LSC-DC-CIK对慢性粒细胞白血病干细胞杀伤作用的体外试验研究

Killing effects of cytokines-induced killer cells on chronic myelocytic leukemia(CML) stem cells in vitro

目的研究慢性粒细胞白血病(CML)患者来源的干细胞体外扩增诱导生成树突细胞(DC),与同来源的细胞因子诱导的杀伤细胞(CIK)共培养,对慢性粒细胞白血病细胞株K562干细胞的杀伤作用。方法提取CML患者的骨髓单个核细胞(BMMC),利用流式细胞术(FCM)分选纯化白血病干细胞(LSC)CD34+CD38-CD44+,体外扩增诱导生成LSC-DC。取同一患者骨髓的BMMC诱导出CIK。LSC-DC与CIK共培养,用光学显微镜观察细胞形态,FCM分析LSC-DC和CIK细胞免疫表型,荧光原位杂交(FISH)检测BCR/ABL融合基因在LSC及LSC-DC中的表达,FCM检测LSC-DC-CIK对慢性粒细胞白血病细胞株K562干细胞的杀伤及凋亡情况。结果白血病干细胞能成功诱导为LSC-DC,其DC成熟免疫表型CD40、CD80、CD83、CD86及受体(HLA-DR)的表达均高于诱导前;共培养后CIK表面标志CD3、CD8、CD56的表达率高于单独培养CIK,差异有统计学意义(P<0.01)。LSC-DC与CIK共培养后对慢性粒细胞白血病细胞株K562干细胞的杀伤率(66.94%)明显高于单独培养的CIK(31.89%),差异有统计学意义(P<0.01)。LSC-DC-CIK可诱导K562细胞凋亡,凋亡率为52.28%±3.71%,与CIK组(37.51%±1.89%)相比,差异有统计学意义(P<0.01);但对其中白血病干细胞无明显的诱导凋亡作用。结论 CML来源LSC-DC保留有干细胞特征,其与CIK共培养能杀伤慢性粒细胞白血病细胞株干细胞,但无明显的诱导干细胞凋亡作用。

Objective To investigate the killing effects of cytokines-induced killer cells （CIK） co-cultured with dendritic cells derived from chronic myeloeytie leukemia （CML） stem cells on CML stem cells （K562）. Methods Bone marrow mononuelear cells （BMMC） were isolated from CML donors. CD34＋CD38-CD44＋ cells （LSC） were isolated and purified by flow eytometry （FCM）, which were cultured and induced to LSC-DC. The BMMCs from the same donors were induced to CIK. LSC-DC and CIK were co-cultured, and co-cultured cells were observed under a light microscope. FCM technique was used to analyze the cellular immuno-phenotype. The expressions of BCR/ABL fusion gene in LSC and LSC-DC were detected by fluorescence in situ hybridization （FISH）. The killing and apoptotic effects of LSC-DC-CIK on CML stem cells （K562） were measure by FCM. Results LSC-DCs were successfully induced from leukemia stem cells. The expression rates of CD40, CD80, CD83, CD86 and HLA-DR in LSC-DC increased significantly, as compared with cells before induction, the expression rates of CD3, CDs and CD56 in co-cultured CIK were significantly higher than those in CIK （P〈0.01）. The killing rate of CIK co-cultured with LSC-DC on K562 cells was 66.94%, which was obviously higher than that （31.89%） ofCIK （P〈0.01）. Moreover, the apoptotic rate of K562 cells induced by LSC-DC-CIK was 52.28 %, which was significantly higher than that （37.51% ） induced by CIK （P〈0.01 ）, but there was no significant apoptotic effect on leukemia stem cells. Conclusion The functions of LSC-DC derived from CML are normal, LSC-DC co-cultured with CIK could effectively kill stern cells of CML, but could not significantly induce the apoptosis of stem cells.