Effects Of Thalidomide On Fetal Hemoglobin Synthesis Bet

沙利度胺已被证明是有效的一些患者骨髓增生异常综合征（MDS），包括增加总血红蛋白和胎儿血红蛋白的比例在一些患者的临床试验。此外，已经证明沙利度胺增加胚状体中的细胞内活性氧（ROS）。反应停的作用机制？的治疗效果仍在确定中。我们假设沙利度胺诱导了？-在成人红细胞生成中的珠蛋白基因表达，这种诱导可能是由ROS形成增加介导的。为了研究这一假设，我们使用培养的原代人CD 34+祖细胞评估增加剂量的沙利度胺（0.01 μ M至100 μ M）对细胞生长、珠蛋白基因表达和ROS产生的影响。不同浓度的沙利度胺对培养的CD 34+细胞的影响表明，在100 μ M的最大沙利度胺浓度下，细胞数量显著增加。真实的实时定量PCR分析？-然后呢？珠蛋白基因表达表明，沙利度胺显着诱导？珠蛋白基因表达呈剂量依赖性。平均？/？+？100 μ M沙利度胺处理组细胞增殖百分率为12.89% ± 0.11%，而Epo处理组细胞增殖百分率为3.21% ± 0.07%（P<0.01）。有趣的是，我们发现用100 μ M沙利度胺处理48小时，从第3天到第5天，细胞内ROS水平增加。我们不能排除沙利度胺对超过第6天的ROS产生的影响，因为此时伴随的血红蛋白形成也诱导H2 DCF-DA染料产生荧光。我们还发现，通过Western blot分析，沙利度胺激活p38 MAPK信号通路的时间和剂量依赖性的方式，并增加组蛋白H4乙酰化。相反，用抗氧化酶过氧化氢酶和细胞内羟基清除剂二甲基硫脲（DMTU）处理废除了沙利度胺诱导的p38活性和组蛋白H4乙酰化。此外，过氧化氢酶和DMTU减少沙利度胺诱导的？珠蛋白基因表达这些数据表明，沙利度胺诱导？-珠蛋白基因的增加通过激活p38 MAPK信号通路以及与ROS产生相关的组蛋白修饰。

Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide?s therapeutic effect are still being defined. We hypothesize that thalidomide induce the ?-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of ?- and ?-globin gene expression demonstrated that thalidomide significantly induces ?-globin gene expression in a dose-dependent manner. The averaged ?/?+? percentage ratio was 12.89% + 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% + 0.07% in Epo alone (P<0.01). Interestingly, we found that intracellular ROS levels were increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. In contrast, treatment with anti-oxidant enzyme catalase and intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide induced p38 activity and histone H4 acetylation. Moreover, the catalase and DMTU diminished thalidomide induced ?-globin gene expression. These data indicate that thalidomide induced the ?-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.