抵抗LAK细胞杀伤的胃癌细胞株的建立及生物学特性的研究

人胃癌细胞系ＭＧＣ－８０３经反复与ＬＡＫ细胞共培养十个周期，获得对ＬＡＫ细胞杀伤抵抗的特性，命名该细胞变株为ＭＧＣ－８０３Ｒ。当效靶比为４０∶１时，对该细胞变株的杀伤率仅为４０％，而对亲代细胞的杀伤率为９０％。用“冷”靶细胞抑制试验、电镜、ＦＡＣＳ及荧光标记技术分析，证实ＭＧＣ－８０３Ｒ细胞膜结合识别位点有改变，ＭＨＣ－Ⅰ表达升高，ＩＣＡＭ－１表达降低，相互粘附的细胞数减少，胞内Ｆ肌动蛋白小体消失，微丝恢复有序排列，细胞生长速率减慢。上述结果提示经免疫筛选对杀伤有抵抗的ＭＧＣ－８０３Ｒ细胞在生物学特性上有所改变。     

细胞因子诱导的杀伤细胞的研究进展

正常人或患者外周血、骨髓单个核细胞中定期加入IFN-γ、IL-1、IL-2、CD3mAb经2-4W的诱导可获得大量的新型肿瘤杀伤细胞—细胞因子诱导的杀伤细胞CIK,此细胞增殖旺盛杀伤活性高。CIK在体内和体外都具有非MHC限制抗肿瘤活性,在临床上已经用于治疗多种恶性肿瘤表现出强大的杀瘤活性,是一种很好的免疫治疗方法。     

细胞因子诱导杀伤（CIK）细胞的大容量扩增与杀伤活性观察

建立大容量细胞因子诱导杀伤(Cytokine-inducedkiller,CIK)细胞培养方法,观察CIK细胞回输后对患者细胞免疫功能的影响。采用1000ml培养袋大量扩增患者自体CIK细胞,用MTT法检测CIK细胞杀伤活性,比较回输前后患者外周血单个核细胞(PBMNC)对靶细胞的杀伤活性及其毒副作用。结果表明大容量培养法使自体CIK细胞扩增总量达1.6×1010以上,回输CIK细胞使患者PBMNC的杀伤活性明显增加,未出现毒副作用。因此CIK细胞大容量扩增方法在治疗肿瘤微小残留病变上有着良好的临床应用前景。     

细胞因子调节人肝癌细胞ICAM—1分子的表达及其对CD3AK杀伤功能…

应用基因重组人干扰素α（ＩＦＮ－α）、干扰素γ（ＩＦＮ－γ）和肿瘤坏死因子（ＴＮＦ）体外处理人肝癌细胞系Ｈ－７４０２，ＡＢＣ－ＣＥＬＩＳＡ法检测各处理组和对照组细胞间粘附分子－１（ＩＣＡＭ－１）的表达水平，用ＬＤＨ释放法观察ＣＤ３单克隆缺本活化的杀伤细胞对各处理条件下Ｈ－７４０２细胞的杀伤活性。     

制备细胞因子诱导的杀伤细胞的方法比较

生物治疗是目前国内外治疗恶性肿瘤常规方法即手术治疗、化疗和放疗之外的一种新型疗法.过继性细胞免疫治疗(adaptive cellular immunotherapy,ACI)是生物治疗的一种,它是一种通过将具有抗肿瘤活性的、能直接或间接介导抗肿瘤效应的免疫细胞输注肿瘤宿主体内进行肿瘤治疗的方法[1].对于促进患者免疫系统的重建、消除残留病变及骨髓净化都具有良好效果[2].细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK细胞)是将人外周血单个核细胞在体外用多种细胞因子共同培养一段时间后获得的一群异质细胞,具有增殖速度快、杀瘤活性高、杀瘤谱广等优点[3-4],已广泛应用于临床,但疗效差异很大,原因可能与制备CIK细胞的方法、所使用细胞因子的种类和剂量密切相关.本研究拟采用3种方法制备CIK细胞,并比较用这3种方法制备的CIK细胞的生物学特性及其杀瘤活性,为制备高活性的CIK细胞提供方法,以提高CIK细胞临床应用的疗效.     

