人脐血CD_3AK细胞生物活性及抗瘤作用的实验研究

目的　应用抗CD3单克隆抗体(CD3McAb)和重组人白细胞介素- 2(rhIL- 2)共同诱导人脐血单个核细胞以制备CD3AK细胞,动态观察其体外增殖能力、杀伤活性、免疫表型变化及分泌细胞因子水平。方法　用台盼蓝活细胞计数计算细胞扩增倍数,MTT法测定细胞杀伤活性,间接免疫荧光法分析细胞免疫表型,ELISA双抗夹心法检测肿瘤坏死因子- α(TNF- α)、干扰素 -γ(IFN- γ)、白细胞介素 -6(IL- 6)的水平。结果　脐血CD3AK细胞在第2周增殖能力最强,增殖倍数为78.56;培养至第12天的脐血CD3AK细胞杀伤活性最高,并对多种靶细胞的杀伤作用显著;表型分析脐血CD3AK细胞属异质性细胞群,培养第7、14 天,群体中 CD3+、CD8+、CD25+、CD38+、CD16+及 CD56+ 细胞较培养前显著增加(P<0.01); CD3 AK细胞培养至第 3 天的上清中 TNF -α、IFN -γ、IL -6 水平明显升高。结论　脐血CD3AK细胞是一种具有广谱杀瘤活力且增殖活性强的免疫细胞。本研究为脐血 CD3 AK细胞的免疫治疗提供了实验和理论依据。

The experimental study of the biological activity and anti-tumor effects of CD3AK cells in human umbilical cord blood

Objective To induce human mononuclear cell of cord blood into CD 3 activating killing (CD 3AK) cells with anti-CD 3 monoclonal antibody (CD 3McAb) and recombinant human interleukin-2 (rhIL-2), so that their proliferative activity, activity of killing action, phenotypes and level of secretory cytokines can be observed dynamically. Methods The increase of the number of cells was counted by Tapan-blue staining. The killing action can be measured by using methyl -thiazolyl-tetrazolium-array. The phenotypes of cells were analysed by using indirect immunofluorescence assay. The levels of IL-6, interferon-γ (IFN-γ) and tumor necrotic factor-α (TNF-α)in culture supernatants were analysed by using enzyme-linked irnmunosorbent assay(ELISA). Results The increase of the number of CD 3AK cells from cord blood was the highest amounting to 78.56 times in the second week. The killing action reached the peak on day 12, and all target cells (malignant cell lines) could be killed significantly. The heterogeneous phenotypes of CD 3AK cells showed that the number of cells with CD 3+, CD 8+, CD 25+, CD 38+, CD 16+ and CD 56+ increased significantly on day 7,14 compared with those of pre-culture (P< 0.01). The levels of IL-6, IFN-γ and TNF-α in CD 3AK cells in culture supernatants increased noticeably on day 3. Conclusion CD 3AK cells from cord blood are very strong proliferative immune cells with wide-spectrum killing activities of tumor cells. The research provides theoretical and experimental basis for the immunotherapy of CD 3AK cells in cord blood.