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背景与目的CIK细胞是过继免疫治疗的重要手段之一,简化体外培养过程从而提高其增殖率和杀瘤活性仍是目前研究的一个热点课题。本研究观察重组人纤维连接蛋白(recombinant human fibronectin,RN)诱导CIK细胞的生物学特性,建立一种高效、简便的体外CIK细胞扩增方法。方法抽取10名健康人外周静脉血各50mL,用淋巴细胞分离液分离单个核细胞,分别采用RN诱导法和传统方法培养CIK细胞,记录细胞增殖数;用流式细胞术测定免疫细胞表型和分泌IFN-γ、IL-4、穿孔素和颗粒酶B细胞的百分比;用MTT法测定CIK细胞对4种人肺癌细胞株的体外杀伤率。结果RN诱导的CIK细胞扩增倍数为传统方法的2.0倍-3.5倍,具有统计学差异(P<0.05);RN诱导组和传统方法组CD3+CD16+CD56+细胞绝对数分别增加了3778倍和2069倍;RN诱导组细胞中CD3+CD8+细胞比例明显高于传统方法组(P<0.05);但CD3+CD4+细胞比例无统计学差异(P>0.05);对4种肺癌细胞株的体外杀伤活性无统计学差异(P>0.05)。RN诱导的CIK较诱导前:分泌IFN-γ的细胞比例明显增加;分泌IL-4的细胞比例略有降低;释放穿孔素、颗粒酶B的阳性细胞比例较诱导前增加。结论RN诱导法是一种高效、简便的体外扩增CIK方法,可以替代传统方法。
BACKGROUND & OBJECTIVE: CIK cells are one of the most important means of adoptive immunotherapy. To simplify the process of in vitro culture to enhance its proliferation rate and tumor-killing activity is still a hot issue in current research. In this study, we observed the biological characteristics of CIK cells induced by recombinant human fibronectin (RN), and established an efficient and simple method for the expansion of CIK cells in vitro. Methods Peripheral venous blood samples from 10 healthy volunteers were collected and 50mL respectively. The mononuclear cells were isolated by lymphocyte separation. The CIK cells were cultured by RN induction method and conventional method respectively. The proliferation and proliferation of CIK cells were measured by flow cytometry. The percentage of secreting IFN-γ, IL-4, perforin and granzyme B cells were determined. The cytotoxicity of CIK cells to four human lung cancer cell lines in vitro was determined by MTT assay. Results The multiplicity of proliferation of CIK cells induced by RN was 2.0- to 3.5-fold higher than that of the conventional method (P <0.05). The absolute numbers of CD3 + CD16 + CD56 + cells in RN-induced group and traditional group increased by 3778-fold and (P <0.05). However, the proportion of CD3 + CD8 + cells in RN-induced cells was significantly higher than that in the traditional methods (P <0.05) No significant difference in activity (P> 0.05). Compared with pre-induction, CIK induced by RN significantly increased the proportion of IFN-γ-secreting cells; the proportion of IL-4-secreting cells decreased slightly; the percentage of perforin and granzyme B-releasing cells increased before induction. Conclusion RN induction method is an efficient and simple method to expand CIK in vitro, which can replace traditional methods.