中国医学科学院学报

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中国医学科学院学报

中国医学科学院学报 ›› 2007, Vol. 29 ›› Issue (3): 374-378.

• 论著 • 上一篇    下一篇

抗凋亡蛋白Bcl-xL在巨核细胞分化和成熟过程中的作用

张 磊,杨仁池,卢士红,刘 斌,任 贺,韩之波,韩忠朝   

  1. 中国医学科学院 北京协和医学院 血液学研究所 血液病医院 实验血液学国家重点实验室, 天津 300020
  • 收稿日期:2006-06-20 修回日期:1900-01-01 出版日期:2007-06-30 发布日期:2007-06-30
  • 通讯作者: 杨仁池

Role of Antiapoptotic Bcl-xL in Megakaryocyte Differentiation and Maturation

ZHANG Lei, YANG Ren-chi, LU Shi-hong, LIU Bin, REN He, HAN Zhi-bo, HAN Zhong-chao   

  1. State Key Laboratory of Experimental Hematology, Hospital of Blood Disease, Institute of Hematology, CAMS and PUMC, Tianjin 300020, China
  • Received:2006-06-20 Revised:1900-01-01 Online:2007-06-30 Published:2007-06-30
  • Contact: YANG Ren-chi

摘要: 摘要:目的 研究抗凋亡蛋白Bcl-xL在巨核细胞分化和成熟过程中的作用。方法 采用PDBu诱导K562细胞向巨核细胞分化,RNA干扰阻断Bcl-xL在K562细胞向巨核细胞诱导分化中的表达,RT-PCR和流式细胞仪技术检测其改变。采用免疫磁珠从正常骨髓中富集CD34+细胞,在无血清培养下用TPO诱导其向巨核细胞分化,免疫组织化学和流式细胞仪技术观察分化过程中Bcl-xL的表达改变。结果 PDBu诱导K562细胞向巨核细胞分化24h后,CD61+细胞百分比迅速增加,并且在72h内维持较高的阳性率;用siBcl-xL干扰后72h内,CD61+细胞百分比只有轻度增加,同时RT-PCR检测显示24h后Bcl-xL mRNA表达显著减少,流式细胞检测显示Bcl-xL蛋白的表达也相应降低。正常骨髓中CD34+细胞经TPO诱导后,在培养5~20d间Bcl-xL蛋白表达阴性的巨核细胞随着培养时间延长逐渐增多,免疫组织化学检测显示未成熟的巨核细胞Bcl-xL蛋白表达呈强阳性,而在成熟的巨核细胞中表达为阴性。结论 抗凋亡蛋白Bcl-xL在巨核细胞分化过程中发挥重要作用,而在巨核细胞发育晚期Bcl-xL蛋白的表达下调可能是巨核细胞成熟的关键,并与成熟巨核细胞的特殊凋亡发生密切相关。

关键词: 巨核细胞, 凋亡, Bcl-xL, 干扰RNA

Abstract: ABSTRACT:Objective To investigate the role of antiapoptotic Bcl-xL protein in megakaryocyte differentiation and maturation. Methods RNA interference was used to block the expression of Bcl-xL when K562 cells were induced to differentiate into megakaryocyte( CD61+ cells) by PDBu, and the expression of Bcl-xL was evaluated with flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). The CD34+ cell fraction was positively isolated by using the MiniMACS system from normal bone marrow. Immunochemical staining and flow cytometry were used to detect the expression of Bcl-xL in the differentiation (CD41+ cells) of CD34+ cells induced by trombopoietin (TPO). Results Among K562 cells induced by PDBu, the percentage of CD61+ cells rapidly increased in 24 hours and maintained at a high positive level in 72 hours. When exposured to si-Bcl-xL, the percentage of CD61+ cells only slightly increased in 72 hours. The expression of Bcl-xL mRNA was significantly decreased after transfection compared with that of control group, and Bcl-xL protein expression decreased correspondingly. After the CD34+ bone marrow cells having been treated with TPO for 5 days to 20 days, the Bcl-xL-megakaryocytes increased as the culture time prolonged, and there was a strong expression of Bcl-xL in immature megakaryocyte and an obviously decreased expression in degenerating mature megakaryocyte. Conclusions Increased expression of antiapoptotic Bcl-xL may be essential to megakaryocytes maturation. The down-regulation of antiapoptotic Bcl-xL in mature megakaryocyte may be crucial to platelets formation.

Key words: mecakaryocyte, apoptosis, bcl-xl, sirna