【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。更多还原

【Abstract】 ObjectiveTo 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.Meth odsThe 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 wereco-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 CIKand the P-gp expressions of K562/A02 and DC were detected by flow cytometry. The expressions of BCR/ABL fusion gene ofLSC and DC were detected by fluorescence in situ hybridization (FISH).ResultsDCs were successfully induced from leuke mia 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 CD56were significantly increased in CIK co-cultured with DC(P< 0.01). There were significant differences in the killing effectson 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.ConclusionThe functions of DC derived from stem cellswere 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.更多还原