自体细胞因子诱导杀伤细胞治疗HBV感染肝硬变患者的临床疗效分析

目的观察自体细胞因子诱导杀伤细胞(CIK细胞)治疗HBV DNA阳性肝硬化患者的近期疗效。方法33例HBV DNA阳性肝硬化患者给予CIK细胞治疗,在体外培养前后以及回输体内后检测CD3 、CD3 CD4 、CD3 CD8 、CD3 CD56 、CD25 细胞以及mDC和pDC。比较治疗前后病毒学指标及肝脏功能的变化。结果培养结束以及回输体内后,CD3 细胞、CD3 CD8 细胞、CD3 CD56 细胞较培养前显著升高,mDC和pDC在回输后也明显增高。12例患者HBV DNA阴转,4例患者拷贝数下降大于2个log。在14例HBeAg阳性患者中有10例阴转,2例出现HBeAb转换。肝脏功能较治疗前有所好转。所有患者均能耐受治疗。结论CIK细胞可明显提高免疫效应细胞数量,具有一定的抗病毒效应作用,毒副作用低。     

-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。     

脐血来源的树突状细胞增强CIK细胞的杀伤作用

为确认体外诱导出的成熟脐血来源树突状细胞 (CBDC )可以引发强大抗肿瘤免疫反应 ,CBDC培养 12d后用电镜分析形态 ,用流式细胞仪检测其表型。在不同培养时间 ,用3 H TdR掺入法检测CBDC和细胞因子诱导杀伤细胞 (CIK )的扩增功能 ;用MTT法检测被CBDC激活的CIK抗肿瘤活性。结果表明 ,体外诱导出形态典型的DC ,高表达主要组织相容性抗原I、II类分子、CD5 4 ,也表达CD80、CD83、CD1a,不表达CD6 8。体外培养 12d成熟的CBDC ,能使CIK以最高的比率扩增 ,2种细胞最佳混合比为 1∶10时被激活的CIK杀伤BEL 74 0 2肝癌细胞的功能最强 ,最佳效靶比为 80∶1     

慢性粒细胞白血病患者DC对细胞因子诱导的杀伤细胞细胞毒作用的影响

目的 观察慢性粒细胞白血病（CGL）患者树突状细胞（DC）对自身细胞因子诱导的杀伤细胞（CIK）细胞毒的影响。方法 从CGL患者和正常人末梢血中分离单个核细胞（PBMC），用细胞因子（rhGM-CSF,rhIL-4和rHTNF-α)联合定向诱导培养DC。用CGL细胞抗原致敏DC，再将致敏的DC与CIK共同培养后，检测CIK对不同靶细胞的杀伤作用。结果 CGL患者和正常人的PBMC经细胞因子联合诱导培养后，DC占20．78％－56．0％。CGL患者的CIK和经CGL细胞抗原致敏的DC作用后的CIK，对自身白血病细胞的杀伤活性分别为56．0％和83．4％；正常人对照组则分别为30％和62％。经CGL患者CGL细胞抗原致敏的DC作用后的CIK, K562和SGC－7901的杀伤活性较未致敏的CIK活性低，而正常人对照组则无此现象。结论 从CGL患者的PBMC中能定向诱导扩增出DC。经CGL细胞抗原致敏的DC，能明显增强CIK对自身CGL细胞的杀伤活性。     

树突状细胞对脐血来源细胞因子诱导杀伤、自然杀伤细胞杀伤活性及CD45RO表达的影响

目的:探索同一来源脐血树突状细胞(DCs)对细胞因子诱导杀伤(CIK)、自然杀伤(NK)细胞杀伤活性和CIK细胞上CD45RO表达的影响。方法:通过细胞因子组合诱导、扩增脐血来源的DCs、CIK和NK细胞,杀伤效应细胞分为两组:A组为接受DCs共孵育6d刺激的CIK、NK细胞,B组为未接受任何刺激的CIK、NK细胞,了解DCs对相应效靶比下CIK、NK细胞杀伤K562、HL-60细胞株细胞毒活性的影响;通过流式细胞术检测两组CIK细胞上CD45RO、CD45RA的表达率。结果:随着效靶比的升高,脐血CIK、NK细胞对肿瘤细胞株的杀伤力增加。在20∶1、10∶1效靶比下,A组CIK、NK细胞对HL-60的杀伤率分别为76.77％±5.76％、55.87％±2.09％,B组为61.14％±3.72％、49.96％±1.51％,A组对HL-60的杀伤明显高于B组;在20∶1效靶比下,A组对K562的杀伤明显高于B组;而在10∶1效靶比下两组之间的杀伤活性未见显著差异;A组CIK细胞上CD45RO表达率显著高于B组。结论:DCs与CIK、NK细胞共孵育可提高CIK、NK细胞的细胞毒活性及增加CIK细胞上CD45RO的表达。     

CIK在无血清培养体系中的增殖、表型变化和抗肿瘤活性

目的:探索细胞因子诱导的杀伤细胞(CIK)在无血清体系中的增殖规律、表型变化及其抗瘤活性。方法:不同培养基培养CIK细胞,采用活细胞计数法观察CIK细胞的增殖,流式细胞仪检测CIK细胞的表型,CytoTox96非放射性细胞毒试剂盒检测CIK细胞的细胞毒活性。结果:经过细胞因子和抗体刺激后,CIK细胞能明显增殖,无血清培养基加自体血浆组最高可扩增473.28±27.53倍,无血清培养基组可扩增218.24±16.86倍,而RPMI1640加FCS只扩增11.52±1.04倍。CD3 CD8 、CD3 CD56 、CD226 CD11a 和CD305 CD11a 细胞随着培养时间的延长而增加,而CD3 CD4 细胞则明显减少。CIK细胞对肿瘤细胞的细胞毒作用明显高于LAK细胞(P<0.01),且其细胞毒活性随着培养时间的延长而增高。结论:CIK细胞在体外扩增能力强,对肿瘤细胞的杀伤活性高,有望成为新一代抗肿瘤过继免疫细胞制剂而应用于临床。     

不同细胞因子诱导脐血来源的CIK、NK细胞对K562细胞杀伤活性的研究

目的：了解不同细胞因子组合扩增的脐血CIK、NK细胞对人K562细胞株的杀伤活性的差异性。方法：通过SCF、FLT3L、IL-2、IL-7及IL-15等细胞因子的不同组合扩增的脐血CIK、NK细胞，分为3组，A组（SCF＋IL-2＋IL-7＋IL-15）、B组（SCF＋FLT3L＋IL-2＋IL-7＋IL-15）和C组（IL-2＋IL-7＋IL-15，对照组）；通过MTT法检测各组CIK、NK细胞在不同效靶比下对K562的杀伤率，计算各组的总杀伤单位。结果：经过21天培养A组体系中CIK＋NK细胞的比例平均超过60％，B组CIK＋NK细胞的比例平均超过70％，而C组约为50％；各组扩增的CB-CIK／NK细胞同组间在效靶比20：1时对K562的杀伤率均显著高于10：1（P〈0．01）。在不同效靶比下A组CIK／NK细胞对K562的杀伤率显著高于B和C组（P〈0．01）；C组CIK／NK细胞对K562的杀伤率稍高于B组，但两组间对K562的杀伤率无显著性差异。A组总杀伤单位显著高于C组，与B组无显著差异。结论：不同细胞因子组合扩增的脐血CIK、NK细胞对人K562细胞株的杀伤活性有差异，考虑到对效应细胞的扩增效果，B组为具有最佳杀伤活性的脐血CIK、NK细胞培养体系，其中的SCF、FLT3L对CIK／NK细胞诱导有协同效果。     

