癌性胸水LAK细胞的特性和抗肿瘤作用的观察

１７例癌性胸水经不连续密度梯度离心分离淋巴细胞，在ｒＩＬ－２诱导下体外培养，观察不同时期的ＬＡＫ细胞增殖速度、细胞表型和细胞毒活性，结果显示，胸水ＬＡＫ细胞在培养第１４天细胞扩增至原代的２０倍～８０倍，２１天后生长速度逐渐减慢。培养７天时对Ｋ５６２细胞和Ｒａｊｉ细胞的杀伤活性分别为７２％和６５％，以后随培养天数增加均呈逐渐下降趋势，但均高于同期培养的外周血ＬＡＫ细胞杀伤活性。培养７天～２１天间的细胞表型分析提示，ＣＤ＋３细胞百分率无明显改变，ＣＤ＋４细胞略增加，ＣＤ＋８细胞略有下降。ｒＩＬ－２／ＬＡＫ联合治疗肺癌胸水和肾癌胸水总有效率分别为５０％和６７％。     

石斛多糖增强脐带血和肿瘤病人外周血LAK细胞体外杀伤作用的研究

目的：探讨石斛多糖（ｄｅｎｄｒｏｂｉｕｍ ｃａｎｄｉｄｕｍ ｐｏｌｙｓａｃｃｈａｒｉｄｅ，ＤＣＰ）与ｒＩＬ－２联合应用对脐带血ＬＡＫ细胞（ＣＢ－ＬＡＫ）和肿瘤病人外周血ＬＡＫ细胞（ＰＢ－ＬＡＫ）体外杀伤肿瘤细胞作用的影响。方法：应用＾３Ｈ－ＴｄＲ释放法测定ＣＢ－ＬＡＫ和ＰＢ－ＬＡＫ细胞的杀伤活性。结果：①ｒＩＬ－２（５００ｕ／ｍｌ）能明显提高ＣＢ－ＬＡＫ对Ｒａｊｉ细胞的杀伤活性，０ｈ的ＣＢ－ＬＡＫ活性为１３．６３％，ｒＩＬ－２（５００ｕ／ｍｌ）诱导４８、７２、９６、１２０ｈ后，ＣＢ－ＬＡＫ活性分别提高至２２．２８％、２６．２３％、２８．５５％和２５．７６％（Ｐ〈０．０１）；②ＤＣＰ（１００ｍｇ／Ｌ或４００ｍｇ／Ｌ）与ｒＩＬ－２（５００ｕ／ｍｌ）联合诱导ＣＢ－ＬＡＫ的活性，比单纯ｒＩＬ－２（５００ｕ／ｍｌ）的作用     

CD3AK细胞和LAK细胞治疗晚期恶性肿瘤的临床和实验研究

将５１例晚期恶性肿瘤患者（男性２３例，女性２８例）分成两组，其中一组（３１例）以ＣＤ３ＭｃＡｂ（ＣＤ３单克隆抗体）和小剂量ＩＬ－２（５００ｕ／ｍｌ）共同诱导的ＣＤ３ＡＫ细胞治疗，另一组（２０例）输注大剂量ＩＬ－２（１０００ｕ／ｍｌ）诱导的常规ＬＡＫ细胞治疗，以探讨降低ＩＬ－２用量、提高杀伤细胞细胞毒活性的可能性。结果显示ＣＤ３ＡＫ组患者生活质量改善、症状缓解均优于ＬＡＫ组。ＣＤ３ＡＫ组ＰＲ＋ＭＲ率较ＬＡＫ组高２９．０％，Ｓ＋Ｐ率和死亡率分别较ＬＡＫ组低１２．４％和９．６％。同时比较了ＣＤ３ＡＫ细胞与ＬＡＫ细胞的体外增殖和细胞毒活性，结果表明ＣＤ３ＡＫ细胞增殖率高于ＬＡＫ细胞（Ｐ＜０．０１），靶细胞抑制率二者在０．０５水平无显著差异。提示ＣＤ３ＭｃＡｂ在刺激杀伤细胞活性，尤其在提高其增殖能力方面，具有显著的作用，ＣＤ３ＡＫ／ＩＬ－２能更有效地治疗晚期恶性肿瘤。     

