CIK细胞对Lewis肺癌细胞凋亡影响的实验研究

目的 观察CIK细胞对Lewis肺癌细胞增殖的抑制作用,探讨其抑瘤机制.方法 常规方法培养CIK细胞,以CIK细胞为效应细胞,Lewis肺癌细胞为靶细胞,以不同效靶比共同培养,MTT法检测细胞增殖情况,同时电镜观察Lewis肺癌细胞形态特征改变;流式细胞仪检测Lewis肺癌细胞的凋亡;免疫细胞化学法检测并比较FasL在单个核细胞和CIK细胞表面的表达;用MTF法检测抗FasL单克隆抗体对CIK细胞杀伤Lewis肺癌细胞的影响.结果 电镜观察:CIK细胞能促进Lewis肺癌细胞凋亡超微结构的改变;流式细胞仪检测:CIK细胞组凋亡率明显高于对照组;FasL在CIK细胞表达增加;抗Fas单抗可抑制CIK杀伤Lewis肺癌细胞的活性.结论 CIK细胞可在体内及体外抑制Lewis肺癌细胞增殖,诱导肿瘤细胞凋亡,Fas/FasL途径在其中发挥一定作用.     

CIK与DC细胞免疫治疗在恶性肿瘤中的研究进展

随着免疫细胞生物学及免疫分子生物学的高速发展,细胞介导的过继免疫治疗已成为肿瘤患者放疗、化疗后辅助治疗的重要手段之一,作为抗肿瘤免疫治疗的重要手段。目前已用于临床的过继免疫治疗效应细胞有:淋巴因子激活的杀伤细胞(lymphokine-activated killercells,LAK),肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TIL),细胞毒性T淋巴细胞(cytotoxic Tlymphocytes,CTL)、细胞因子诱导的杀伤细胞(cytokine-induced killercells,CIK)和树突状细胞(dendritic cells,DC)等。CIK是     

源于健康人与乳腺癌患者CIK细胞的生物学特性及抗癌活性的研究

目的探讨健康人与乳腺癌患者CIK细胞在体外增殖能力及抗肿瘤作用的差异。方法分离健康人和乳腺癌患者外周血单个核细胞(PBMC)加入细胞因子,体外诱导CIK细胞,观察CIK细胞增殖能力,用MTT法检测其对不同肿瘤细胞株的杀伤活性。结果与健康人CIK细胞相比,乳腺癌患者CIK细胞的增殖速度较慢,其最大增殖倍数低(P<0.05);健康人CIK细胞对乳腺癌细胞株(MCF-7)、肝癌细胞株(SMMC-7721)、肺癌细胞株(SPC-A-1)的杀伤活性明显高于乳腺癌患者来源的CIK细胞(P<0.05)。结论健康人CIK细胞的体外增殖力及对肿瘤细胞的杀伤活性均高于乳腺癌患者的CIK细胞。健康人CIK细胞对3种肿瘤细胞株均有较强的杀伤效果。     

CIK细胞联合化疗治疗晚期非小细胞肺癌临床观察

目的观察CIK细胞联合化疗治疗晚期非小细胞肺癌的疗效、生活质量及毒副反应。方法 48例晚期非小细胞肺癌患者随机分成两组,研究组23例(TP方案+CIK细胞免疫治疗)与对照组25例(TP方案化疗),评价两组的疗效及毒副反应,并采用肺癌特异性量表QLQ-LC13对治疗前后生活质量进行评估。结果研究组有效率(39.1%)高于对照组(36.0%,P0.05),差异无统计学意义;LC13肺癌特异量表评分,研究组优于对照组(P0.05);CIK细胞回输过程中的主要毒副反应为发热。结论 CIK细胞回输安全、副作用小,CIK细胞联合化疗治疗晚期非小细胞肺癌能有效改善患者生活质量,有望成为治疗的安全、有效候选方案。     

