侧群细胞对NOD/SCID小鼠NK细胞杀伤的抵抗作用

Side population cells isolated from hepatic carcinoma cell lines escape being killed by NK cells from NOD/SCID mice

目的:探讨人肝癌细胞株Huh7、PLC/REF/5中以侧群(SP)细胞为标志的肿瘤干细胞抵抗NOD/SCID小鼠来源的NK细胞杀伤的作用.方法:分选所得NK细胞制成细胞悬液接种于培养皿中,经小鼠重组IL2和IL15活化后,同肝癌细胞株Huh7、PLC/REF/5经FACSAria分选后所得主群(MP)、SP细胞共培养,使NK细胞:肿瘤靶细胞为6:1.经AnnexinV和7AAD染色后,流式细胞仪检测.结果:单独培养Huh7或PLC/PRF/5细胞中MP与SP细胞比较,其细胞活力(A值)及细胞凋亡比例无明显差别(P>0.05);而与NK共培养的MP细胞中AnnexinV+7AAD+细胞较单独培养的MP和与NK共培养的SP细胞明显升高(Huh7:8.43±1.56vs4.33±1.36,4.41±0.70;PLC/PRF/5:7.89±1.53vs5.47±1.04,4.94±0.95,P<0.05).结论:通过细胞凋亡检测发现,Huh7和PLC/REF/5中SP能明显抵抗NK细胞诱导的杀伤作用,提示肝癌细胞株Huh7、PLC/REF/5中SP细胞在体外具有主动的免疫逃逸能力.

AIM：To examine whether side population（SP） cells isolated from hepatic carcinoma cell lines can escape being killed by NK cells from NOD/SCID mice.METHODS：Sorted natural killer（NK） cells from NOD/SCID mice were seeded in dishes and cultured for three days in the presence of 25 ng/mL IL-2 and IL-15 to activate cells.SP cells and main population（MP） cells were sorted with the FACSAria from Huh7 and PLC/REF/5 cell lines and co-cultured with activated NK cells for 12 hours.Cell apoptosis was evaluated by annexin V/7AAD staining and flow cytometry.RESULTS：There are no significant differences in cell viability and apoptosis between MP cells and SP cells from Huh7 and PLC/REF/5 cell lines（all P 〉0.05）.However,the proportions of annexin V＋/7-AAD＋ cells in MP cells co-cultured with NK cells were significantly higher than those in MP cells cultured alone and SP cells co-cultured with NK cells（Huh7：8.43 ± 1.56 vs 4.33± 1.36,4.41 ± 0.70;PLC/PRF/5：7.89 ± 1.53 vs 5.47 ± 1.04,4.94 ± 0.95,all P〈 0.05）.CONCLUSION：SP cells isolated from Huh7 and PLC/REF/5 cells could probably escape being killed by activated NK cells from NOD/SCID mice.