CD3AK细胞的体外诱导及免疫生物学特征的实验研究

目的：研究ＣＤ３ＡＫ细胞的体外诱导方法及其免疫生物学特征。方法：采用抗单克隆抗体（ａｎｔｉ－ＣＤ３ＭｃＡｂ）和基因重组人白细胞介素２（ｒＩＬ－２）、植物血凝素（ＰＨＡ０共同诱导人外周血单核细胞（ＰＢＭＣｓ）制备ＣＤ３ＡＫ细胞,应用ＡＰＡＡＰ法、吉姆萨染色法分析ＣＤ３ＡＫ细胞的表型、核型等免疫生物学特征。结果：微量的ａｎ－ｔｉ－ＣＤ３ＭｃＡｂ（终浓度３０～５０００ｎｇ／ｍｌ）辅以少量的ｒＩＬ－２（１     

CD_3单克隆抗体活化的杀伤细胞对人胃癌细胞株MNK_（45）体外杀伤及裸鼠体

用人的外周血单个核细胞（ＰＢＭＣ）与抗ＣＤ３单克隆抗体和基因重组的人ＩＬ－２，共同培养制备ＣＤ３ＡＫ细胞（ＣＤ３ＭｃＡｂＡｃｔｉｖａｔｅｄＫｉｌｌｅｒＣｅｌｌｓ），ＰＢＭＣ与ｒＩＬ－２共同培养制备ＬＡＫ细胞，用单纯ＰＢＭＣ作为对照细胞。对这三种细胞的增殖及在体外和裸鼠体内的抗人胃癌细胞株ＭＮＫ４５作用进行了实验研究。体外试验结果表明，ＣＤ３ＡＫ对ＭＮＫ４５有明显杀伤作用，而ＬＡＫ细胞杀伤率较低，二者差异有非常显著性意义（Ｐ＜０．０１）；用的ＭＴＴ法和台盼蓝活细胞计数法测定这三种细胞的增殖能力和细胞数量的增加，表明ＣＤ３ＡＫ的增殖能力和扩增数量均明显高于ＬＡＫ细胞（Ｐ＜０．０１）。裸鼠体内试验结果显示，对照组及ＬＡＫ组全部长出瘤结节，而ＣＤ３ＡＫ组无一例长出皮下结节，且生存期明显长于前者。     

CD3单克隆抗体活化的杀伤细胞对人胃癌细胞株MNK45体外杀伤及…

用人的外周血单个核细胞（ＰＢＭＣ）与抗ＣＤ３单克隆抗体和基因重组的人ＩＬ－２，共同培养制备ＣＤ３ＡＫ细胞，ＰＢＭＣ与ｒＩＬ－２共同培养制备ＬＡＫ细胞，用单纯ＰＢＭＣ作为对照细胞。对这三种细胞的增殖及在体外和裸鼠体内的抗人胃癌细胞株ＭＮＫ４５作用进行了实验研究。体外试验结果表明，ＣＤ３ＡＫ对ＭＮＫ４５有明显杀伤作用，而ＬＡＫ细胞杀伤率较低，二者差异有非常显著性意义（Ｐ＜０．０１）；用ＭＴＴ法和台盼蓝     

CD＿3AK与LAK细胞生物活性的比较

采用正常人外周血单个核细胞,经抗ＣＤ３单克隆抗体预刺激后,加入白细胞介素２继续激活的ＣＤ３ＡＫ细胞,与常规ＬＡＫ细胞的生物活性进行了比较。结果表明,ＣＤ３ＡＫ细胞的体外增殖能力,存活时间及细胞活性明显高于ＬＡＫ细胞细胞表型的检测发现ＣＤ３ＡＫ细胞的ＣＤ３＋、ＣＤ８＋表型高于ＬＡＫ细胞,而ＣＤ１６＋表型低于ＬＡＫ细胞（ｐ＜０．０ｌ）。认为ＣＤ３ＡＫ细胞是一种非ＮＫ型ＹＭ细胞毒活性的肿瘤杀伤细胞,为应用ＣＤ３ＡＫ细胞过继免疫疗法提供了有价值的参考依据。     