不同激活方式影响细胞因子诱导杀伤细胞的扩增与活性

目的：研究不同激活方式对CIK细胞(Cytoline-induced killer cells，CIK)体外扩增的影响。方法：使用植物血球凝集素(pHYTOHEMAGGLUTININ，PHA)或抗CD3单抗刺激细胞增殖，从脐血单个核细胞定向诱导CIK细胞，用乳酸脱氢酶(LDH)分析法分析细胞毒活性，用流式细胞分析法对不同培养条件下CIK细胞的纯度进行分析。结果:使用抗CD3 mAb刺激生成的CIK细胞在扩增能力、纯度和细胞毒性方面都优于使用PHA刺激生成的CIK细胞。在细胞因子和有丝分裂原加入3d后，将其洗脱，但仍加入IL-2，有利于提高细胞的扩增能力。另外，使用包被的方法将抗CD3 mAb固定在孔板上比悬浮加入效果好。结论：这一结果对于CIK细胞的大量扩增和临床应用具有指导意义。     

-80℃冰箱直接冻存对细胞因子诱导的杀伤细胞杀伤活性的影响

目的:研究-80℃冰箱直接冻存对细胞因子诱导的杀伤细胞(CIK)杀伤活性的影响。方法采用非程序降温方法-80℃冰箱冻存CIK,于冻存后6、12、18周复苏,通过LDH释放法分别测定其对K562细胞的杀伤活性,与新鲜未经冻存的CIK的杀伤活性进行比较。结果:未冻存的CIK在培养至第11天时,有较多贴壁成团细胞,而冻存后复苏培养的CIK贴壁成团细胞较少。比较各组细胞杀伤活性,冻存6、12周后复苏的CIK与未冻存的CIK,对K562细胞的杀伤活性无显著性差异(P>0.05),冻存18周后复苏的CIK的杀伤活性与未冻存的CIK对K562细胞的杀伤活性有显著性差异(P<0.05)。结论:采用-80℃冰箱直接冻存CIK,在12周内复苏应用于临床治疗较好。     

细胞因子诱导杀伤细胞转MDR1基因诱导耐药研究

目的细胞因子诱导杀伤细胞(cytokine-induced-killer,CIK)进行临床免疫治疗能够杀伤肿瘤细胞,将MDR1基因转入CIK细胞,观察其是否产生耐药性并且保持其肿瘤杀伤活性.方法用Ficoll密度梯度离心法获得外周血单个核细胞,分别加入IFN-γ,CD3Mab,IL-2,IL-1等细胞因子体外培养获得CIK细胞.在细胞呈对数生长期时,采用电穿孔方法将多药耐药基因PHAMDR质粒转入细胞.转染后72h提取细胞总RNA,RT-PCR鉴定耐药基因表达;流式细胞仪方法检测细胞膜泵蛋白P-gp.四唑蓝比色法(MTT Assay)检测转基因CIK细胞对阿霉素和秋水仙碱的耐药性,同时检测转染前后CIK细胞对人类乳腺癌细胞系(MCF7)的杀伤活性变化.结果转染后CIK细胞出现MDR1基因的转录产物mR-NA;流式细胞仪检测,转染后的CIK细胞表达耐药蛋白P-gp,阳性细胞约为21%.阿霉素对转染后CIK细胞的IC50为0.11mg/ml,转染后CIK细胞对阿霉素的耐受性较转染前CIK细胞(IC50为0.0024mg/ml)增强了45.8倍;秋水仙碱对转染后CIK细胞的IC50为1.34ng/ml,转染后耐受性较转染前CIK细胞增强了11.35倍(IC50为0.118ng/ml).比较转染前后CIK细胞对MCF7肿瘤细胞的杀伤活性无显著性差异(P＞0.05).结论用电穿孔方法可以成功地将MDR1基因转入CIK细胞,并使其表现出多药耐药性,转染前后细胞杀伤活性无变化.     

