[Establishment of A Novel Method to Activate and Expand Human NK Cells Ex Vivo].

To develop a new method to activate and expand human NK cells ex vivo by using sodium hyaluronate as a major activating agent and to explore its related mechanism.Mononuclear cells were isolated from 3 samples of peripheral blood from three healthy donors. New NK cell culture method and the control method were used to culture NK cells from each samples separately for 14 days. Flow cytometry was used to analyze the ratio of NK cells and CD69 expression. To measure the in vitro cytotoxicity of NK cells cultured by the two methods, the K562 cells were used as the targeting cells and flow cytometry combined with CFSE marker was used as the testing method.After culturing for 14 days, the number of NK cells obtained by new culture method for NK cells expanded by 188.63±3.83 times while the number of NK cells cultured by control method expanded by 152.77±5.77 times. The ratio of NK cells in new cell culture method was above 90%, while the ratio of NK cells in control method was about 70%. The ratio of CD69+ NK cells in new cell culture method was 32.37%±3.22%, while the ratio of CD69+ NK cells in control method was 17.29%±3.79%. The results of cytotoxicity experiment in vitro showed that NK cells cultured by the new method had a higher killing ability to the target cells as compared with NK cells cultured by the control method.New NK cell culture method using sodium hyaluronate as a major activating agent can expand NK cells more efficiently as compared with the cells cultured by control method, which may be related to the direct and/or indirect activation of sodium hyaluronate to NK cells, further causing the dominant expansion of the NK cells.一种体外活化扩增人NK细胞新方法的建立.建立一种以透明质酸钠作为活性成分的新型人NK细胞体外扩增培养方法, 并简要阐明其相关机制.采集3位健康志愿者的静脉血共3份, 分离外周血单个核细胞。每份静脉血分别用新型NK细胞培养法和对照NK细胞培养法各自培养14 d。采用流式细胞术分析NK细胞比例及CD69表达, 应用CFSE细胞标记结合流式细胞术, 以K562细胞为靶细胞, 观察2种培养方法获得细胞的体外杀伤能力.经过14 d的细胞培养, 采用新型NK细胞培养法培养的NK细胞总数扩增188.63±3.83倍, 而采用对照培养法培养的NK细胞扩增152.77± 5.77倍。新型培养方法所获得的NK细胞比例为90%以上, 而对照培养法获得NK细胞比例为70%。在新型培养法组中CD69+ NK细胞比率为32.37%±3.22%, 而在对照培养法组中CD69+ NK细胞比率为17.29%±3.79%。体外杀伤结果表明, 经新型培养法扩增的NK细胞杀伤能力更高.以透明质酸钠作为活性成分的新型人NK细胞体外扩增培养法可使外周血来源的NK细胞有效扩增, 可能与透明质酸促进NK细胞活化, 从而导致NK细胞优势扩增有关.