mdr1基因转染的CIK细胞耐药性及杀伤活性的研究

目的:将多药耐药基因(mdr1)转入CIK细胞,观察转染前后其对紫杉醇的耐药性及对Lewis肺癌细胞杀伤活性的影响。方法:常规方法:培养CIK细胞,在细胞对数生长期,将mdr1的重组质粒转染CIK,通过RT-PCR鉴定耐药基因表达;MTT法检测CIK细胞对紫杉醇敏感性的变化,同时检测转染前后CIK细胞对Lewis肺癌细胞的杀伤活性变化;Western blot检测细胞中mdr1编码的P-gp蛋白的表达的变化;通过计算瘤重抑制率(TWI)检测转染前后的CIK细胞对Lewis肺癌移植瘤的抑制作用。结果:转染mdr1后的CIK细胞mdr1 mRNA阳性,同时P-gp的表达较转染前及转染空质粒的CIK细胞均有显著增高(P<0.05),转染后的CIK细胞对紫杉醇的耐药性有显著提高,但对Lewis肺癌细胞的杀伤活性无明显变化(P>0.05)。结论:将mdr1基因转入CIK细胞后,细胞获得了多药耐药性,同时保持了原有的对肿瘤细胞的杀伤活性。     

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.     

慢性乙型肝炎患者外周血细胞因子诱导的杀伤细胞的动态观察

目的:动态观察慢性乙型肝炎患者细胞因子诱导的杀伤(CIK)细胞的增殖及杀伤活性。方法:抽取10例健康人及20例慢性乙型肝炎患者的外周血,常规分离单个核细胞(PB-MC),加IFN-γ、IL-2及抗人CD3单克隆抗体(mAb)后培养,诱导CIK细胞形成。于培养后3、6、12、24及30 d,取培养的 细胞,用流式细胞仪检测CIK细胞表面CD3、CD4和CD8以及CD4、CD25、CD3、CD56和CD95、CD28分子的表达水平、增殖及杀伤活性。结果:慢性乙型肝炎患者CIK细胞的增殖及杀伤活性均低于正常对照组。培养不同时间乙肝患者CIK细胞的增殖倍数、杀伤活性和上述表面标志的表达水平,均较培养前明显增高,于培养后12 d达高峰。结论:慢性乙型肝炎患者CIK细胞的增殖倍数、杀伤活性均低于正常人,但明显高于培养前;这可能是导致HBV感染持续发展的原因之一。     

A phase I trial of autologous cytokine-induced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma.

We have previously reported on the ex vivo generation of cytotoxic effector cells, termed cytokine-induced killer (CIK) cells, that have both in vitro and in vivo antitumor activity in murine models. We now report on our efforts for the large-scale expansion of CIK cells and also present preliminary results from a phase I clinical trial. Nine patients with advanced Hodgkin disease (n = 7) and non-Hodgkin lymphoma (n = 2), all of whom had relapsed after an autologous transplantation, were treated with escalating doses of CIK cells (3 patients at each dose level of 1 x 10(9) , 5 x 10(9) , or 1 x 10(10) cells). The CIK cells were produced by culturing unselected cells from steady-state apheresis products with interferon gamma, OKT3, and interleukin 2. After 21 days in culture, with the addition of fresh media and interleukin 2 every 3 to 4 days, the median culture was 97% viable (range, 61%-100%), 98% CD3 + (range, 66%-99%), 76% CD8 + (range, 27%-96%), 23% CD4 + (range, 6%-78%), 20% CD3 + CD56 + (range, 8%-58%), and <1% CD16 + 56 + (range, 0.2%-7.7%). The CD3 + CD56 + cells have previously been shown to exhibit the most cytotoxic activity. The absolute number of CD3 + CD56 + cells typically expanded 290-fold (range, 3- to 4000-fold) under these culture conditions. In vitro cytotoxic activity was measured against a human B-cell tumor line (OCI-Ly8). At a 40:1 effector-target cell ratio, CIK cells killed 32% (range, 2%-69%) of the target cells. A total of 21 infusions were administered to 9 patients. The number of CIK cells infused ranged from 1.0 x 10(9) to 1.0 x 10(10) per treatment. Toxicity was minimal, and there were no immediate adverse reactions to the infusions. Two patients had partial responses, and 2 patients had stabilization of disease: 1 for more than 18 months. Considering that these were heavily pretreated patients with advanced hematologic malignancies, we believe that CIK cells expanded in this fashion may have utility for the treatment of high-risk patients with evidence of minimal residual disease after autologous transplantation.     