脐血T淋巴细胞亚群、LAK细胞表型和杀伤活性研究

应用单克隆抗体技术活细胞间接免疫荧光法及乳酸脱氢酶释放法分别检测了３０例脐血和３０例成人外周血Ｔ淋巴细胞亚群、ＬＡＫ细胞表型及杀伤活性，结果表明：脐血ＣＤ３、ＣＤ２及ＨＬＡ－ＤＲ的表达均明显低于成人外周血，而ＣＤ４、ＣＤ８、ＣＤ４／ＣＤ８、ＣＤ１６、ＣＤ２５及ＣＤ５６的表达与成人外周血相似。经ＩＬ－２诱导后，脐血ＬＡＫ细胞表型较诱导前ＣＤ８、ＣＤ１６、ＣＤ２５及ＨＬＡ－ＤＲ表达均明显增加，脐血ＬＡＫ细胞杀伤活性较成人外周血ＬＡＫ细胞低。     

骨肉瘤病人外周血淋巴细胞体外相关瘤细胞致敏和rIL-2诱导的杀伤细胞

对骨肉瘤病人外周血淋巴细胞体外相关瘤细胞致敏和ｒＩＬ┐２诱导的杀伤细胞（ｒＩＬ┐２┐ＣＬ）进行探讨。方法采用２种培养方法：１．淋巴细胞／肿瘤细胞混合培养；２．单独ｒＩＬ┐２激活ＬＡＫ细胞，借助５１Ｃｒ释放实验对２种杀伤细胞的抗瘤效力作比较性分析。结果ｒＩＬ┐２活性高峰在ｒＩＬ┐２诱导第１２天，２周后仍可维持相当强杀伤水平，而ＬＡＫ细胞高峰出现于３～５天，１２天后消失。前者比后者对病人肿瘤组织学相关骨肉瘤细胞系具有更强烈特异杀伤效力，且对自体淋巴母细胞无毒副反应。结论ｒＩＬ┐２┐ＣＬ主要是一种特异性较高，杀伤活性强烈ＣＴＬ细胞群，对抗ＬＡＫ细胞骨肉瘤细胞具有明显杀伤效应     

粘附性淋巴因子活化的杀伤细胞抗自体白血病细胞作用的研究

建立了一套培养和处理粘附ＬＡＫ细胞（Ａ－ＬＡＫ）的系统，将１２例缓解期急性髓系白血病（ＡＭＬ）患者（ＲＰＳ）的Ａ－ＬＡＫ细胞与常规制备ＬＡＫ细胞（ＲＴ－ＬＡＫ）进行了对照研究，结果显示ＲＰＳ－Ａ－ＬＡＫ细胞于培养第１０天时其扩增指数（２０．１±１３．９）较ＲＴ－ＬＡＫ细胞的扩增指数（７．５±２．１）明显提高（Ｐ＜０．０５）。形态学研究显示Ａ－ＬＡＫ细胞主要由大颗粒淋巴细胞组成，免疫表型分析显示其主要由ＣＤ１６＋的ＮＫ细胞组成，ＲＴ－ＬＡＫ细胞主要由ＣＤ３＋的Ｔ细胞组成。其杀伤活性与ＣＤ１６＋ＮＫ细胞之间有很好的相关性（ｒ＝０．８２，Ｐ＜０．０５）。这提示ＣＤ１６＋ＮＫ细胞是Ａ－ＬＡＫ细胞的主要组成细胞，代表了杀伤功能最强的亚群。     

干细胞、癌症与癌症干细胞

干细胞被认为是一种未分化的细胞,具有永远增殖和自我更新能力.大量研究证明,癌症中存在对化疗更具抵抗性的癌症干细胞.识别癌症干细胞和普通癌症细胞之间的差异,可以发展更有效的癌症分类、诊断和治疗方法.     

