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目的采用费城染色体阳性急性淋巴细胞白血病(Ph~+ALL)伊马替尼耐药细胞株SUP-B15/R研究伊马替尼耐药的可能机制。方法通过基因芯片分析法对比找出伊马替尼耐药株SUP-B15/R与敏感株SUPB15/S之间表达差异的基因,筛选出可能与耐药相关的基因SLC2A5,分别采用实时荧光定量PCR(qPCR)和Western blot进一步验证SLC2A5及其编码蛋白葡萄糖转运体5(Glut5)在SUP-B15/R与SUP-B15/S之间表达的差异。采用MTT实验检测果糖对SUP-B15/S细胞伊马替尼敏感性的影响,以及qPCR检测相关信号通路的改变,探究Glut5表达增加在SUP-B15细胞伊马替尼耐药中的作用。结果基因芯片结果发现,与细胞代谢相关的SLC2A5基因在SUP-B15/R中高表达,qPCR和Western blot实验进一步验证了上述结果。而果糖处理后SUPB15/S细胞对伊马替尼的敏感性下降,IC50由(44.50±2.38)μmol/L增加到(64.71±1.69)μmol/L,同时Glut5、PI3 K、AKT mRNA表达增强。结论 SUP-B15/R细胞高表达SLC2A5,Glut5高表达促进细胞对果糖的吸收,激活伊马替尼作用下受到抑制的PI3K/AKT信号通路,导致SUP-B15细胞对伊马替尼耐药。
Objective To investigate the possible mechanism of imatinib resistance by using imatinib-resistant cell line SUP-B15 / R in Philadelphia-positive acute lymphoblastic leukemia (Ph ~ + ALL). Methods By gene chip analysis, the gene expression difference between SUP-B15 / R and SUPB15 / S was detected. The gene SLC2A5, which may be related to resistance, was screened by real-time fluorescence quantitative PCR (qPCR) and Western blot to further verify the difference of SLC2A5 and its encoded protein glucose transporter 5 (Glut5) expression between SUP-B15 / R and SUP-B15 / S. MTT assay was used to detect the effect of fructose on the sensitivity of imatinib to SUP-B15 / S cells and the changes of qPCR-related signal pathways, so as to explore the role of Glut5 in increasing imatinib resistance in SUP-B15 cells. Results Gene microarray results showed that SLC2A5 gene related to cell metabolism was highly expressed in SUP-B15 / R, and qPCR and Western blot experiments further verified the above results. After fructose treatment, the sensitivity of SUPB15 / S cells to imatinib decreased. The IC50 increased from (44.50 ± 2.38) μmol / L to (64.71 ± 1.69) μmol / L, while the expression of Glut5, PI3 K and AKT mRNA increased. CONCLUSIONS: SLC2A5 is overexpressed in SUP-B15 / R cells, and Glut5 overexpression promotes the uptake of fructose by cells and activates the PI3K / AKT signaling pathway inhibited by imatinib, resulting in the resistance of SUP-B15 cells to imatinib.