TRAIL净化自体造血干细胞移植物中白血病细胞的实验研究

The investigation of using TRAIL in vitro to remove leukemia cells from the autologous hemopoietic stem cell transplants

目的探讨TRAIL作为自体造血干细胞移植物体外净化剂的可行性。方法常规分离骨髓单个核细胞，分别以PE—DcR1、PE—DcR2荧光染色，荧光显微镜观察观察TRAIL诱骗受体DcR1和DcR2在骨髓单个核细胞的表达及定位。200ng／ml的TRAIL作用骨髓单个核细胞、白血病Jurkat细胞系18h，FITC-Annexin V／PI标记流式细胞仪定量凋亡率，并将经上述处理的骨髓单个核细胞行成纤维细胞集落（CFU—F）培养。将绿色荧光蛋白标记的Jurkat细胞和骨髓单个核细胞混合经200ng／ml TRAIL作用24h，在GM—CSF和EPO作用下行集落培养，第7天计数Jurkat细胞所形成的荧光集落及骨髓单个核细胞所形成的非荧光集落数CFU—GM和BFU—E。结果TRAIL诱骗受体DcR1和DcR2在骨髓单个核细胞均有表达，且主要定位于胞浆和胞膜。200ng／mlTRAIL作用18h，骨髓单个核细胞、Jurkat细胞系凋亡率分别为（5．95±1．23）％、（33．42±2．28）％，2组间差异有统计学意义（P〈0.01）。TRAIL组每4×10^6个骨髓单个核细胞形成CFU—F集落数为235．67±33．56，与对照组249．33±42．72比较差异无统计学意义（P〉0．05）。200ng／mlTRAIL明显抑制Jurkat细胞所形成荧光集落，但基本不影响骨髓单个核细胞CFU—GM和BFU—E的形成。结论TRAIL能选择性地诱导Jurkat细胞凋亡，而对骨髓单个核细胞无明显毒性作用，可用于自体造血干细胞移植物体外净化。

Objective To explore the feasibility of TRAIL to be used to remove the leukemia cells from the autologous hemopoietic stem cell transplants. Methods The expression of decoy receptor 1 and decoy receptor 2 on the bone marrow mononuclear cell were routinely isolated and observed by fluorescence microscope after PE-DcR1 or PE-DcR2 stain. The apoptosis rates of mononuclearcell and Jurkat cells interfered by 200ng/ml TRAIL for 18h were determined by flow cytometry after Annexin V/PI stain. The interfered mononuclear cells were cultured to count the number to form colony-forming unit-fibroblast（CFU-F）. The Jurkat ceils labeled by green fluorescent protein were incorporated into the mononuclear cells and affected by 200ng/ml TRAIL for 24h. The incorporated cells were cultured in the system with GMCSF and EPO and the numbers of the CFU-GM, BFU-E and fluorescence colony were counted on the seventh day. Results Both decoy receptor land decoy receptor 2 of TRAIL can be detected on membrane or in cytoplasm of the bone marrow mononuclear cell. The apoptosis rate of mononuclear cell interfered by 200ng/ml TRAIL for 18h was （5.95 ± 1.23）% , which was markedly lower than that of Jurkat cells （33.42 ± 2. 28） %. The number of CFU-F of TRAIL group and control group were 235.67 ± 33.56 and 249.33 ± 42. 72, respectively. No marked difference can be found between the mentioned two groups. Moreover, TRAIL decreased the number of fluorescence colony formed by Jurkat cells without significant decreasing the number of CFU-GM and BFU-E formed by bone marrow mononuclear cells. Conclusion TRAIL can selectively induce apoptosis in Jurkat cells without marked toxic effect on the bone marrow mononuclear cells, which means that TRAIL can be used to remove the leukemia cells from the autologous hemopoietic stem cell transplants in vitro.