细胞因子激活的杀伤细胞治疗晚期肺癌的研究

目的探讨细胞因子激活的杀伤细胞(CIK)过继性免疫治疗对晚期肺癌患者近期免疫指标及生活质量(QOL)的影响,并观察CIK细胞治疗的安全性。方法 41例晚期肺癌的患者,体外在富细胞因子——干扰素-γ(IFN-γ)及白细胞介素-2(IL-2)的环境下,经1周左右的时间培养、增殖CIK细胞,细胞数量达到(2～5)×109后分次回输给患者。分别于CIK细胞回输前1 d及回输结束后4周抽取患者的外周血,行T细胞亚群及NK细胞活性的检测,同时分别于CIK细胞回输前1 d及回输结束后至下一疗程前评价患者的Karnofsky(KPS)状态。该组患者治疗前后的免疫指标及KPS状态进行自身对比。结果 41例晚期肺癌患者行CIK细胞治疗后的CD3+CD4+百分率上升(P<0.05),CD3+CD8+百分率下降(P<0.05),CD3+CD4+/CD3+CD8+值上升(P<0.05),NK细胞百分率升高(P<0.05)。该组患者CIK细胞治疗后KPS评分提高率为75.6%。41例肺癌行CIK细胞治疗后,1例患者出现超过39℃的高热,1例患者出现皮疹,余患者未见明显副作用出现。结论 CIK细胞治疗能改善晚期肺癌患者近期细胞免疫功能,提高患者生活质量,且安全可靠,值得临床进一步的研究。     

托盘根乙醇提取物的抗肿瘤抗转移作用研究

目的:观察托盘根乙醇提取物(ERC)对肿瘤生长的影响。方法:用MTT法观察ERC体外对人肿瘤SPC-A-1细胞的影响,体内抑瘤实验观察ERC对Lewis肺癌的作用。结果:ERC体外明显抑制SPC-A-1细胞的生长,体内明显抑制Lewis癌块增大,减少Lewis肺转移结节数,增强小鼠对Lewis肺癌的相伴免疫反应。结论:ERC对肺癌细胞系有明显抗肿瘤作用     

CIK细胞对人肺腺癌AS49／DDP耐药细胞株的抗增殖及诱导凋亡形态学研究

目的研究多种细胞因子诱导的杀伤细胞（CIK）对A549／DDP肺癌耐药细胞株的抗增殖和诱导凋亡作用。方法应用细胞计数法设计细胞浓度,通过相差显微镜下观察CIK细胞对A549／DDP肺癌耐药细胞株形态学影响。结果随着CIK细胞与肺癌细胞效靶比增加及作用时间延长,抑制率明显增强,CIK细胞作用于肺癌细胞24h后,形态学观察肺癌细胞发生凋亡或坏死。结论体外制备的CIK细胞对肺癌耐药细胞株A549／DDP具有抑制增殖和促进凋亡作用。     

CIK细胞诱导及对K562细胞毒作用的研究

目的 体外诱导细胞因子诱导的杀伤细胞(CIK),并研究其生物学活性.方法 从外周血分离单个核细胞(PBMC),经过细胞因子诱导、培养并扩增CIK细胞,以LAK细胞作比较,检测CIK的增殖能力,流式细胞仪检测CIK细胞表面标志CD3、CD56,MTT法检测对K562细胞系杀伤活性.结果 CIK细胞第二周进入快速增殖期,到第21d扩增倍数超过120倍,CD3 、CD56 细胞扩增倍数达15倍以上;CIK对K562细胞的杀伤能力明显优于LAK细胞.结论 CIK细胞是一种具有很强杀瘤活性的免疫活性细胞,具有临床应用前景.     

树突状细胞诱导的淋巴因子激活的杀伤细胞对肺癌细胞系A549的体外杀伤作用

目的:探讨负载肺癌抗原的树突状细胞(dendritic cells,DC)诱导淋巴因子激活的杀伤(LAK)细胞对肺癌细胞A549的杀伤作用.方法:采用肺癌患者外周血单个核细胞(PBMC)经rhGM-CSF、rhIL-4、rhTNF-a诱导和肿瘤冻融抗原刺激诱导获得的DC与LAK细胞按1:20比例共培养2 d,获得Ag-DC-LAK细胞作效应细胞,分别用Ag-Dc-LAK、DC-LAK、Ag-LAK和LAK对肺癌细胞系A549细胞进行杀伤实验.结果:以30μg/mL蛋白的肿瘤抗原负载的DC其表型CD1a、CD80、CD86、HLA-DR均显著升高(P<0.05),高于单纯细胞因子诱导成熟的DCs表型,由其刺激增殖的LAK细胞对肺癌细胞A549杀伤率为(71±5)%,明显高于对照组(P<0.05).结论:经肿瘤抗原刺激诱导成熟的DC可有效传递抗原,增加T细胞对肿瘤细胞的杀伤作用.     

树突状细胞诱导的淋巴因子激活的杀伤细胞对肺癌细胞系A549的体外杀伤作用

目的:探讨负载肺癌抗原的树突状细胞(dendritic cells,DC)诱导淋巴因子激活的杀伤(LAK)细胞对肺癌细胞A549的杀伤作用.方法:采用肺癌患者外周血单个核细胞(PBMC)经rhGM-CSF、rhIL-4、rhTNF-a诱导和肿瘤冻融抗原刺激诱导获得的DC与LAK细胞按1:20比例共培养2 d,获得Ag-DC-LAK细胞作效应细胞,分别用Ag-Dc-LAK、DC-LAK、Ag-LAK和LAK对肺癌细胞系A549细胞进行杀伤实验.结果:以30μg/mL蛋白的肿瘤抗原负载的DC其表型CD1a、CD80、CD86、HLA-DR均显著升高(P<0.05),高于单纯细胞因子诱导成熟的DCs表型,由其刺激增殖的LAK细胞对肺癌细胞A549杀伤率为(71±5)%,明显高于对照组(P<0.05).结论:经肿瘤抗原刺激诱导成熟的DC可有效传递抗原,增加T细胞对肿瘤细胞的杀伤作用.     

Antitumor activity of cytokine-induced killer cells against human lung cancer

Lung cancer is the leading cause of cancer-related death among men and women in the world. Despite the aggressive treatment with surgery, radiation and chemotherapy, the long term survival for lung cancer patients remains low. In this study, the anti-tumor activity of cytokine-induced killer (CIK) cells against human lung cancer was evaluated in vitro and in vivo. Although CD3(+)CD56(+) CIK cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1000-fold on day 14 in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 98% of NCI-H460 human lung cancer cells, which was determined by the (51)Cr-release assay. In addition, CIK cells at doses of 3 and 30 million cells per mouse inhibited 57% and 77% of NCI-H460 tumor growth in nude mouse xenograft assay, respectively. This study suggests that CD3(+)CD56(+) CIK cells may be used as an adoptive immunotherapy for patients with lung cancer.     

耐药乳腺癌裸鼠模型免疫治疗初探

目的 耐药乳腺癌表现为治疗效果差,转移率高,死亡率高的一种恶性程度极高的肿瘤性疾病。有研究表明,细胞因子诱导的杀伤细胞（CIK）在树突细胞（DC）辅助下,对耐药肿瘤细胞的杀伤能力可能超过T细胞。本实验利用DC与CIK联合培养,比较在不同条件下对乳腺癌多药耐药细胞株的杀伤效应。 方法 分离健康人外周血获得单个核细胞,分别诱导为树突细胞（DC）和CIK细胞,将人类乳腺癌耐药细胞株MCF-7/ADR细胞的冻融物抗原冲击DC（AP-DC）,分别将DC与CIK细胞共培养（AP-DC+CIK 、DC+CIK）,CIK细胞单独培养作对照。用流式细胞仪分析细胞表型,酶联免疫吸附法（ELISA）测定分泌IL-12、TNF-α、IFN-γ和IL-2水平,MTT法测定细胞毒效应。结果 DC和CIK细胞共孵育,使CIK细胞的CD3CD56双阳性细胞的比例及分泌IFN-γ、TNF-α和IL-2的水平增高,DC的成熟表型和分泌IL-12的水平增加,其中均以AP-DC+CIK组增高最为明显,和其他组间比较差异有统计学意义。对乳腺癌耐药细胞MCF-7/ADR的细胞毒效应,AP-DC+CIK组杀伤效应最强, 与DC+CIK组和CIK组比较差异均有统计学意义;结论 经耐药肿瘤抗原负载的DC与CIK共同作用后,其对耐药乳腺癌细胞MCF-7/ADR的抗瘤免疫能力最强,为临床治疗耐药肿瘤带来希望。     

