胞中NKG2D阳性表达率为25.41%±8.47%,高于治疗前的8.07%±2.02%(P<0.05)。结论 DC-CIK共培养用于AL化疗后的免疫治疗效果较好,能更有效地清除微小残留病灶,提高外周血中NKG2D的表达,纠正AL细胞对自体NK细胞的免疫编辑作用。     

新型免疫活性细胞CIK体外对肝癌细胞的杀伤

目的 观察人体CIK(cytokine-induced killer)细胞的体外增殖力及杀伤肝癌细胞活性.方法 采取健康自愿者的成分血,用淋巴细胞分离液分离获得外周血单个核细胞(PBMC),在体外培养条件下,通过加入不同细胞因子(如IFN-γ,IL-1,IL-2,CD3 mAb)培养诱导成CIK细胞和LAK细胞,观察不同培养细胞的增殖能力;用流式细胞仪对培养细胞做细胞表型动态分析,观察培养细胞的表面标志;与LAK细胞作对比,用MTT法测定并比较不同效应细胞的体外抗肝癌细胞活性.结果 CIK细胞在培养14 d后开始大量增殖且在含人血清的培养基中增殖数量最多,可达500多倍.经表型分析表明,CIK细胞属于异质细胞群,在培养过程中,CD3+CD56+双阳性细胞的绝对数量获得了大量增殖,至56 d时可占细胞总数的51.26%,是CIK主要的效应细胞;实验表明,CIK细胞体外杀伤肝癌细胞的活性明显优于LAK细胞.结论 CIK细胞是一种具有较强杀伤肝癌细胞的免疫活性细胞,有可能应用于抗肿瘤的生物治疗.     

Lymphokine activated killer cells

Summary Various subpopulations of human leukocytes may be induced by lymphokines to exert cytotoxic activity. In man major histocompatibility complex non-restricted tumor cell lysis by interleukin-2 (IL-2) induced peripheral blood lymphocytes is attributed mainly to natural killer cells. These T cell receptor negative large granular lymphocytes are called lymphokine activated killer (LAK) cells. In order to explore the potential of LAK cells in tumor therapy, several clinical studies have been conducted, using IL-2 alone or in combination with ex vivo IL-2-activated peripheral blood lymphocytes. Objective responses have reproducibly been achieved only in renal cell carcinoma and malignant melanoma and were associated with considerable toxicity. In view of restricted efficacy and increasing doubts as to whether LAK cells indeed account for the in vivo observed responses, more recent strategies focus on tumor antigen specific cytotoxic T cells or tumor infiltrating lymphocytes. Successful translation of this approach into clinical practice, however, may be dependend on some basic problems of tumor immunology to be solved which were thought to be by-passed by the LAK cell approach.     

自然杀伤T淋巴细胞与肝脏疾病

自然杀伤T淋巴细胞（natural killer T cells，NKT细胞）是一类同时表达恒定的T淋巴细胞受体（TCR）αβ（人为Vα24／Vβ11，鼠为Vα14／Vβ8）和NK细胞标志的T细胞亚群；主要起源于胸腺，发育成熟过程大多依赖于MHC-Ⅰ类样分子CD1d；主要分布于肝脏、骨髓、胸腺等器官，约占小鼠肝脏内成熟T淋巴细胞（T细胞）的30％，占人类肝脏T细胞的10％，外周血也有少量分布；主要经抗原提呈细胞表面CD1d提呈的糖脂类抗原活化，并迅速地释放大量的IFNγ和IL-4等细胞因子，通过调节Th1／Th2细胞间的平衡，在自身免疫性疾病、抗感染、抗肿瘤等方面发挥重要的作用。近年研究表明，NKT细胞与肝脏疾病关系密切，可能参与了肝炎、肝硬化、肝癌等诸多肝脏疾病的发生与发展。     

Natural killer T cells and haemopoiesis

Invariant natural killer T (iNKT) cells are a small but powerful subset of regulatory T cells involved in the modulation of a variety of normal and pathological immune responses. In contrast to conventional or other types of regulatory T cells, they are activated by glycolipid and phospholipid ligands that are presented to them by the non-polymorphic, major histocompatibility complex class I-like molecule CD1d. The in-depth understanding of their function has resulted in successful, iNKT cell-centred experimental therapeutic interventions including prevention of graft-versus-host disease and anti-leukaemia effects. Extending these successes into the clinical arena will require better understanding of their contribution to the pathogenesis of human, including haematological, diseases.     

Cytokine-induced killer cells/dendritic cells and cytokine-induced killer cells immunotherapy for the treatment of esophageal cancer: A meta-analysis

