Notch基因在慢性淋巴细胞白血病细胞的表达和介导抗凋亡、耐药机制的研究

Expression of Notch Gene and Its Role of Anti-apoptosis and Drugresistance of Cells in Chronic Lymphocytic Leukemia

目的:检测原代慢性淋巴细胞白血病细胞Notch基因的表达情况;研究阿糖胞苷及地塞米松作用后肿瘤细胞Notch蛋白的变化,探讨Notch基因介导慢性淋巴细胞白血病抗凋亡及耐药机制。方法:采集慢性淋巴细胞白血病24例初治患者的骨髓血或外周血单个核细胞,以健康体检者14例作为对照组,采用实时荧光定量PCR(qRTPCR)检测Notch及BCL-2、NF-κB基因的转录水平;应用Western blot检测L1210细胞株在化疗药物阿糖胞苷、地塞米松作用后Notch蛋白的变化。结果:CLL组的Notch1、Notch2、BCL-2、NF-κB基因的mRNA水平表达明显高于正常对照组,分别为0.8556±0.8726和0.6731±0.5334(P=0.0182)、1.2273±0.8207和0.6577±0.6424(P﹤0.0001)、8.0960±7.5661和0.5969±0.4976(P﹤0.0001)、1.0966±0.6925和0.5373±0.7180(P﹤0.0001),而2组的Notch3和Notch4基因的mRNA水平未见明显差异,分别为1.1914±2.4219和0.8713±0.7937(P=0.3427)、0.8174±1.0869和0.9752±1.3446(P=0.2402);L1210细胞在低浓度、中浓度阿糖胞苷作用24 h后,Notch1蛋白表达明显下降,在高浓度阿糖胞苷或延长中浓度阿糖胞苷组的作用时间,Notch1蛋白表达量增高。L1210细胞在地塞米松作用后,Notch1蛋白表达下降,但不随地塞米松的浓度及作用时间的变化而变化。结论:慢性淋巴细胞白血病细胞Notch基因转录水平明显高于正常人。Notch1蛋白在阿糖胞苷和地塞米松抑制肿瘤细胞增殖过程中表达下调。Notch信号通路可能介导慢性淋巴细胞白血病细胞抗凋亡及耐药过程。

Objective : To investigate the expression of Notch gene in chronic lymphocytic leukemia cells and to explore the change of Notch protein after the therapy with cytosine arabinoside or dexmethasone,and the mechanism of Notch mediated anti-apoptosis and drug-resistance in chronic lymphocytic leukemia cells. Me thods: The mononuclear cells from bone marrowor peripheral blood of chronic lymphocytic leukemia patients( 24 cases) and healthy donors( 14cases) were collected,then the expression of Notch gene,BCL-2,as well as NF-κB gene were detected by real-time fluorescent quantitative PCR( qRT-PCR) at the level of transcription. The change of Notch protein in L1210 cell lines after therapy with cytosine arabinoside and dexmethasone was determined by Western blot. Re sults: mRNA expression levels of Notch1,Notch2,BCL-2 and NF-κB gene in CLL group were significantly higher than those in healthy control group( 0. 8556 ± 0. 8726 vs 0. 6731 ± 0. 5334,P = 0. 0182; 1. 2273 ± 0. 8207 vs 0. 6577 ± 0. 6424,P ﹤ 0. 0001; 8. 0960± 7. 5661 vs 0. 5969 ± 0. 4976,P ﹤ 0. 0001; 1. 0966 ± 0. 6925 vs 0. 5373 ± 0. 7180,P ﹤ 0. 0001,respectively),but no significant difference was found between Notch3 and Notch4 gene( 1. 1914 ± 2. 4219 vs 0. 8713 ± 0. 7937,P = 0. 3427;0. 8174 ± 1. 0869 vs 0. 9752 ± 1. 3446,P = 0. 2402,respectively). Notch1 protein expression in L1210 cells were significantly decreased after treating with cytosine arabinoside of lowand middle concentrations,but increased after treating with cytosine arabinoside of high concentration or prolonging time of cytosine arabinoside of middle concentration. Notch1 protein expression in L1210 cells dereased after treating with dexamethasone,but did not be changed with the different concentrations and different times of dexmethason. Conclusion: The transcription level of Notch gene in CLL patients significantly higher than that in normal controls. The Notch1 protein expression is down-regulated in process of inhibiting L1210 cell proliferation by Ara-C and dexmethason. Notch signaling pathway may mediated antiapoptosis and drug resistance of CLL cells. Notch molecule possibly plays an important role in the anti-apoptosis and drug-resistance of CLL cells.