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Lycorine上调p53非依赖的p21转录机制的研究
The Study on the Transcription Mechanism of Up-regulating p21 Via p53-independent Pathway by Lycorine
【作者】 王淑玲；
【导师】 刘静；
【作者基本信息】 中南大学 ， 生物化学与分子生物学， 2011， 硕士
【摘要】 白血病是严重危害人类健康的造血组织恶性疾病,目前化学治疗仍是白血病治疗的主要途径。Lycorine(石蒜碱)是从我国广泛分布的多年生草本植物石蒜鳞茎中分离出的一种单体化合物,属于异喹啉类(isoquinoline)生物碱,具有广谱的抗白血病作用。本研究以人急性早幼粒白血病细胞株HL-60和人慢性粒细胞白血病细胞株K562作为实验对象,研究lycorine在白血病细胞中上调p53非依赖的p21转录过程中相关蛋白表达的变化,以及lycorine对p21转录的靶向特异性,进一步阐明lycorine对白血病p53非依赖的p21转录的分子机制。本课题用不同浓度的lycorine作用HL-60细胞和K562细胞24h后,提取总蛋白,通过Western blotting检测lycorine作用后p53蛋白的表达变化及调控p21转录相关蛋白：E2F、Rb、c-myc、HDAC1、HDAC3的表达变化。结果发现,在p53野生型的K562细胞中,lycorine上调p21的表达不是通过上调p53来实现的,表明lycorine对p21的调控是p53非依赖的；随着lycorine药物作用浓度的逐渐增大,HL-60细胞和K562细胞中E2F和Rb蛋白表达量上调,c-myc蛋白表达量下调,而HDAC1和HDAC3蛋白表达量没有明显差异,表明Rb/E2F途径及c-myc途径参与了p21的转录调控,HDACs途径不参与p21的转录调控。另外,为检测p21靶标的特异性,以p21 Si RNA封闭p21基因的表达后,用2.5μM lycorine药物干预p21封闭前后的细胞,采用流式细胞术检测p21封闭后的细胞凋亡变化。结果发现,p21 siRNA+lycorine组细胞凋亡百分率较阴性序列+lycorine组有明显减少。综上所述,lycorine能够通过上调E2F,下调c-myc的表达来调控p53非依赖的p21的转录；阐明了p21是lycorine抗白血病的关键效应分子。
【Abstract】 Leukemia is a hematological malignancy, which is one of the greatest health challenges facing the world today. Chemotherapy is still considered one of the most effective methods or the basis of other treatments. Previous studies in our group had found lycorine activated p21 transcription in a p53-independent mechanism after treatment in leukemia. To further elucidate anti-leukemia action of lycorine, this research focused on the expression change of proteins associated with p21 transcription in p53-independent pathway in HL-60 and K562 cells and specificity of p21 transcription after treatment of lycorineAfter HL-60 and K562 cells were treated with different concentrations of lycorine for 24h, Western blotting was used to identify the effect of lycorine on the expression of E2F、Rb、c-myc、HDAC1 and HDAC3. The results showed that expression of E2F and Rb were up-regulated, c-myc was down-regulated in HL-60 and K562 cells. But the expression of HDAC1 and HDAC3 was no changed in HL-60 and K562 cells. Furthermore, RNAi was performed to interfere the expression of p21 gene, then flow cytometry was used to detect the cell apotosis rate after treated by 2.5μM lycorine. Compared to the scrambled sequence+lycorine group, the apoptosis rate in p21 siRNA+lycorine group was significantly decreased. This result suggested that lycocrine was specificly targeted to p21.