摘要
背景:慢性髓细胞白血病因为伊马替尼的应用出现了重大转机。但由于伊马替尼的耐药性,应用该药治疗的白血病患者预后较差,特别是对于急变期白血病患者。目的:观察肿瘤干细胞在伊马替尼ST1571耐药的慢性髓细胞白血病细胞系K562中的表征,阐明白血病细胞系对伊马替尼ST1571耐药的机制。设计:观察性对比实验。单位:河南省肿瘤医院,河南省血液病研究所。材料:选用5周龄BALB/c-nu/nu小鼠30只,雌性,由中国医学科学院动物中心提供。ST1571由北京诺华制药有限公司提供。足叶乙甙(Vp16)购自德国Bristol-Myers Squibb;抗P-gp购自美国Santa Cruz公司。抗ab1购自美国BD Biosciences公司。实验过程中对动物的处置符合动物伦理学标准。方法:实验于2003-09/2005-11在河南省血液病研究所完成。经过对K562细胞系长期的足叶乙甙(Vp16)诱导和克隆筛选,建立了一株耐药细胞系K562Np16;利用于细胞高效能将Hoechst 33342荧光染料泵出细胞的特性,采用流式细胞分选方法从K562/Vp16细胞系中分选出一小群细胞,即边缘细胞(SP),称为K562/Vp16 SP细胞,余下部分为K562/Vp16非SP细胞。为了分析白血病细胞对伊马替尼ST1571耐药的机制,分别检测K562,K562/Vp16非SP细胞或K562/Vp16 SP细胞中MDR1,Bcr-Abl和P-gp的表达。另外,按每只BALB/c-nu/nu小鼠1000个K562、K562/Vp16非SP细胞或K562/Vp16 SP细胞的数量分别对其进行腹腔注射。主要观察指标:K562/Vp16非SP细胞或K562/Vp16 SP细胞对ST1571的耐药性和致瘤性。结果:Bcr/Abl和Abl蛋白在K562细胞、K562/Vp16 SP细胞及K562/Vp16非SP细胞中的表达水平差异无显著性意义(P>0.05);P-gp在K562细胞中不表达,在K562/Vp16 SP及K562Np16非SP细胞中均高表达且表达水平一致(P>0.05);与K562/Vp16非SP细胞比较,K562/Vp16 SP细胞对伊马替尼的耐药性更强,并且这种抗性几乎不能被多种多药耐药逆转剂逆转;另外,体内外实验显示,K562/Vp16细胞的致瘤性几乎全部来源于K562/Vp16 SP细胞。结论:Bcr/Abl基因的扩增、过度表达和多药耐药基因及其蛋白表达产物P-gp的高表达,也许并不是白血病细胞产生对伊马替尼临床耐药的重要机制;白血病细胞对伊马替尼具有一定的抗性.可能与数量极少的白血病干细胞有直接的关系。因此,这类数量极少的干细胞样的肿瘤细胞应当成为有效治疗肿瘤的靶细胞。
BACKGROUND: The treatment of chronic myelogenous leukemia (CML) is revolutionized by the tyrosine kinase inhibitor imatinib mesylate (imatinib). However, resistance to imatinib is increasingly recognized as a clinical problem, the prognosis of patients who develop imatinib resistance is poor, particularly in acute transformation phase of leukemia.
OBJECTIVE : To characterize a novel CML cell line and to further elucidate the mechanisms of resistance to STI571.
DESIGN: An observational comparative experiment.
SETTING: Henan Institute of Haematology, Henan Tumor Hospita MATERIALS: Thirty female BALB/c nu/nu mice with 5 weeks old were purchased from Animal Center, Chinese Academy of Medical Sciences. STI571 was kindly provided by Novartis (Nuremberg, Germany). VP-16 was purchased from Bristol-Myers Squibb (Munich, Germany); anti-P-gp from Santa Cruz Company, USA; anti-abl from BD Biosciences Company, USA. The disposal of experimental animal was coincidence with the ethical standard.
METHODS: The experiment was performed in the Henan Institute of Haematology from September 2003 to November 2005. A novel K562 cell line (K562/VP16) was achieved after exposure of the K562 cells to VP16. A small subpopulation (SP K562/VP16) that was capable of excluding Hoechst 33342 in the K562/VP16 cell line was isolated by flowcytometry sorting. The rest of the K562/VP16 cells were classified as non-SP K562/VP16. In order to elucidate the mechanisms involved in K562/VP16 SP cells which became resistant to STI571, the expression of multidrug-resistant gene 1 (MDR1), Bcr-Abl and P-gp was detected in K562, non-SP K562/VP16, or K562NP16 SP cells, respectively. Furthermore, one thousand cells of K562, K562/VP16 SP and non-SP cells were injected, respectively, intrapontoneally into the right flanks of ten 5-week-old female BALB/c nu/nu mice. The same experiment was repeated twice.
MAIN OUTCOME MEASURES: Comparison of STI571 resistance and oncogenicity of nonoSP K562NP16 and K562NP16 SP cells.
RESULTS: The MDR-1 gene expression of the M, 170 000 P-gp was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562/VP16 cell lines were similar (P 〉 0.05). The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562/VP16 and K562NP16 SP cells (P 〉 0.05). Compared with non-SP K562/VP16, the K562/VP16 SP cells were more resistant to STI571, and this resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced oncogenicity in mice, while the K562/VP16 non-SP cells failed to do so.
CONCLUSION: Bcr/abl gene amplification and MDR1 overexpression may not be an important clinical mechanism in the diversity of resistance development to imatinib treatment, and the development of drug resistance by leukemia cells may be at least partly due to a rare SP cells which drives leukemia occurrence and maintenance. So, these SP cells need to be targeted for effective cancer therapy.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第50期10194-10199,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
河南省杰出青年基金(0612000900)~~