【作者】 李晓红；

【导师】 达万明；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 目的意义：供者异源反应性NK细胞在异基因造血干细胞移植中可以发挥移植物抗白血病(graft-versus-leukemia,GVL)效应,并减少移植物抗宿主病(graft-versus-host disease, GVHD)的发生；在实体瘤(如肾细胞癌、黑色素瘤)也有裂解肿瘤细胞的抗实体瘤作用。故输注供者异源反应性NK细胞已经成为临床移植及抗肿瘤辅助治疗的新策略。但外周血中NK细胞含量少,目前尚缺乏有效的体外扩增体系。本研究目的在于探讨从人外周血中分选及扩增高纯度NK细胞的方法,尤其是自体DCs作为饲养细胞的可行性及效果,并对扩增后NK细胞功能进行评价,以期筛选出有效的NK细胞体外扩增方法,为进一步的临床应用奠定基础。材料和方法：先采用miniMACS免疫磁珠分选方法,从外周血单个核细胞(PBMNC)得到纯化的CD14+单核细胞及NK细胞,随后体外经GM-CSF及IL4诱导单核细胞5天,成为非成熟DCs。在干细胞培养基条件下,NK细胞经IL2和IL15培养5天后,培养体系中加入不同比例的DCs,根据与DC混合的比例及方式将培养体系分为5组：A组：NK／DC=10:1,B组：NK／DC=5:1;C组：NK／DC=2:1,D组：NK／DC=1:1 transwel1非接触共培养,E组(对照组)：不加DC。培养15天,每3天半量换液并补充细胞因子；检测分选及扩增前后(CD3~-CD56’)NK细胞含量、扩增倍数及扩增前后各组NK细胞功能的变化(从以下三方面进行)：在不同效靶比下对K562细胞的杀伤作用；realt ime-PCR方法定量检测IFN-γ、穿孔素、颗粒酶B mRNA的表达水平；流式检测NK细胞表面KIR3DL1. NKG2D表达的变化,并探寻可能的miRNA调控机制。结果：(1)经miniMACS阴性免疫磁珠分选后(CD3~-CD56~+)NK细胞含量由分选前的11.19±5.25%提高到94.23±3.50%。培养15天后除C组NK细胞纯度略有下降外,其余四组与扩增前无显著性差异(P>0.05)；(2)在各培养体系中A、B、C、D组细胞的扩增倍数分别为168±64.4、170.5±82.6,244.8±148,和70.8±17.5,均显著高于对照组(未加DC)的50.46±4.3(P<0.01)；(3)培养后NK细胞对K562细胞的杀伤活性呈A组≈B组(P>0.05)>C组>D组>E组(P<0.05)。(4)各扩增体系NK细胞IFN-γ、Perforin及GranzymeB基因的表达量均较扩增前(NKO组)明显增加(P<0.01)。(5)细胞因子扩增后CD158e+的细胞下降约10%(P<0.05)。荧光素酶报告实验显示miR-146b可与KIR3DL13’UTR在靶位点特异结合,很有可能调控KIR3DL1的基因表达。结论：经miniMACS免疫磁珠阴性分选得少量高纯度NK细胞后,在含IL2(200U／mL)+IL15(20ng／mL)的SCGM(含5%人AB血清的)培养条件下,以人外周血单核细胞诱导的未成熟DC作为饲养细胞,当NK细胞与DC比例为10：1时,我们可以获得纯度高、细胞毒活性强、增值倍数高的NK细胞。荧光素酶报告实验显示miR-146b很可能参与调控KIR3DL1的表达.更多还原

【Abstract】 [Objective] Adoptive immunotherapy using allogeneic natural killer (NK) cells may prove useful in recipients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and in patients with solid tumor. But it has been limited by the inability to obtain sufficient numbers of pure NK cells. The goal of our study was to optimize the expansion of high purity NK cells from human peripheral blood by cytokines IL2,15 and autologous DC Cells, and evaluate the changes in biological functions after ex vivo expansion.[Methods] First, we isolated CD14+cells and NK cells from PBMNC by using miniMACS (Magnetic cell-selection) (Miltenyi Biotec, Germany). DCs were induced f rom CD14~+cells by cytokines GM-CSF and IL4 for 5 days. The isolated NK cells were cultured in SCGM supplemented with 5% human AB serum and combination of interleukin IL2 and IL15 for 5 days, then DCs come from CD14~+cells was add to NK cells at different ratio. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, and biological functions at the end of the culture period. The biological functions of NK cells were detected by 3 ways:(1) cytotoxicity to target K562 cells; (2) IFN-y, perforin and granzyme B mRNA expressions were assayed by realtime-PCR; (3)Expression of KIR3DL1 (CD158e) and NKG2D of NK cells were analyzed by flow cytometry. stimulate NK cells proliferation and strengthen NK cells vitality. When NK/DCs ratio were 10 to 1,5 tol,2 to l,or 1 to 1 but separated by transwell, cells were expanded 168±64.4,170.5±82.6,244.8±148 and 70.8±17.5 fold, respectively. But when NK/DCs ratio was 2 to 1, CD3~-CD56+NK cells purity dropping significantly, while in other groups was over 92%. The cytotoxicity of expanded NK cells cultured with DCs was significantly higher than the no DCs control, and NK/DCs ratio 10:1 group was slightly higher than other groups. The expressions of IFN-Y, perforin and granzyme B mRNA of expanded NK cells was significantly higher than the starting population (P<0.01). In NK/DCs ratio≤2:1 groups, IFN-Y mRNA expression level were higher, and in NK/DCs ratio≥5:1 group, perforin and granzyme B mRNA expression level were higher. After culture with cytokins for 15 days, CD158e~+NK cells number drop 10%, while there was no change in NKG2D expression. Using a luciferase reporter assay, we found miRNA 146b maybe regulate KIR3DL1 expression.[Conclusion]:Our data suggest that high purity NK cells could be efficiently expanded in culture with IL2+IL15 and autologous DCs at 10:1 NK/DCs ratio, and its biological functions were enhanced in this condition.The miRNA 146b maybe regulate KIR3DL1 expression..更多还原