Antitumor effect of 24R,25-dihydroxyvitamin D3

24R,25(OH)2D3, one of the endogenous active metabolites of vitamin D3, showed suppressive effects on the proliferation of various tumor cells in vitro and a prolonging effect on the survival of P-388 bearing mice in vivo. Lewis lung carcinoma was found to cause hypercalcemia in tumor bearing mice. K-DR (24R,25(OH)2D3 (prepared by Kureha Chemical Ind.) significantly prolonged the survival of mice with Lewis lung carcinoma. K-DR also showed a suppressive effect on the growth of human osteosarcoma transplanted in nude mice.     

Inhibition of human ovarian tumor growth by cytokine-induced killer cells

Despite the recent improvement in the treatment of ovarian cancer, this disease is still leading cause of cancer death in women. In this study, the anti-tumor activity of cytokine-induced killer (CIK) cells against human ovarian cancer was evaluated in vitro and in vivo. Although CD3+CD56+ cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1,000-fold on day 14 in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 45% of SK-OV-3 human ovarian cancer cells, which was determined by the 51Cr-release assay. In addition, CIK cells at a dose of 23 million cells per mouse inhibited 73% of SK-OV-3 tumor growth in nude mouse xenograft assay. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with ovarian cancer.     

肿瘤干细胞来源的DC-CIK 对同源肿瘤细胞的杀伤作用

【摘要】目的 观察干细胞负载树突细胞诱导的杀伤细胞（CSC-DC-CIK）作为效应细胞对同源肿瘤细胞的杀伤作用,探讨CSC 抗原参与肿瘤杀伤作用的可行性。方法 培养肾癌细胞株A498 和肺癌细胞株A549,用流式分选术分离纯化CD133+细胞,分别作为肾癌干细胞（KSC）和肺癌干细胞（LSC）,冻融法制备抗原。提取健康产妇脐带血的单个核细胞,体外扩增诱导生成DC 和CIK 细胞。分别用上述CSC 抗原负载DC,与CIK 共培养（CSC-DC-CIK）,流式细胞术分析DC 和CIK 细胞免疫表型,ELISA 法检测细胞因子分泌水平,用乳酸脱氢酶（LDH）释放法检测CSCDC- CIK 对同源肿瘤细胞的杀伤效率。结果CSC-DC 的DC 免疫表型CD40+、CD80+、CD86+及HLA-DR+的表达均高于单纯DC 的相应免疫表型的表达（P < 0.01）;DC、CSC-DC 与CIK 共培养后的DC 免疫表型CD40+、CD80+、CD86+ 及HLA-DR+的表达均高于共培养前（P < 0.01）;CSC-DC 与CIK 共培养后的DC 免疫表型CD40+、CD80+、CD86+及 HLA-DR+的表达高于DC 与CIK 共培养后的相应免疫表型（P < 0.01）;DC、CSC-DC 与CIK 共培养后的CIK 免疫表型CD3+、CD8+、CD56+的表达高于共培养前（P < 0.01）;CSC-DC 与CIK 共培养后的CIK 免疫表型CD3+、CD8+、CD56+ 的表达高于DC 与CIK 共培养后的CIK 相应免疫表型（P < 0.01）;DC、CSC-DC 与CIK 共培养后的IFN-γ、TNF-α和 IL-2 分泌水平高于共培养前（P < 0.01）;CSC-DC 与CIK 共培养后的IFN-γ、TNF-α和IL-2 分泌水平高于DC 与CIK 共培养后的相应细胞因子表达（P < 0.01）;KSC-DC-CIK 组和LSC-DC-CIK 组对靶细胞的杀伤率为（50.21±4.24）% 和（49.32±3.89）%,明显高于DC-CIK 组的（30.25±3.11）%（F=89.157,P < 0.01）。结论 CSC 抗原负载DC 活化CIK （CSC-DC-CIK）对同源肿瘤细胞有更好的杀伤作用,对其作用机制和临床应用的可能性尚需深入研究。     