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。     

Clinical expansion of cytokine induced killer (CIK) cells

Cytokine induced killer (CIK) cells are polyclonal T cells that can be expanded from marrow or peripheral blood lymphocytes with potent non-MHC-restricted cytotoxicity against a variety of tumor target cells. Earlier work had established the superiority of CIK cells over LAK cells in terms expansion and cytotoxicity. It has been studied extensively both in vitro and in murine experiments with promising killing activity against a wide range of haematological malignancies. Early clinical trials of CIK cells have reported safety and feasibility data as well as possible efficacy and have provided the framework for further clinical studies. As a robust and easily expandable cell population, its role in clinical adoptive immunotherapy should be investigated for translation into a good manufacturing practice (GMP) manufacturing process. Large scale culture to generate adequate number of CIK cells for clinical use involves up-scaling from flasks to culture bags carried out in a GMP compliant cell therapy facility, use of GMP compliant materials and reagents, efficient harvesting procedures to remove foreign elements and strict release criteria with respect to viability, sterility and potency of the product. All the steps should preferably to be done in a closed system, in order to minimize the chance of contamination. A phase I/II clinical study on the use of allogeneic CIK cells has been conducted to define the role of CIK cells in the management of relapse post stem cell transplant with the aim of looking at feasibility of GMP expansion, toxicity and efficacy. A separate trial looking at autologous CIK cells was also conducted. Allogeneic CIK cells were successfully generated for a total of 24 patients including from patients’ own leukepheresed cells in 5 patients who have no access to further donor cells. The median CD3 + T cell expansion was 9·33 (1·3–38·97) fold and CD3 + CD56 + NK-like T cells expansion was 27·77 (2·59–438·93) fold. CIK cells were infused into 16 patients who have either failed or progressed after initial response to various individualized chemotherapy regimen and donor lymphocyte infusion (DLI), for a total of 55 infusions at doses ranging from 10 to 200 million CD3/kg. Evidence of efficacy as defined by a demonstrable response attributable to CIK cell infusion was observed in five patients with minimal toxicity. Other published examples to augment CIK efficacy include co-culture of CIK with myeloma idiotype or CA19-9 peptide-pulsed autologous dendritic cells. Redirecting CIK cells by bispecific antibodies to target has been shown for ovarian carcinoma and B cell lymphoma, as well as B cell ALL by engineering CIK cells to express anti-CD19 receptor. Exploiting the heterogeneous subsets within the bulk CIK cell culture is another avenue for enhancing its potency.     

Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway

CIK cells are a subset of effector lymphocytes endowed with a non-MHC restricted anti-tumor activity making them an appealing and promising cell population for adoptive immunotherapy. CIK are usually generated ex-vivo by initial priming with Interferon-γ (IFN-γ) and monoclonal antibody against CD3 (anti-CD3), followed by culture in medium containing Interleukin-2 (IL-2). Interleukin-15 (IL-15) shares with IL-2 similar biological functions and recently it has been reported to induce CIK with increased anti-leukemic potential.The aim of the study was to compare the killing efficacy of CIK generated by IL-2 alone or IL-2 and IL-15 toward tumor targets of different origins, leukemic cells and malignant cells from epithelial solid tumors. CIK bulk cultures were examined for cell proliferation, surface phenotype and cytotoxic potential against tumor cell lines K562, HL60, HeLa and MCF-7. The results showed that IL-15 is able to induce a selective expansion of CIK cells, but it is less effective in sustaining CIK cell proliferation compared to IL-2. Conversely, our data confirm and reinforce the feature of IL-15 to induce CIK cells with a potent cytotoxic activity mostly against tumor cells from epithelial solid malignancies via NKG2D-mediated mechanism.     

γ氨基丁酸对人IL-2和IFN-γ诱导的杀伤细胞的作用研究

为研究γ氨基丁酸(GABA)及A受体激动剂(THIP)对人细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)的增殖、T细胞亚群和杀瘤活性的影响和作用机制;在体外用不同浓度的GABA和THIP与人CIK细胞作用后,用MTT比色法和流式细胞仪(FCM)以及乳酸脱氢酶(LDH)法分别检测人CIK细胞的增殖能力、T细胞亚群和杀伤活性的变化。结果显示,GABA能显著的抑制CIK细胞的生长(P<0.01),并具有浓度依赖性,THIP对GABA的抑制细胞增殖有明显促进作用;GABA显著降低CIK细胞表面分子CD28分子的表达,降低CD8+T细胞的百分比,抑制CIK细胞对人结肠癌细胞株SW480的杀伤活性,THIP能够增强GABA的作用。上述结果提示,GABA能显著抑制CIK细胞的增殖,降低其细胞表面CD28分子的表达,降低CD8+T细胞的百分比,抑制CIK细胞的杀伤活性,这些作用主要是通过T淋巴细胞上的GABAA受体介导。     