胃癌干细胞抗原负载树突细胞与细胞因子诱导的杀伤细胞

BACKGROUND:As the existence of tumor stem cells, it is difficult to completely eliminate tumors in clinic. OBJECTIVE:To explore the tumor-killing effect of gastric cancer stem cells as antigen to stimulate dendritic cells combined with cytokine-induced killer cells. METHODS:Side population cells from human gastric cancer cell lines were isolated, and tumor antigen was prepared by freeze thawing method. After coculture with dendritic cells, dendritic cells combined with cytokine-induced killer cells, gastric cancer cell antigen, and gastric cancer stem cell antigen, killing rates of gastric cancer cells were detected using MTT assay. Expression rate of CD83, a mature dendritic cell surface marker, was also detected. RESULTS AND CONCLUSION:The CD83 expression level and killing rate of gastric cancer cells were both significantly lower in the gastric cancer stem cell antigen group than the other groups (both P < 0.05). These results indicate that gastric cancer stem cells as antigen to stimulate dendritic cells combined with cytokine-induced killer cells can promote the proliferation of gastric cancer cells and elevate the ability to killing gastric cancer cells.     

淋巴因子激活杀伤（LAK）细胞杀伤肝癌细胞的超微结构研究

Co-cultured subcloned LAK cell and human liver cancer cell (H7402) were fixed in situ for scanning electron microscopic and transmission electron microscopic examinations. The SEM and TEM findings gave a new evidence in answering how LAK cells kill the cancer cells. First, the activated killer cells recognized and made close contact with the target cell. Then some kind of lethal hit produced by intact killer cells was delivered, injuring the target cell, boring holes and pole-like tunnels on the cell surface or even penetrating into the cytoplasm of the cancer cells. Eventually, cell death in the manner of apoptosis and necrosis occurred as the end result of the damage to the cancer cell. These findings strongly support the idea that the death of the target cells is mediated by LAK cells.     

抗原致敏DC与CIK共培养体外抗肾癌效应的研究

目的： 探讨肾癌(RCC)抗原致敏树突状细胞(DC)与同源细胞因子诱导杀伤细胞(CIK)共培养后的DC-CIK细胞对RCC的杀伤活性。 方法： 将健康成人外周血单个核细胞（PBMC）来源的DC经RCC(786-0细胞株)抗原致敏后与同源CIK细胞共培养，实验分3组：RCC抗原致敏DC与CIK共培养组（A组），未致敏DC与CIK共培养组（B组），单纯CIK组（C组）。流式细胞仪检测DC及CIK免疫表型。MTT法检测3组效应细胞对786-0细胞杀伤活性。 结果： 效靶比 20∶1 时，A、B、C组对786-0细胞杀伤活性分别为（70.64±8.26）%、（53.40±7.33）%、（46.64±6.01）%，各组比较差异显著（P<0.05）；以前列腺癌PC3细胞作靶细胞对照，A组对786-0及PC3细胞的杀伤活性有显著差异（P<0.05）。 结论： RCC抗原致敏DC与CIK共培养后的DC-CIK细胞可明显提高CIK细胞对RCC的杀伤特异性和杀伤活性。     

淋巴瘤患者外周血TCRVα24+Vβ11+自然杀伤T细胞体外活化特性研究

目的： 研究淋巴瘤患者外周血TCRVα24+Vβ11+自然杀伤T（NKT）细胞的数量以及体外活化后的功能状态，与正常人外周血NKT细胞的数量及功能状态进行比较。方法: 制备30例淋巴瘤患者和30例年龄及性别匹配的正常对照外周血单个核细胞（PBMNCs），以流式细胞术（FACS）检测TCRVα24+Vβ11+NKT细胞数量，以α-半乳糖神经酰胺（α-Galcer）及白细胞介素-2（IL-2）从PBMNCs中扩增活化NKT细胞，采用细胞内细胞因子流式细胞术检测手段，测定NKT细胞中胞内白细胞介素-4（IL-4）、干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）阳性细胞的比例。结果: 淋巴瘤患者与正常对照PBMNCs中TCRVα24+Vβ11+NKT细胞的细胞比率分别为0.17%±0.10%、0.28%±0.18%(P<0.05)。PBMNCs培养体系中加入α-Galcer及IL-2，将NKT细胞扩增活化7d后，淋巴瘤患者与正常对照的NKT细胞的扩增倍数分别为101.37±44.61、129.66±56.31(P<0.05)。扩增活化后，淋巴瘤患者与正常对照的NKT细胞中胞内细胞因子IFN-γ阳性细胞的比例分别为41.96%±15.06%、52.48%±18.85%(P<0.05)；TNF-α阳性细胞的比例分别为46.30%±16.03%、71.37%±17.28%(P<0.05)；IL-4阳性细胞的比例分别为36.19%±11.74%、33.12%±12.95%(P>0.05)。不同病理分型及分期的淋巴瘤患者之间上述各指标无显著差异。结论: 淋巴瘤患者外周血TCRVα24+Vβ11+NKT细胞数量较正常对照明显减少，经α-Galcer扩增活化后扩增倍数较正常对照降低，分泌细胞因子IFN-γ、TNF-α的功能较正常降低，此数量及功能的降低与淋巴瘤的分型及分期无关。但其仍保持有对α-Galcer刺激后的扩增活化反应能力。     

