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目的:探讨间变性淋巴瘤激酶（ALK）阳性间变性大细胞淋巴瘤（ALCL）中自噬介导的克唑替尼耐药对肿瘤干细胞样细胞亚群的影响。方法:本课题组前期通过植入Sox2报告基因已将ALKn + ALCL Karpas299细胞株分为报告基因非反应型（RU）细胞和报告基因反应型（RR）细胞，其中RR细胞具备干细胞特性。通过慢病毒感染技术构建荧光标记的LC3过表达RR和RU细胞（分别为RR-LC3、RU-LC3），蛋白质印迹法及流式细胞术验证感染效率。RU-LC3和RR-LC3细胞使用不同浓度（0、250、500、1 000 nmol/L）克唑替尼处理后，采用双信号流式细胞术检测RED、GEN信号（RED代表下一代远红色荧光蛋白TagFP635 mKate的红色信号B695，GEN代表来自pH敏感的GFP变体pHluorin的绿色信号B530），以RED/GEN表示自噬通量。实时荧光定量聚合酶链反应（qRT-PCR）检测细胞自噬相关基因ULK1、WIPI1、LC3B mRNA表达水平。MTS法检测不同浓度（250、500、1 000 nmol/L）克唑替尼联合氯喹（5、10 μmol/L）对细胞存活的影响。n 结果:成功构建过表达LC3的RR-LC3和RU-LC3细胞。250、500、1 000 nmol/L克唑替尼诱导下，RU-LC3细胞中RED/GEN分别为1.135±0.017、1.453±0.017和1.755±0.021，RR-LC3细胞中分别为1.193±0.018、2.116±0.013和3.307±0.189，RU-LC3细胞和RR-LC3细胞中RED/GEN均呈剂量依赖性增加；相同浓度克唑替尼作用的RR-LC3细胞中RED/GEN均高于RU-LC3细胞，差异均有统计学意义（均n P<0.01）；表明RR-LC3细胞较RU-LC3细胞自噬通量变化大。未经克唑替尼处理时，RR细胞中ULK1、WIPI1、LC3B mRNA相对表达量均高于RU细胞（1.69±0.05比1.01±0.02，n t=-1.62，n P<0.01；1.24±0.04比1.03±0.05，n t=-2.11，n P<0.01；1.70±0.22比1.02±0.05，n t=-1.74，n P=0.033）。无氯喹作用时，RR细胞克唑替尼半数抑制浓度（n IC50）高于RU细胞（950 nmol/L比709 nmol/L）。氯喹作用后，RU细胞n IC50无变化；RR细胞n IC50随氯喹浓度增加而降低。n 结论:与RU细胞相比，具有肿瘤干细胞特征的RR细胞自噬反应更快速、强烈，这可能是其对克唑替尼耐药的重要原因之一。“,”Objective:To investigate the effects of autophagy-mediated crizotinib resistance on cancer stem-like cell subsets in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALKn + ALCL).n Methods:The preliminary research of our group divided ALKn + ALCL Karpas299 cell line into two subgroups: reporter unresponsive (RU) and reporter responsive (RR) cells through the implantation of Sox2 reporter genes, among which the RR cells had the characteristics of stem cells. Fluorescent labeled LC3 overexpressing RR and RU cells (RR-LC3 and RU-LC3) were constructed by lentiviral transfection technique, and the transfection efficiency was verified by using Western blotting and flow cytometry. RU-LC3 and RR-LC3 were treated with crizotinib at different concentrations (0, 250, 500, 1 000 nmol/L). The RED and GEN signals were detected by using double-signal flow cytometry to observe autophagy flux (RED represents the red signal B695 of the next generation of far-red fluorescent protein TagFP635 mKate; GEN represents the green signal from pH-sensitive GFP variant pHluorin B530), and the RED to GEV ratio represents autophagy flux. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect autophagy related genes ULK1, WIPI1 and LC3B mRNA expression levels in cells. The effects of different concentrations of crizotinib (250, 500, 1 000 nmol/L) combined with chloroquine (5, 10 μmol/L) on the cell survival were detected by using MTS assay.n Results:RU-LC3 and RR-LC3 cells with overexpression of LC3 were successfully constructed. After induction of 250, 500 and 1 000 nmol/L crizotinib, the RED to GEN ratio in RU-LC3 cells was 1.135±0.017, 1.453±0.017 and 1.755±0.021, respectively; the RED to GEN ratio in RR-LC3 cells was 1.193±0.018, 2.116±0.013 and 3.307±0.189, respectively; the RED to GEN ratio in RU-LC3 cells and RR-LC3 cells showed a dose-dependent manner. The RED to GEN ratio in RR-LC3 cells was higher than that in RU-LC3 cells when treated with same concentrations of crizotinib, and the differences were statistically significant (all n P < 0.01). The autophagy flux of RR-LC3 cells was larger than that of RU-LC3 cells. When treated without crizotinib, mRNA relative expression levels of ULK1, WIPI1 and LC3B in RR cells were higher than those in RU cells (1.69±0.05 vs.1.01±0.02, n t = -1.62, n P < 0.01; 1.24±0.04 vs. 1.03±0.05, n t = -2.11, n P < 0.01; 1.70±0.22 vs. 1.02±0.05, n t = -1.74, n P = 0.033). In the absence of chloroquine, the half-inhibitory concentration (n IC50) of crizotinib in RR cells was higher than that of RU cells (950 nmol/L vs. 709 nmol/L). After treated with chloroquine, n IC50 of RU cells did not change, while n IC50 of RR cells was decreased with the increase of chloroquine concentration.n Conclusions:Compared with RU cells, autophagy reaction of cancer stem-like RR cells is more rapid and intense, which is considered to be one of the important reasons for their resistance to crizotinib.