侧群细胞与肺癌肿瘤干细胞

侧群(side population,SP)细胞是一小群能够将染料Hoechst 33342排出从而在流式细胞图上呈现Hoechst 33342低染的细胞,研究者已在多种肿瘤组织和细胞系中检测出了SP细胞并验证这些SP细胞具有强干细胞活性。现有试验结果证实多种肺癌组织和肺癌肿瘤细胞系中也存在具有类干细胞特性的SP细胞,这些SP细胞的鉴别与分离对于肺癌肿瘤干细胞的研究有何意义?本文将整合现有研究资料就这一问题进行阶段性综述。     

CAR-T Cells

The recent approval of two autologous CAR-T cell products for the treatment of relapsed/refractory diffuse large B cell lymphoma (DLBCL) and similar histologies has provided a new treatment paradigm for patients who were not cured by current combination chemoimmunotherapy and in some cases by high dose therapy and autologous hematopoietic transplantation (HCT). The two commercial agents tisagenlecleucel (Novartis) and axicabtagene ciloleucel (Kite/Gilead) are both directed against the pan B     

NCI-H446人小细胞肺癌细胞株细胞亚群克隆与肿瘤干细胞的实验研究

目的 探讨NCI-H446人小细胞肺癌细胞株细胞不同克隆细胞异质性机制与肿瘤干细胞的关系.方法 采用有限稀释法克隆培养NCI-H446人小细胞肺癌细胞株细胞,免疫组化染色检测干细胞标志物CD133、ABCG2,流式细胞术测定各克隆细胞周期.结果 15个单细胞克隆生长形成克隆,形态上大致分为2种克隆.克隆A共染色6片次,CD133 、ABCG2表达阳性率为66.67%(4/6)、33.33%(2/6);克隆B共染色26片次,CD133 、ABCG2表达阳性率为15.38%(4/26)、7.69%(2/26).克隆A细胞处于G0/G1者多于克隆B,分别是70.13%和54.61%.结论 NCI-H446人小细胞肺癌细胞株细胞存在异质性肿瘤细胞克隆,可能来源于肿瘤干细胞的分化.     

抗原致敏DC联合CIK细胞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）联合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，及肺癌细胞杀伤作用中细胞因子水平。[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；ELISA法检测杀伤试验中细胞因子IL-2、IL-12、IFN-γ的分泌水平。[结果]DC经肿瘤抗原冲击后在镜下呈典型成熟形态；DC—CIK细胞对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，DC—CIK细胞对肺癌细胞的杀伤效应随之增强（P〈0．05）；细胞杀伤试验中DC—CIK细胞组中IFN-γ、IL-12的分泌量明显高于其它3组（P〈0．05）：而IL2的分泌量以DC-LAK细胞组为最高，DC—LAK和LAK细胞组明显高于其他2组（P〈0．05）。[结论]肿瘤抗原致敏的DC可诱导特异性CIK细胞，显著提高CIK细胞对肺腺癌原代细胞的杀伤作用，其杀伤作用可能与IFN-γ、IL12的分泌量有关。     

Effects of Glioma Cells on Maturation of Dendritic Cells

We investigated whether glioma cells affected the maturation of dendritic cells (DCs). First, DCs phenotypes were analyzed using FACScan. Incubation of both human and mouse immature DCs with glioma cells altered their expression of cell surface molecules, whereas expression of cell surface molecules by mature mouse and human DCs was not affected by exposure to glioma cells. Subsequently, phagositosis was assessed by uptake of FITC-dextran. Coculture with glioma cells did not inhibit phagositosis of either mature or immature DCs relative to the controls. We also analyzed the concentration of IL-12 secreted by mature DCs using enzyme-linked immunoabsorbent assay (ELISA). IL-12 production by DCs was inhibited by coculture with glioma cells. However, stimulation with lipopolysaccharide restored the production of IL-12 by both human and mouse DCs. These data suggest that glioma cells suppress the maturation of DCs.     