CIK细胞对胃癌OCUM-2MD3细胞体内外杀瘤活性的实验研究

目的：研究正常人细胞因子激活的杀伤细胞（Cytokine-induced killer,CIK)对人胃癌细胞株OCUM－2MD3的体外细胞毒活性以及对胃癌腹膜移植模型的体内抗肿瘤作用。方法：取正常人外周血单个核细胞（Peripheral blood mononuclear cell,PBMC)，加入γ－IFN、IL－2、抗－CD3单抗和IL－1，体外诱导成CIK细胞，用流式细胞仪对细胞作动态表型分析，用MTT法测定CIK细胞体外对OCUM－2MD3的细胞毒活性，并与CD3AK作对比。在Balb/c裸小鼠腹腔内接种OCUM－2MD3细胞，观察CIK细胞对荷瘤鼠的抑瘤作用。结果：CIK细胞在培养2周左右获得大量增殖，CD3^ CD56^ 双阳性细胞大量增殖达1000倍以上；体外实验证明，CIK细胞对OCUM－2MD3有明显的细胞毒活性，对比CD3AK其抑瘤率与85％与62．8％（P＜0．05）。体内实验表明，CIK细胞能够显著抑制癌性腹水的生长，其抑瘤率可达100％；肿瘤标志物CEA含量明显降低。结论：CIK细胞是一种新型、高效的免疫活性细胞，具有较强的体内外抗胃癌细胞活性，具有明显的抑制癌性腹水的生长的作用，有可能用于临床上晚期胃癌的过继性免疫治疗。     

Expansion of cytotoxic CD3+ CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation.

Immunotherapy may potentially improve the outcome of autologous hematopoietic cell transplantation (HCT). Poor effector cell proliferation and marginal antitumor activity limit attempts to use immunotherapy. We have characterized the ex vivo expansion, up to 1000-fold, of CD3+ CD56+ lymphocytes from the peripheral blood lymphocytes (PBL) of healthy donors. Expanded cells termed cytokine-induced killer (CIK) cells induce non-major histocompatibility complex-restricted lysis of tumor cells and demonstrate cytolytic activity superior to lymphokine-activated killer cells without the requirement of interleukin (IL)-2 treatment in vivo. To determine whether cytolytic cells could be expanded from patient material, we evaluated samples of peripheral blood progenitor cells (PBPCs) from 25 patients undergoing autologous HCT. The PBPCs were expanded by priming with interferon-gamma followed by anti-CD3 monoclonal antibody and IL-2 the next day. Fluorescence-activated cell sorting analysis was performed on days 0, 15, 21, and 28 of cell culture. The median T-cell content rose from 15.3% (range, 1.1% to 89.7%) on day 0 to 97.2% (range, 83.6% to 99.5%) by day 15. By day 21, T cells expanded 21.8-fold (range, 1.7- to 420.0-fold) and CD3+ CD56+ cells expanded 44.8-fold (range, 5.1- to 747.0-fold). CIK cells were used as effector cells against B-cell lymphoma targets (OCI-Ly8) with a median of 24% (range, 3% to 67%) and 42% (range, 6% to 96%) specific lysis of target cells on days 21 and 28, respectively. CIK cells derived from PBL of 2 additional patients with acute myelogenous leukemia demonstrated 39% and 78% specific lysis of OCI-Ly8 and 26% and 58% specific lysis of autologous leukemic blasts at an effector:target ratio of 40:1. CIK cells may be expanded from granulocyte colony-stimulating factor-mobilized PBPCs of patients undergoing autologous HCT. CIK cells may provide a potent tool for use in posttransplantation adoptive immunotherapy.     