Objectives:This meta-analysis was designed to systematically evaluate whether autologous cytokine-induced killer cells (CIK) or dendritic cells and cytokine-induced killer cells (DC-CIK) immunotherapy combined with chemotherapy can improve the therapeutic effect and safety of chemotherapy in esophageal cancer (EC).Materials and methods:Randomized controlled trials (RCTs) were electronically searched databases including CNKI, WanFang, WeiPu, CBMDisc, PubMed, Web of Science, EMbase, the Cochrane Library, and Clinical Trials. The databases were searched for articles published until June 2019. Two researchers independently screened the literature, extracted data, and evaluated the quality of the included literature. Meta-analysis was performed using RevMan5.3.Results:Seventeen studies (1416 participants) were included. The differences between CIK/DC-CIK combination chemotherapy and chemotherapy alone were significant. The results displayed that the number of CD3⁺, CD4⁺, CD4⁺/CD8⁺, and NK cells was significantly increased after 1 to 2 weeks of treatment with CIK/DC-CIK cells in the treatment group (all P < .05). In addition, the results shown that 1-year overall survival was significantly prolonged (P < .0001) and quality of life was improved (P = .001) in EC chemotherapy combined with immunotherapy groups compared with conventional treatment. Furthermore, cytokine expression levels of interleukin 2 (IL-2), tumor necrosis factor α (TNF-α), and interleukin 12 (IL-12) were significantly increased (P = .0003) as well as the levels of immunoglobulins were elevated (P < .00001). Serum levels of tumor marker molecules, carcinoembryonic antigen (CEA), carbohydrate antigen (CA)-199, and CA-125 were lower in treatment groups than that of control groups (P < .00001). No fatal adverse reactions were noted (P = .04).Conclusions:It is safe and effective for patients to use chemotherapy combined with CIK/DC-CIK immunotherapy. Immunotherapy can simultaneously improve the antitumor immune response. Specifically, DC-CIK cells can increase T lymphocyte subsets, CIK cells, NK cells, and immunoglobulins in peripheral blood to enhance antitumor immunity. Therefore, combination therapy enhances the immune function and improves the therapeutic efficacy of patients with EC.     

不同细胞因子联合诱导K562细胞向杀伤细胞转化的研究

目的 探讨肿瘤细胞系K562向杀伤细胞转化的可能性。方法 通过rhIL-2、rhIL-15和A23187的不同组合诱导K562细胞，动态监测细胞形态学、免疫表型及杀伤功能变化。结果 经过40d的诱导，K562细胞在各细胞因子组合下均可以被诱导成具有杀伤功能的CD3^-CD36^＋NK细胞和CD3^＋CD56^＋NKT细胞，以IL-2＋IL-15组的诱导率最高，杀伤功能最强；IL-2＋A23187组的免疫标记出现最早。结论 IL-2可将K562细胞诱导成杀伤细胞dL-15可提高K562细胞向杀伤细胞转化的诱导率；A23187可能加速K562细胞向杀伤细胞的转化。     

Alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation

Natural killer (NK) cell-mediated, donor-vs.-recipient alloresponses occur following transplantation of human leukocyte antigen (HLA) haplotype-mismatched hematopoietic stem cells (HSCs). NK cell alloreactivity reduced the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-vs.-host disease (GvHD). NK cells are primed to kill by several activating receptors. NK killing of autologous cells is prevented because NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) MHC class I alleles. As KIRs are clonally distributed, the NK population in any individual is constituted of a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and in some cases, functional assessment of donor NK clones will identify haploidentical donors who are able to mount donor-vs.-recipient NK alloreactions.     

Highly activated and expanded natural killer cells for multiple myeloma immunotherapy

Background Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS: OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS: Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold, respectively, without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred, expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion, persisted up to 4 weeks, were readily detectable in the human bone, inhibited myeloma growth and protected bone from myeloma-induced osteolysis. Conclusions These studies provide the rationale for testing expanded natural killer cells in humans.     

Immunotherapy with dendritic cells and cytokine-induced killer cells for hepatocellular carcinoma: A meta-analysis

BACKGROUND Hepatocellular carcinoma(HCC) has been revealed as the second most common cause of cancer-related deaths worldwide. The introduction of cell-based immunotherapy, including dendritic cells(DCs) and cytokine-induced killer cells(CIKs), has brought HCC patients an effective benefit. However, the efficacy and necessity of cellular immunotherapy after different interventional therapy remains to be further explored.AIM To investigate the efficacy of cellular immunotherapy, involving DCs and CIKs,combined with different conventional treatments of HCC.METHODS We performed a literature search on PubMed and Web of Science up to February15, 2019. Long-term efficacy(overall survival and recurrence) and short-term adverse effects were investigated to assess the effectiveness of immunotherapy with DCs and/or CIKs. Review Manager 5.3 was used to perform the analysis.RESULTS A total of 22 studies involving 3756 patients selected by eligibility inclusion criteria were forwarded for meta-analysis. Combined with the conventional clinical treatment, immunotherapy with DCs and/or CIKs was demonstrated to significantly improve overall survival at 6 mo [risk ratio(RR) = 1.07; 95%confidence interval(CI): 1.01-1.13, P = 0.02], 1 year(RR = 1.12; 95%CI: 1.07-1.17, P 0.00001), 3 years(RR = 1.23; 95%CI: 1.15-1.31, P 0.00001) and 5 years(RR =1.26; 95%CI: 1.15-1.37, P 0.00001). Recurrence rate was significantly reduced by cellular immunotherapy at 6 mo(RR = 0.50; 95%CI: 0.36-0.69, P 0.0001) and 1 year(RR = 0.82; 95%CI: 0.75-0.89, P 0.00001). Adverse effect assessment addressed that immunotherapy with DCs and/or CIKs was accepted as a safe,feasible treatment.CONCLUSION Combination immunotherapy with DCs, CIKs and DC/CIK with various routine treatments for HCC was evidently suggested to improve patients' prognosis by increasing overall survival and reducing cancer recurrence.