Tumor-specific T Cells which Form Clusters with Dendritic Cells and Tumor Cells and Deliver Macrophage-activating Factors

T cells prepared from tumor (Meth A)-bearing mice were cocultured with homologous tumor cells and splenic dendritic cells to enrich tumor-specific T cells by the separation of clusters. T blasts generated from clusters were capable of inhibiting the in vivo tumor cell growth. The culture supernatant of clustering cells (CLSN) was effective in activating macrophages (MØ) to be cytostatic and cytocidal against tumor cells. Moreover, it was found that CLSN contains at least 3 distinct factors; one was identified as interferon-γ (IFN-γ), and the others are so far unidentified, but one acts synergistically with IFN-γ, possibly as the second signal, and the other cooperates with lipopolysaccharide but not with IFN-γ. We propose that the tumor-specific T cells secrete soluble mediators which cooperate with each other in MØ activation against tumor cells.     

DC与CIK共培养后对白血病细胞杀伤活性的研究

目的研究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CIK细胞的增殖活性、表型的变化,及其对K562、HL-60白血病细胞细胞毒作用的影响。方法采集健康供者的外周血单个核细胞(MNC),置于37℃,5%CO2培养箱培养2 h,收集非贴壁细胞用于诱导培养CIK细胞,贴壁细胞诱导分化出成熟DC,将成熟DC和CIK细胞按1 5的比例混合培养3天,用MTT法检测DC-CIK共培养细胞杀伤K562和HL-60白血病细胞株的活性。结果DC-CIK共培养后增殖速度明显快于单纯CIK细胞组(P<0.05);培养第14天,CIK中CD3+CD8+、CD3+CD56+双阳性细胞的比率分别为58.6%±7.3和26.5%±6.2,DC-CIK的CD3+CD8+、CD3+CD56+的比率分别为72.5%±4.2和38.4%±6.1,表达差异显著(P<0.05);在2.5∶1~20∶1的效靶比范围内,DC-CIK对K562、HL-60细胞的杀伤活性较单纯CIK细胞组的杀伤活性要高(P<0.05)。结论DC与CIK共培养细胞是增殖活性和细胞毒活性均高于CIK细胞的免疫活性细胞。     

Interaction between Colon Cancer Cells and Human Liver Sinusoidal Endothelial Cells Promotes Liver Metastasis of Tumor Cells

OBJECTIVE To investigate the effect of co-culture between colon cancer cells(SW1116)and human liver sinusoidal endothelial cells (HLSECs)on cancer cell metastasis, and to provide a novel model for studying the mechanism of colon cancer liver metastasis.METHODS HLSECs and SW1116 were co-cultured for 21 rounds in vitro. Transwell migration,gelatin-zymography,CCK-8 proliferation and colony formation assays were used to examine the invasion, proliferation, and colony forming ability of cancer cells.Assays were carried out to examine tumor growth ability and liver metastasis. The associated molecular change was examined by western blotting.RESULTS After 21 selection rounds, colon cancer cells SW1116P21 displayed a clear boundary. Compared with the SW1116 cells,SW1116P21 cells had a greater invasive ability, cell proliferation and colony formation in soft agar.A gelatin-zymography assay showed that the ability of SW1116P21 cells to secrete matrixmetaIloproteinase-2/9 was significantly greater than that ofSW1116 cells. Additionally, the capacity for subcutaneous tumorformation of SW1116P21 was significantly increased. It was found that mice injected with SW1116P21 cells developed significantly morevisually observable liver nodules than mice injected with SW1116cells.Western blotting showed increased vimentin expression anddecreased E-cadherin expression in the SW1116P21 cells, comparedwith the SW1116 cells.CONCLUSION The interaction between SW1116 and HLSECs maypromote tumor cell invasion, proliferation and colony formation in vitro, and tumor formation and liver metastasis in vivo. Anepithelial-mesenchymal transition occurs in SW1116P21 cells, whichcontributes to the change in the characteristics of tumor cells.