紫杉醇顺铂方案联合自体CIK细胞输注治疗晚期食道癌的临床分析

目的:晚期食道癌的的治疗以化疗为主,紫杉醇顺铂方案治疗晚期食道癌因疗效确定,在临床应用日益推广。自体CIK细胞治疗晚期恶性肿瘤能起到延缓或阻止肿瘤的转移与复发,并可提高晚期食道癌患者的细胞免疫功能及改善生活质量等综合作用。本组应用紫杉醇顺铂方案联合自体CIK细胞输注治疗晚期食道癌患者,旨在探讨采用自体CIK细胞输注同步化疗的综合治疗方法,是否能提高晚期食道癌的临床疗效。方法:本科自2008年1月至2010年1月共入选59例患者。均为Ⅳ期食道癌病人。化疗方案剂量及方法设定:自体CIK治疗操作流程:采血前一天需对患者进行血常规检测,对于白细胞数低于8×109L-1的患者应于采血前24小时注射1～2支集落刺激因子。采血前急查血常规,确认患者白细胞数达到或高于8×109L-1时方可采血,采集患者静脉血50 ml。培养至14天左右细胞成熟,经检验科和实验室测量CIK细胞数量达1×109～11L-1时,对细胞培养物进行无菌检测,当检验科和实验室自身均检测无菌后,安排进行回输。自体CIK细胞输注+紫杉醇顺铂方案治疗组:Cik采血,d1,紫杉醇PTX 175 mg/m2,d2,顺铂DDP 20 mg/m2,静滴d3-7,紫杉醇使用前严格按照说明书进行预处理。CIK回输:d14。21天为1个周期,2个周期后评价疗效。结果:全组59例患者总有效率为32.2%(19/59),其中完全缓解率为1.7%(1/59),部分缓解率为30.5%(18/59),稳定52.5%(31/59),进展15.2%(9/59)。中位进展时间7.6个月,中位生存时间11.7个月。主要毒性反应为外周血细胞下降、脱发及外周神经毒性。结论:应用紫杉醇顺铂方案联合自体CIK细胞输注治疗Ⅳ期食道癌,疗效突出,毒性反应轻微,治疗顺应性好,提高了患者的生存质量,值得进一步推广。     

抗CD28单抗和IL-15对CIK增殖和杀肿瘤作用的研究

CIK是肿瘤过继性细胞免疫治疗中的免疫效应细胞。为使CIK在实验室里能被更有效地诱导增殖并赋予其更强的杀肿瘤效应,我们在CIK常规培养环境中加入抗CD28单抗和IL-15,探讨抗CD28单抗和IL-15对CIK增殖和杀肿瘤效应。取人外周血单个核细胞(PBMC),预先以常规方法诱导CIK,然后加入抗CD28单抗和IL-15与CIK共培养。用全自动五分类血液分析仪计数CIK增殖率;用流式细胞术测定CIK中粒酶B、穿孔素和CD107a等分子的表达率;用ELISA方法检测CIK分泌IL-10、IL-12、INF-γ和TNF-α水平;用乳酸脱氢酶释放法测定CIK对人肺癌细胞株(A549)、乳腺腺癌细胞株(MFC-7)和人黑素瘤细胞株(HME1)的杀伤活性。PBMC经常规CIK诱导培养以后再加入抗CD28单抗和IL-15与对照组比较,前者细胞增殖率明显增强(P<0.05);在CIK培养体系中加入抗CD28单抗和IL-15可促进颗粒酶B、穿孔素和CD107a等分子的表达率进一步增强(P<0.05);加入抗CD28单抗和IL-15,培养8d后CIK对A549、MFC-7和HME1细胞杀伤活性分别为82.2%、59.3%和70.6%,与对照组(分别为60.9%、49.6%和48.4%)相比差异有统计学意义(P<0.05);在培养体系中加入抗CD28单抗和IL-15,培养8d后其细胞因子IFN-γ、TNF-α分泌水平显著高于对照组(P<0.05),组间IL-10和IL-12的分泌量未见显著差异(P>0.05)。实验说明在CIK培养体系中加入抗CD28单抗和IL-15可增加CIK增殖率并提高其抗肿瘤效应。