Inhibition of curcumin on myeloid-derived suppressor cells is requisite for controlling lung cancer

Lung cancer remains the leading cause of cancer mortality. Myeloid-derived suppressor cells (MDSCs) are potent immune-suppressive cells and present in most cancer patients. Recently, several studies have shown that curcumin inhibits the expansion of MDSCs in some cancers. However, it is not clear how curcumin modulates the suppressive function of MDSCs, and whether curcumin achieves anti-tumor effects via regulating the expansion of MDSCs in lung cancer. Here, our results showed that curcumin significantly inhibited tumor growth in a Lewis lung carcinoma (LLC) isogenic tumor model. Curcumin reduced the accumulation of MDSCs in spleen and tumor tissue in LLC isogenic model. And curcumin promoted the maturation and differentiation of MDSCs in tumor tissue. Notably, curcumin inhibited the expression level of immune suppressive factors of MDSCs, arginase-1 (Arg-1) and ROS, in purified MDSCs from tumor tissue in vivo. Expectedly, curcumin also inhibited the immunosuppressive function of isolated MDSCs from tumor tissue and spleen of tumor bearing mice in vitro. Moreover, curcumin decreased the level of IL-6 in the tumor tissue and serum from LLC-bearing mice. Taken together, curcumin indeed possesses anti-cancer effect and inhibits the accumulation and function of MDSCs. And curcumin reduces the level of IL-6 in tumor-bearing mice to impair the expansion and function of MDSCs. These results suggest that inhibition of MDSCs in tumor is requisite for controlling lung cancer.     

Anti-tumor activity of ex vivo expanded cytokine-induced killer cells against human hepatocellular carcinoma

Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with natural killer cell phenotypes and functions. In this study, the anti-tumor activity of CIK cells against hepatocellular carcinoma was evaluated in vitro and in vivo. In the presence of anti-CD3 antibody and IL-2 for 14 days, human peripheral blood mononuclear cell population changed to heterogeneous CIK cell population, which comprised 96% CD3(+), 3% CD3( inverted exclamation mark(c))CD56(+), 32% CD3(+)CD56(+), 11% CD4(+), 75% CD8(+), and 30% CD8(+)CD56(+). CIK cells produced significant amounts of IFN-gamma and TNF-alpha; however, produced only slight amounts of IL-2, IL-4, and IL-5. At an effector-target cell ratio of 30:1, CIK cells destroyed 33% of SNU-354 human hepatocellular carcinoma cells, which was determined by the (51)Cr-release assay. In addition, a dose of 1x10(6) CIK cells per mouse inhibited 60% of SNU-354 tumor growth in irradiated nude mice. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with hepatocellular carcinoma.     

托盘根乙醇提取物的抗肿瘤抗转移作用研究

目的：观察托盘根乙醇提取物（ERC）对肿瘤生长的影响。方法：用MTT法观察ERC体外对人肿瘤SPC－A－1细胞的影响，体内抑瘤实验观察ERC对Lewis肿瘤的作用。结果：ERC体外明显抑制SPC－A－1细胞的生长，体内明显抑制Lewis癌块增大，减少Lewis肺转移结节数，增强小鼠对Lewis肺癌的相伴免疫反应。结论：ERC对肺癌细胞系有明显抗肿瘤作用。