脐血CIK细胞的体外增生及其抗肿瘤效应

 【摘要】　目的　研究脐血CIK细胞体外增生活性及对肿瘤细胞的杀伤活性，为CIK细胞在肿瘤过继免疫治疗中的应用提供实验依据。方法　脐血经密度梯度离心获得单个核细胞，以CD3单抗、重组人白细胞介素-2（rhIL-2）、重组人白细胞介素-1（rhIL-1）和重组人干扰素-γ（rhIFN-γ）为诱导剂制备多种细胞因子诱导的杀伤细胞（CIK细胞）。并设只加IL-2诱导成的LAK细胞和未加细胞因子的脐血单个核细胞（CBMNC）作为对照。培养前后分别用显微镜观察细胞形态，流式细胞术测定细胞表型的改变，锥虫蓝拒染法测定细胞增生水平，四甲基偶氮唑盐（MTT）法测定对肺癌细胞的杀伤活性。结果　实验发现，通过细胞因子的联合，可大量诱导出高活性的CIK细胞，CIK细胞在培养的第5天开始增生，第14天达到高峰，增生的细胞表型以CD+3 CD+56细胞为主。而LAK细胞的增生高峰出现在第7天，随后增生不明显；CBMNC表型无明显变化，增生不明显。CIK细胞针对肿瘤细胞的体外杀伤活性高于LAK细胞和CBMNC。结论　在体外细胞因子的联合作用下脐血能成功地诱导出大量的CIK细胞。脐血CIK细胞体外扩增快，杀伤活性强，对肿瘤细胞的作用优于传统的LAK细胞，为肿瘤的过继性免疫治疗提供了一个新的方向。

In vitro proliferation of CIK cells from the cord blood and the experimental research of their anti-tumor effect

【Abstract】　Objective　To build the experimental basement for the clinical use of cytokines induced killer (CIK) cells from the cord blood mononuclear cells(CBMNC) in tumor adoptive cellular immunotherapy, an effective protocol for their proliferation in vitro and cytotoxicity of CIK cells was established. Methods　The lymphocytes from umbilical cord blood were isolated by density gradient centrifugation and suspended in medium with CD3 mAb, rIL-2 , rIL-1 and IFN-γ as inducing agents to prepare CIK cells. At the same time, the lymphokine activated killer (LAK) and CBMNC were set as controls, which were only added IL-2 and not any cytokines during the whole culture. The changes of CIK cells before and after induction were observed with microscope and the phenotypes of the cells were analyzed by using flow cytometry. The proliferation of CIK cells were determined by trypan blue exclusion assay and the cytotoxic activity to lung cancer cell were tested with MTT method. Results　According to the experiment, combining use of four types of cytokines could generate a great deal of CIK cells possessing highly cytotoxicity. From day 5 CIK cells became to prolif－erate and reached the peak at day 14. During the whole period, the relative percentage of CD＋3 CD+56 cells in－creased significantly. Compared with LAK cells, which reached the proliferation peak at day 7 and then showed no evident proliferation. The control cells (CBMNCs) showed no evident change of phenotypes and proliferation. CIK cells showed a higher antitumor activity on the tumor cells than LAK cells and CBMNCs in vitro. Conclusion　Umbilical cord blood can generate a great deal of CIK cells combining used with cy－tokines. Compared with classic LAK cells, umbilical cord blood CIK cells have the advantages of rapid prolif－eration speed and powerful cytotoxicity. CIK cells will be promising as a new strategy for the adoptive cellular immunotherapy of tumor.