脐血源MHC限制性杀伤T细胞的扩增及功能研究

Expansion and Function of MHC Restricted Killer T Cells Derived from Umbilical Cord Blood

目的:本研究通过不同细胞因子的组合定向诱导脐血单个核细胞分化扩增杀伤性T细胞(CTL)并探讨其功能。方法:利用密度梯度离心法获得脐血单个核细胞(MNK),添加IL-2、IL-15、PHA-P和IFN-γ等多种因子,诱导分化并扩增CTL细胞;通过流式细胞术、细胞杀伤实验及定量PCR技术检测其生物学功能。结果:经过15 d的培养细胞数量明显增多,细胞增殖率平均为(1 522±137)%。流式细胞仪检测显示,直接分离的脐血单个核细胞流式分型的结果为95%以上属CD3-CD8-,体外培养15 d后CD3+CD8+细胞的比例高达82.77±5.61,具有良好的杀瘤活性,直接提取的MNC细胞对He La细胞及K562细胞的杀伤效果平均值为(61.88±1.08)%,培养后的CTL细胞杀伤效果均能达到80%以上,平均值为(90.33±2.02)%,与对照组相比有显著提高;同时颗粒酶(Granzyme)A、颗粒酶B、GM-CSF、颗粒溶素(Granulysin)、IFN-γ、TGF-β、TNF-α和穿孔素(perforin))等相关细胞因子的mRNA表达水平均有不同程度增加,与对照组相比IFN-γ和TGF-β的表达显著上调(P<0.05),其余因子的表达极显著上调(P<0.01)。结论:脐血造血单个核细胞能体外分化为杀伤性T淋巴细胞,并可以大量扩增,脐血可以作为肿瘤过继免疫治疗所需T淋巴细胞的来源。

Objective： This study was to expand the cytotoxic T lymphocytes （CTL） through inducing the differentiation of umbilical blood rnonomuclear cells （UBMNC） by using various combination of cytokines, and to investigate the functions of expanded CTL. Methods ： The MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real- time fluorescence quantitative PCR. Results： After expansion for 15 days, the cell number increased by 1522%± 137%. The content of CD3 - CD8 - cells in uncultured cord blood MNC was 95%, and the CD3 ＋ CD8 ＋ CTL cells reached 82. 77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ＋- 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ＋ 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-~/, TGF-[3, TNF-a and perforin. Compared with the control group, the expression of IFN-~/ and TGF-[3 significantly increased （ P 〈 0.05 ）, and the other factors dramatically increased （P 〈 O. O1 ）. Conclusion： The cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotheraov.