天津医药

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干细胞来源DC与CIK共培养抗K562/A02干细胞的体外研究

梁芳芳1,庞华1,司玉玲1,张丽莉1,2   

  1. 1. 天津市第四中心医院
    2. 天津医科大学
  • 收稿日期:2012-09-11 修回日期:2012-12-18 出版日期:2013-04-15 发布日期:2013-04-15
  • 通讯作者: 梁芳芳

Cytotoxicity effect of DC derived from stem cells co-culture with CIK on leukemia stem cells of K562/A02 cells

LIANG Fang fang1,PANG Hua 2, lili ZHANG4,4   

  • Received:2012-09-11 Revised:2012-12-18 Published:2013-04-15 Online:2013-04-15
  • Contact: LIANG Fang fang

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摘要：

【摘要】目的 研究慢性粒细胞白血病（CML）源干细胞体外诱导生成树突细胞（DC）与同来源的细胞因子诱导的杀伤细胞（CIK）共培养，对白血病耐药株K562/A02干细胞（LSC）的杀伤作用。方法 提取BCR/ABL阳性CML患者骨髓单个核细胞（BMMC），流式分选技术分离纯化CD34+CD38-干细胞，体外扩增诱导生成DC。取同来源的CML患者 BMMC诱导出CIK。DC与CIK共培养，流式细胞术（FCM）检测其对K562/A02干细胞的凋亡及杀伤情况。光镜下观察细胞形态，FCM分析DC和CIK免疫表型、K562/A02及DC的P-糖蛋白（P-gp）表达情况，荧光原位杂交（FISH）检测 BCR/ABL融合基因在LSC及DC中的表达。结果 LSC能成功诱导为成熟DC，且LSC来源DC的P-gp阳性表达率约为51.8%。DC免疫表型CD40、CD80、CD83、CD86以及HLA-DR的表达率均高于共培养前；共培养后CIK免疫表型 CD3、CD8、CD56的表达率高于单独培养的CIK，差异均有统计学意义（P < 0.01）。DC与CIK共培养后对CD34+CD38- 干细胞的杀伤效应明显高于单独培养的CIK。CIK、DC-CIK均可诱导K562/A02细胞凋亡，凋亡率分别为(32.56± 3.20)%、(40.21±3.09)%，且2组比较差异有统计学意义（P < 0.01），但对CD34+CD38-干细胞无明显的诱导凋亡作用。结论 来源LSC的DC功能正常，同时该来源DC联合CIK能杀伤K562/A02干细胞，但对其无明显凋亡作用。

关键词: 白血病, 髓系, 慢性, BCR-ABL阳性, 干细胞, 树突细胞, 杀伤细胞, P糖蛋白

Abstract:

[Abstract] Objective  To investigate the killing effects of dendritic cell (DC) derived from stem cells of chronic myelocytic leukemia (CML) co-cultured with cytokine induced killer (CIK) on leukemia stem cells of K562/A02 cells. Methods  The marrow mononuclear cells were isolated from CML donors. CD34+CD38- cells were sorted by flow cytometry, cultured and induced to DC. The bone marrow mononuclear cells from the same donors were induced to CIK. DC and CIK were co-cultured to determine the apoptosis proapoptosis and killing effect on leukemia stem cells of K562/A02 cells by flow cytometry. Morphologies of cultured cells were examined under optical microscopy．The immune phenotypes of DC and CIK and the P-gp expressions of K562/A02 and DC were detected by flow cytometry. The expressions of BCR/ABL fusion gene of LSC and DC were detected by fluorescence in situ hybridization (FISH). Results  DCs were successfully induced from leukemia stem cells. The positive expression rate of DC’s P-gp was about 51.8%. The expression rates of CD40, CD80, CD83, CD86 and HLA-DR were increased in DC co-cultured with CIK. The mature immune phenotypes of CD3, CD8 and CD56 were significantly increased in CIK co-cultured with DC（P < 0.01）. There were significant differences in the killing effects on CD34+CD38- cells between DC-CIK group and CIK group. DC-CIK induced apoptosis of K562/A02 cells，with the apoptot? ic rate increased from (32.56±3.20)% to (40.21±3.09)%，compared with that of CIK, the difference was significant（P < 0.01）, but it showed no evident effect on apoptosis of CD34+CD38-cells．Conclusion  The functions of DC derived from stem cells were normal. DC co-cultured with CIK can effectively kill stem cells from multidrug resistant cells，but it has no evident apoptosis effect on stem cells．

Key words: leukemia, myelogenous, chronic, BCR-ABL positive, stem cells, dendritic cells, killer cells, P糖蛋白