摘要
目的:探讨Ca2+-活化T细胞核因子(nuclear factors of activated T cells,NFAT)信号通路在骨髓基质细胞介导的费城染色体阳性(Ph+)急性淋巴细胞白血病(ALL)耐药中的作用。方法:聚合酶链式反应检测Sup-B15细胞及Ph+ALL原代细胞NFAT mRNA的转录水平;流式细胞术检测Sup-B15细胞P-糖蛋白表达;Western blot检测Sup-B15细胞NFAT蛋白的变化;Annexin V/7-AAD标记细胞,流式细胞术检测细胞凋亡;Fluo 3-AM染料标记细胞,流式细胞术检测共培养后白血病细胞Ca2+浓度变化。结果:Sup-B15细胞及Ph+ALL原代细胞中均可检测到NFAT表达;流式细胞术未检测到Sup-B15细胞表达P-糖蛋白;临床应用的治疗浓度(2.5和5μmol/L)的环孢素(CAS)可明显抑制NFAT蛋白表达,其中5μmol/L CAS抑制作用更明显;临床治疗浓度CAS(2.5和5μmol/L)对Sup-B15细胞的凋亡无明显影响,而较高浓度CAS(10μmol/L)可诱导Sup-B15细胞的凋亡。骨髓基质细胞OP9可使Sup-B15细胞及Ph+ALL原代细胞对伊马替尼的敏感性下降;与骨髓基质细胞OP9共培养后Sup-B15细胞Ca2+浓度升高,总NFAT蛋白水平及核蛋白水平均增加;在共培养体系中加入环孢素抑制Ca2+-NFAT信号通路,可降低OP9对Sup-B15细胞的保护作用。结论:Ca2+-NFAT信号通路有助于Ph+ALL细胞的存活,骨髓基质细胞+可通过活化Ca2+-NFAT信号通路介导Ph+ALL细胞对IM的耐药。
Objective:To explore the role of Ca2+-NFAT signaling pathway in Ph+-ALL drug resistance mediated by bone marrow stromal cells.Methods:The transcription level of NFAT mRNA in Sup-B15 cells and Ph+ALL primary cells was detected by polymerase chain reaction.The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry.The change of NFAT protein in Sup-B15 cells was detected by Western blot.AnnexinV/7-AAD was used to label cells.Flow cytometry was used to detect cell apoptosis;Fluo 3-AM dye was used to label cells,and flow cytometry used to detect changes of Ca2+concentration in leukemia cells.Results:NFAT expression could be detected in both Sup-B15 and Ph+ALL primary cells;P-glycoprotein could not be detected by flow cytometry;CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations(2.5,5μmol/L);Clinically applied concentration of CAS(2.5,5μmol/L)has no significant effect on the apoptosis of Sup-B15 cells,while higher concentration of CAS(10μmol/L)could induce apoptosis of Sup-B15 cells.Bone marrow stromal cells OP9 could,decrease the sensitivity of Sup-B15 cells and Ph+ALL primary cells to imatinib(IM);After co-culture with bone were marrow stromal cells,the Ca2+concentration in Sup-B15 cells was enhanced,the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced.The addition of CAS in co-culture system could inlibit the Ca2+-NFAT signaling pathway,reduce the protective effect of OP9 on Sup-B15 cells.Conclution:The Ca2+-NFAT sigualing pathway,contributes to the survival of Ph+ALL cells.Bone marrow stromal cells can mediate the resistance of Ph+ALL cells to IM by activating Ca2+-NFAT signaling pathway.
作者
张焕新
韩雅慧
邱婷婷
姚瑶
朱升云
牛铭山
曾令宇
李振宇
闫志凌
徐开林
ZHANG Huan-Xin;HAN Ya-Hui;QIU Ting-Ting;YAO Yao;ZHU Sheng-Yun;NIU Ming-Shan;ZENG Ling-Yu;LI Zhen-Yu;YAN Zhi-Ling;XU Kai-Lin(The First Clinical Medical College,Nanjing Medical University,Nanjing 210029,Jiangsu Province,China;Department of Hematology,The Affiliated Hospital of Xuzhou Medical University,Xuzhou 221002,Jiangsu Province,China.)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2019年第3期717-722,共6页
Journal of Experimental Hematology
基金
国家自然科学基金:(81500088,81300399)
江苏省重点研发计划(BE2015625)
江苏省自然科学基金(BK20161178)
江苏省卫生计生委科研课题(Q201506)
中国博士后科学基金(2015113010)
江苏省博士后基金(1601096B)
江苏省高校基金(16KJB320013)
关键词
Ph^+急性淋巴细胞白血病
白血病细胞耐药
活化T细胞核因子
骨髓基质细胞
Ph^+acute lymphoblastic leukemia
leukemic cell chemoresistance
nuclear factors of activated T cells(NFAT)
bone marrow stromal cells