抗CD3单抗包被技术获得CD3AK细胞的体外实验研究

Study of CD3AK cell acquired by precoated with CD3mAb in vitro

目的抗CD3单抗包被技术与传统方法培养获得的抗CD3单克隆抗体激活的杀伤细胞(anti-CD3monoclonal antibody activated killer cells,CD3AK)、细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK)的体外扩增、体外杀伤活性及免疫表型的比较,以期为选择增殖更快、抗癌效应更强的生物活性细胞供临床治疗。方法健康人外周血单个核细胞,分别采用抗CD3单抗包被技术与传统方法培养获得CD3AK细胞、常规方法培养获得CIK细胞,检测各免疫活性细胞对K562耐药细胞株(K562/ADR)的杀伤活性、细胞膜P-糖蛋白(P-glucoproteins,P-gp)表达以及收获时细胞表型、上清液细胞因子含量。结果培养第13天时,包被技术获得CD3AK细胞和CIK细胞计数分别为非包被培养的1.41倍、2.96倍;3组细胞免疫表型差异均无统计学意义(P<0.05);但上清液中白细胞介素2(IL-2)水平3组分别为(47.97±3.24)ng/L vs(40.14±2.26)ng/L vs(35.90±3.33)ng/L,IL-12为(128.50±14.82)ng/L vs(110.69±10.02)ng/L vs(98.24±10.30)ng/L,肿瘤坏死因子α(TNF-α)为(154.42±16.99)ng/Lvs(143.67±14.24)ng/L vs(121.56±13.62)ng/L,γ干扰素(IFN-γ)为(143.79±12.97)ng/L vs(115.87±13.24)ng/L vs(102.50±10.47)ng/L,包被培养CD3AK细胞组高于其他两组(P<0.05)。3组细胞在不同效价比对K562/ADR细胞杀伤活性差异无统计学意义(P>0.05),与K562/ADR细胞作用后P-gp蛋白表达均下调,但差异无统计学意义(P>0.05)。结论抗CD3单抗包被技术可以在短时间内获得更大数量的免疫活性细胞,抗肿瘤细胞活性肯定,是一种更为有效的培养方式。

Objective To analyze the growth in vitro,the cytotoxicity and the immunological properties of CD3AK cells precoated with CD3Mab,CD3AK acquired by traditional method and CIK cells,to seek better immune cells for biotherapy.Methods Mononuclear cells（MNCs） were isolated from peripheral blood of healthy persons,CD3AK cells were differently cultured with and without precoated with CD3mAb,and CIK cells in the presence of combined cytokines.The change of phenotypic characterization and secretion of cytokines of three types of cells were determined.The effect of cytotoxicity was determined by MTT assay,and the flow cytometry was used to determine the expression of P-glycoproteins.Results The number of CD3AK precoated with CD3mAb was higher than that of CD3AK cells without precoated with CD3mAb and CIK cells,seperately 1.41 times,2.96 times.The level of IL-2 in the three groups was（47.97±3.24） ng/L vs（40.14±2.26） ng/L vs（35.90±3.33） ng/L,IL-12 was（128.50±14.82） ng/L vs（110.69±10.02） ng/L vs（98.24±10.30） ng/L,TNF-α was（154.42±16.99） ng/L vs （143.67±14.24） ng/L vs（121.56±13.62） ng/L,IFN-γ was（143.79±12.97） ng/L vs（115.87±13.24） ng/L vs（102.50±10.47） ng/L.But there were no significant difference in phenotypic characterization,cytotoxicity against K562/ADR and the change of P-glycoproteins in three groups.Conclusion The method of acquiring CD3AK cell precoated with CD3mAb may become more effective way for bio-therapy.