抗凋亡蛋白Bcl-x_L在巨核细胞分化和成熟过程中的作用

Role of Antiapoptotic Bcl-x_L in Megakaryocyte Differentiation and Maturation

目的 研究抗凋亡蛋白Bcl-xL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-xL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-xL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-xL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-xLmRNA表达显著减少，流式细胞检测显示Bcl-xL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-xL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-xL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-xL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-xL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。

Objective To entiation and maturation. Methods investigate the role of antiapoptotic Bcl-xL RNA interference was used to block the protein in megakaryocyte differexpression of Bcl-xL when K562 cells were induced to differentiate into megakaryocyte （ CD61 ＋ cells） by PDBu, and the expression of Bcl-xL 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-xL 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-XL, the percentage of CD61 ＋ cells only slightly increased in 72 hours. The expression of Bcl-xL mRNA was significantly decreased after transfection compared with that of control group, and Bcl-xL protein expression decreased correspondingly. After the CD34 ＋ bone marrow cells having been treated with TPO for 5 days to 20 days, the Bcl-xL-megakaryocytes increased as the culture time prolonged, and there was a strong expression of Bcl-xL in immature megakaryocyte and an obviously decreased expression in degenerating mature megakaryocyte. Conclusions Increased expression of antiapoptotic Bcl-xL may be essential to megakaryocytes maturation. The down-regulation of antiapoptotic Bcl-xL in mature megakaryocyte may be crucial to platelets formation.