正常人外周血TCRVα24+ NKT细胞体外活化特性观察

目的:探讨正常人外周血自然杀伤T(NKT)细胞的数量以及体外活化后表达CD69、IFN-γ和IL-4的规律并与CD3⁺ T细胞进行比较。方法:取正常成人外周血,直接三色荧光标记后溶血获取有核细胞,或以佛波醇酯(PDB)+离子霉素(Ion)刺激并培养6h,经三色荧光标记后溶血并获取有核细胞,以流式细胞术分析NKT细胞和T细胞的数量以及表达CD69和IL-4、IFN-γ的情况。结果:NKT细胞约占外周血T淋巴细胞总数的(1.34±0.42)%(x±s);PDB+Ion活化6h后NKT细胞CD69表达率为(96.71±1.33)%,明显高于对照组(11.47±2.86)%(P＜0.05);同样条件下CD3⁺T细胞CD69表达率分别为(98.60±0.47)%和(1.07±0.45)%(P＜0.05);当莫能菌素(monensin)存在时以PDB+Ion刺激6h后,IL-4阳性NKT细胞的百分比为48.62±2.44,明显高于对照组31.57±3.31(P＜0.05);IFN-γ阳性NKT细胞百分比为46.65±11.91,也高于对照组13.45±6.29(P＜0.01)。CD3⁺T细胞在刺激后表达IL-4和IFN-γ均明显升高,但IL-4表达率远远低于NKT细胞;而且对照组CD3⁺T细胞两种细胞因子表达率都明显低于NKT细胞。结论:正常成人外周血含有少量的NKT细胞,这些细胞IL-4和IFN-γ的表达率明显高于CD3⁺T细胞,是特定微环境里的重要免疫调节细胞。     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

淋巴因子激活的NK细胞杀伤肿瘤细胞的免疫电镜观察

用胶体金标记的扫描与透射免疫电镜术观察ＣＤ１６＾＋淋巴因子激活的杀伤细胞杀伤肺腺癌细胞系ＬＴＥＰａ２或人红白血病细胞Ｋ５６２的过程。发现ＣＤ１６＾＋ＬＡＫ细胞能伸出分支的指状突起较深地插入肿瘤细胞浆内，造成靶细胞表面大小深浅不等的隐窝，及陷窝内局部细胞膜损伤，在ＣＤ１６＾＋ＬＡＫ细胞这种指状突起基底部附近的浆内，有大量胞浆颗粒与囊泡聚集。靶细胞被攻击后常发生凋落型死亡。同时可见坏死型死亡。说明ＣＤ     

小鼠子宫uNK细胞的分离及其特性研究

目的：建立小鼠子宫淋巴细胞分离方法，并初步观察小鼠子宫uNK细胞的特性。方法：分别用机械法和酶消化法制备单细胞悬液分离小鼠子宫淋巴细胞，用二色或三色荧光标记技术检测子宫uNK细胞的比例及相关特征。结果：机械法分离淋巴细胞技术相对稳定、纯度较高；酶消化法未降低DX5 细胞所占的比例，但酶的消化过程容易使淋巴细胞活化水平升高，表现为活化分子CD69的表达上调。子宫中存在大量的uNK细胞(NK1．1 CD3-或DX5 CD3-细胞)，小鼠怀孕早期和中期，uNK细胞占子宫淋巴细胞的比例随着怀孕天数的增长而增长，到怀孕的第10天达到高峰(uNK细胞比例达到48.74％)，之后比例开始迅速下降。结论：成功建立了小鼠子宫uNK细胞分离方法，子宫uNK细胞作为重要的天然免疫淋巴细胞，可能参与妊娠子宫对半同种异体胚胎的保护性免疫反应。     

