重组人纤维连接蛋白诱导的CIK细胞的生物学特性和对肺癌细胞株杀伤活性的体外研究

Biological Characteristics and Antitumor Activity of CIK Cells Activated by Recombinant Human Fibronectin for Human Lung Cancer Cell Lines In Vitro

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 and objective The CIK cell is one of the most important means of the adoptive cellular immunotherapy,and it is a hotspot of which to simplify its culture procedure and to promote its inhibition rate.The aim of this study is to observe the biological function of the CIK cells cultivated by the recombinant human fibronectin （RN） and to establish an effective and simple way of cells expansion.Methods We separated the mononuclear cells （PBMCs） in 50 mL peripheral blood from 10 healthy persons with density gradient centrifugation in the lymphocyte-separating medium,and the PBMCs were divided into two groups,of which were cultivated by RN-introduced and conventional method separately.Then we estimated the proliferation ability,and analyzed the immunologic type,IFN-γ,IL-4,perforin and granzyme B of them with flow cytometry.Besides that,we tested the inhibition rate of CIKs cells to four kinds of human lung cancer cell lines in vitro by MTT assay.Results The RN-induced group had a higher proliferation rate that was 2.0-3.5 times of the conventional group,and there was an obvious statistical difference between the two （P0.05）.The proliferation rates of CD3＋CD16＋CD56＋T cells in each group were 3 778 and 2 068 times of the initial number,respectively.There was also a higher percentage of CD3＋CD8＋ T cells in RN-induced group （P0.05）,while the percentage of CD3＋CD4＋T cells had no significant statistical difference （P0.05）.We found a similar inhibition rate of the CIK cells to all this human lung cancer cell lines （P0.05）.The cells which secreted IFN–γ increased,while the cells which secreted IL-4 did not.The cells which secreted granzyme B and perforin were positive.Conclusion It is an effective and simple way to cultivate the CIK cells with RN,which should be adopted.