中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (6): 1062-1066.doi: 10.3969/j.issn.1673-8225.2012.06.025

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

组蛋白去乙酰化酶抑制剂SAHA诱导骨髓瘤细胞的凋亡★　

于  涛，张  鹏，鹿全意，刘银梅   

  1. 厦门大学附属中山医院血液科，福建省厦门市 361004
  • 收稿日期:2011-08-16 修回日期:2011-10-21 出版日期:2012-02-05 发布日期:2012-02-05
  • 通讯作者: 张鹏，主任医师，厦门大学附属中山医院血液科，福建省厦门市 361004 xm2981@163.com
  • 作者简介:于涛★，男，1983年生，湖南省安化县人，汉族，厦门大学在读硕士，主要从事血液病学方面的研究。 mdmqmdmq@163.com

Novel histone deacetylase inhibitor SAHA induces the apoptosis of myeloma cells in vitro 

Yu Tao, Zhang Peng, Lu Quan-yi, Liu Yin-mei     

  1. Department of Hematology, Xiamen University Zhongshan Hospital, Xiamen 361004, Fujian Province, China
  • Received:2011-08-16 Revised:2011-10-21 Online:2012-02-05 Published:2012-02-05
  • Contact: Zhang Peng, Chief physician, Department of Hematology, Xiamen University Zhongshan Hospital, Xiamen 361004, Fujian Province, China xm2981@163.com
  • About author:Yu Tao★, Studying for master’s degree, Department of Hematology, Xiamen University Zhongshan Hospital, Xiamen 361004, Fujian Province, China mdmqmdmq@163.com

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摘要：

背景：SAHA是一种新型的组蛋白去乙酰化酶抑制剂，目前有关其对多发性骨髓瘤细胞作用的研究还少见报道，而且其诱导细胞凋亡的分子机制还不十分清楚。
目的：观察SAHA对多发性骨髓瘤细胞株U266细胞增殖和凋亡的影响，并分析其可能机制。
方法：采用锥虫蓝拒染法、四氮唑蓝比色法检测SAHA 对U266细胞增殖的影响。AllllexinV和PI染色后应用流式细胞仪检测SAHA 作用U266细胞的凋亡率，Hoechst33342染色法检测凋亡细胞的形态。Western-blot方法检测信号转导通路Ras/Raf/Mek/Erk相关蛋白的表达水平。
结果与结论：锥虫蓝拒染法和四氮唑蓝比色法均显示，SAHA可明显抑制U266细胞增殖，且具有时间剂量依赖性。0.5，2，4 μmol/L SAHA作用U266细胞48 h后，经流式细胞仪检测细胞凋亡率分别为 (17.61±1.30)%，(43.13±3.80)%和(74.01±4.39)%，呈剂量依赖性(P < 0.05)。Hoechst33342染色荧光显微镜下可见，SAHA组细胞胞核出现明显的核固缩、核碎裂，而对照组改变不明显。Western-blot结果显示U266细胞经SAHA处理后，Raf-1和Erk蛋白的磷酸化水平受到明显抑制，药物作用48 h时出现显著降低。提示SAHA抑制多发性骨髓瘤细胞株U266细胞增殖并诱导凋亡，信号转导通路Ras/Raf/Mek/Erk阻断是机制之一。

关键词: 组蛋白去乙酰化酶抑制剂, 多发性骨髓瘤, SAHA, 凋亡, 细胞外调节蛋白激酶

Abstract:

BACKGROUND: SAHA is a new kind of histone deacetylase inhibitors (HDACI), the research about the effect of HDACI on myemola cells is rare, and the molecular mechanism of its induction of apoptosis is still poorly understood. 
OBJECTIVE: To investigate the effects of SAHA on proliferation and apoptosis of multiple myeloma cell line U266 cells in vitro and its possible mechanism.
METHODS: The proliferation of U266 cells was measured by trypan blue exclusion method and colorimetric tetrazolium; the AlllexinV and PI staining was used to detect the apoptosis rates of U266 cells by flow cytometry, and the apoptotic morphological change was observed with Hoechst33342 staining; the expression of cell signaling proteins Ras/Raf /Mek /Erk were detected by Western-blot analysis.
RESULTS AND CONCLUSION: The trypan blue exclusion method and colorimetric tetrazolium showed that SAHA could inhibit the proliferation rate of U266 cells and in a time-dose dependent manner. After treated with 0.5, 2 and 4 μmol/L SAHA for 48 hours, the apoptosis of U266 cells were (17.61±1.30)%, (41.13±3.80)% and (74.01±4.39)% respectively detected by flow cytometry, and in a dose dependent manner (P < 0.05). Distinct morphology changes between SAHA-treated group and control group were observed through fluorescence microscope by Hoechst 33342 staining; cell apoptosis such as karyopyknosis and nuclear fragmentation were significant in SAHA-treated group while in control group such changes were not obvious. The Western blot analysis showed that the phosphorylation of Raf-1 and its downstream ERK kinases were inhibited obviously after treated with SAHA and remarkable down-regulated when treated with the agent for 48 hours. SAHA can inhibit the proliferation of multiple myeloma cell line U266 cells and induce their apoptosis, the intercept of the signal pathway Ras/Raf /Mek /Erk is one of the underlying mechanism.

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