T淋巴细胞表达杀伤细胞抑制性受体的研究进展

Only in recent years,attentions have been drawn to the significance of expressing killer cell inhibitory receptors(KIR)in T cells.KIRs specifically bind to the corresponding region of the MHC class I molecules and transmit negative signals to prevent cytotoxity of T cells.When the ligands of KIRs are missing,the lysis of the target cells can‘t be avoided.Perhaps the existence of KIRs is the main mechanism for preventing T cells from attacking autologous tissues.The recognition mechanism of the interaction between the KIR^ donor T cells and the recipient‘s MHC class I molecule expressing tissue cells might shed light on the establishment of the immunotolerance for the prevention of allo-graft rejection and graft-versus-host disease.     

TLR-mediated stimulation of APC: Distinct cytokine responses of B cells and dendritic cells

In addition to their role in humoral immunity, B lymphocytes are important antigen-presenting cells (APC). In the same way as other APC, B cells make cytokines upon activation and have the potential to modulate T cell responses. In this study, we investigated which mouse B cell subsets are the most potent cytokine producers, and examined the role of Toll-like receptors (TLR) in the control of secretion of IL-6, IL-10, IL-12 and IFN-gamma by B cells. Production of some cytokines was restricted to particular subsets. Marginal zone and B1 cells were the predominant source of B cell IL-10 in the spleen. Conversely, follicular B cells were found to express IFN-gamma mRNA directly ex vivo. The nature of the activating stimulus dramatically influenced the cytokine made by B cells. Thus, in response to combined TLR stimulation, or via phorbol esters, IFN-gamma was secreted. IL-10 was elicited by T-dependent activation or stimulation through TLR2, 4 or 9. This pattern of cytokine expression contrasts with that elicited from dendritic cells. QRT-PCR array data indicate that this may be due to differential expression of TLR signalling molecules, effectors and adaptors. Our data highlight the potentially unique nature of immune modulation when B cells act as APC.     

干式融化箱与水浴锅复苏细胞因子诱导的杀伤细胞的比较

目的对比干式融化箱与水浴锅复苏冻存细胞因子诱导的杀伤细胞（CIK细胞）的效果。方法提取10份健康志愿者外周血单个核细胞分别诱导培养CIK细胞并冻存于一80℃冰箱。1个月后从每份CIK细胞中取出相同的两个冻存管,分别采用干式融化箱和水浴锅进行复苏。记录复苏时间,检测复苏后细胞存活率及对K562细胞的杀伤作用。结果干式融化箱较水浴锅复苏时间较长[（4．33±0．19）min、（2．47±0．30）rain],差异有统计学意义（P〈0．05）。但两种方法复苏的CIK细胞存活率[（89．55＋5．36）％、（89．86±4．63）％]和对K562细胞的杀伤活性[（44．76±9．53）％、（46．97±10．17）％]差异无统计学意义（P〉0．05）。结论干式融化箱可以代替水浴锅进行冻存CIK细胞复苏。     

细胞因子诱导杀伤细胞分泌因子影响人肝癌干细胞的凋亡

BACKGROUND: Immunotherapy with autologous immune cells has been developed as a major adjuvant therapy for malignant tumors, but its mechanism of action has not been elucidated. OBJECTIVE: To investigate the relationship between cytokine-induced killer cell secretion and apoptosis in human liver cancer stem cells. METHODS: Human liver cancer stem cells, HepG2 cells, were isolated and enriched using serum-free suspension method. The peripheral blood mononuclear cells from patients with liver cancer were induced by γ-interferon, CD3 monoclonal antibody and recombinant human interleukin-2 to form killer cells. Passage 1 liver cancer stem cells were divided into control group (culture alone) and experimental group (co-culture of cytokines-induced killer cells and human liver cancer stem cells). At 48 hours after culture, apoptosis in human liver cancer stem cells was detected using flow cytometry, and expression of caspase-3 mRNA and protein was detected using RT-PCR and western blot, respectively. RESULTS AND CONCLUSION: The apoptotic rate in the control group was significantly lower than that in the experimental group (P < 0.05). The expressions of caspase-3 at mRNA and protein levels were both higher in the experimental group than the control group (P < 0.05). Experimental findings show that cytokines-induced killer cells can significantly promote apoptosis in human liver cancer stem cells, and up-regulate the caspase-3 mRNA and protein expressions dramatically.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程