天津医药

• 细胞与分子生物学 • 上一篇    下一篇

肿瘤干细胞来源的DC-CIK 对同源肿瘤细胞的杀伤作用

庞冲1,张腾月2,王长利1   

  1. 1. 天津医科大学肿瘤医院，国家肿瘤临床医学研究中心，天津市“肿瘤防治”重点实验室
    2. 天津市眼科医院
  • 收稿日期:2014-09-11 修回日期:2014-09-17 出版日期:2014-10-15 发布日期:2014-10-15
  • 通讯作者: 庞冲

Effect of Tumor Stem Cell Derived CSC-DC-CIK on Destructing Homologous Tumor Cells

Teng Chong ,Zhang Teng yue,Wang Chang li   

  • Received:2014-09-11 Revised:2014-09-17 Published:2014-10-15 Online:2014-10-15
  • Contact: Teng Chong

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摘要：

【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。

关键词: 肿瘤干细胞, 树突细胞, 免疫疗法, 细胞因子诱导的杀伤细胞

Abstract:

[Abstract] Objective  To investigate the destructive effect of CSC-DC-CIK who were induced by cytokine induced killer (CIK) cells co-cultured with dendritic cells (DCs) on homologous tumor cells and to explore the possibility of CSC anti? gen involving in killing tumor. Methods Kidney cancer stem cells (KSCs) and lung cancer stem cells (LSCs) were isolated through FACS using CD133 + as a selection marker from cultured kidney cancer cell line A498 and lung cancer cell line A549 respectively. Freeze-thaw method was used to obtain the cancer stem cells（CSCs）antigens. DC cells and CIK cells were collected by in vitro expansion and inducted from the mononuclear cells isolated from human cord blood. The CIK cells were co-cultured with the DCs which were pulsed with the CSCs antigens（CSC-DC-CIK）mentioned above. Immunopheno? types of DC and CIK were analyzed by flow cytometry; cytokines levels were detected by ELISA kits and the destructive ef? fects of two kinds of CSC-DC-CIKs were tested by lactate dehydrogenase (LDH) release assay. Results  The expression of phenotypes CD40+, CD80+, CD86+ and HLA-DR+ were higher in CSC-DC than in CD（P < 0.01）; the expression of pheno? types CD40+, CD80+, CD86+ and HLA-DR+ of DC and CSC-DC were higher after co-culture than those before co-culture（ P < 0.01）;the expression of phenotypes CD40+, CD80+, CD86+ and HLA-DR+ of CSC-DC after been co-cultured with CIK were higher than those of DC after been co-cultured with CIK（P < 0.01）. The CIK phenotypes: CD3+, CD8+, CD56+ were in? creased in CIK co-cultured with both CSC-DC and DC than those before co-culture (P < 0.01); the expression of pheno? types CD3+, CD8+, CD56 +were higher in CSC-DC co-cultured with CIK than in DC co-cultured with CIK. DC-CIK and CSC-DC-CIK groups were more capable to express IFN-γ, TNF-α, IL-2 than they were before co-cultured with CIK (P < 0.01). CSC-DC-CIK group can secrete more above cytokines than DC-CIK group does（P < 0.01）. The destructive rates of KSC-DC-CIK and LSC-DC-CIK on target cells were (50.21±4.24)% and (49.32±3.89)% respectively which were much higher than that in DC-CIK（30.25±3.11）%（F = 89.157，P < 0.01）. Conclusion CSC-DC-CIKs have destructive effects on homologous tumor cells. More researches are needed to explore the mechanism and to evaluate the clinical applications.

Key words: Immunohistochemistry, dendritic cells, immunotherapy, cytokine induced killer