干细胞来源DC与CIK共培养抗K562／A02干细胞的体外研究  

Cytotoxicity Effects of DC Derived from Stem Cells Co-Cultured with CIK on K562/A02 Cells

作　　者：梁芳芳[1] 庞华[2] 司玉玲[2] 孙雯雯[3] 窦金霞[3] 

机构地区：[1]天津医科大学研究生院,300070 [2]天津市第四中心医院肿瘤血液科 [3]天津医科大学研究生院中心实验室

出　　处：《天津医药》2013年第4期289-292,I0001,共5页Tianjin Medical Journal

基　　金：天津市自然科学基金资助项目(项目编号:12JCYBJC16500);天津市卫生局科技基金资助项目(项目编号:11KG114)

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。Objective To investigate the killing effects of dendritic cell （DC） derived from stem cells of chronic my elocytic leukemia （CML） cocultured with cytokine induced killer （CIK） on leukemia stem cells of K562/A02 cells. Meth ods The marrow mononuclear ceils were isolated from CML donors. CD34＋CD38 cells were sorted by flow cytometry, cul tured and induced to DC. The bone marrow mononuclear cells from the same donors were induced to CIK. DC and CIK were cocultured to determine the apoptosis proapoptosis and killing effect on leukemia stem cells of K562/A02 cells by flow cy tometry. Morphologies of cultured cells were examined under optical microscopy. The immune phenotypes of DC and CIK and the Pgp 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 leuke mia stem cells. The positive expression rate of DC＇ s Pgp was about 51.8%. The expression rates of CD40, CD80, CD83, CD86 and IILADR were increased in DC cocultured with CIK. The mature immune phenotypes of CD3, CD8 and CD56 were significantly increased in CIK cocultured with DC （P 〈 0.01）. There were significant differences in the killing effects on CD34CD38cells between DCCIK group and CIK group. DCCIK 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-CD38cells. Conclusion The functions of DC derived from stem cells were normal. DC cocultured with CIK can effectively kill stem cells from muhidrug resistant cells, but it has no evident apop tosis effect on stem cells.

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 细胞凋亡 K562细胞 

分 类 号：R733.7[医药卫生—肿瘤]