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背景:慢性髓细胞性白血病是以造血干细胞的恶性克隆性增殖为特征。慢性期的慢性髓细胞性白血病患者如果不予以有效的治疗,必然演进为急变期,其预后往往非常差。因而,从全基因组水平上阐明慢性髓细胞性白血病的发生机制显得尤为重要,目的:应用AppliedBiosystems表达芯片系统对慢性髓细胞性白血病患者骨髓单个核细胞的基因表达谱进行观察。设计:观察对比分析。单位:南方医科大学基因工程研究所。对象:两例骨髓样品(1例慢性髓细胞性白血病患者,1例健康者)来自于广州军区广州总医院血液科。方法:实验于2004-10/2005-09在南方医科大学基因工程研究所完成。分别从一例慢性髓细胞性白血病患者与一例健康人的骨髓样品中分离单个核细胞,提取总RNA,纯化mRNA。通过逆转录体外线性扩增的方法对mRNA样品进行标记,将标记好的cRNA样品与芯片杂交。利用ABI1700化学发光芯片分析仪对骨髓单个核细胞的基因表达谱差异情况进行分析。通过比较同一样品不同芯片间的数据信息,对芯片结果的可重复性进行评估。主要观察指标:①总RNA及标记后的cRNA质量的评定。②芯片可重复性验证。③芯片杂交结果。④半定量反转录-聚合酶链反应结果。结果:①利用统计学数据分析工具,通过比较慢性髓细胞性白血病患者与健康人单个核细胞的基因表达差异,总共发现了6706个差异基因。其中,与慢性髓细胞性白血病密切相关的差异基因为68个,上调的有17个,下调的有51个。②位于C/EBPalpha信号通路和CD40受体信号通路中的大部分差异表达基因,其表达水平降低。③通过对重复实验结果的相关性及检测一致性分析,证实了芯片结果的可重复性较好。而两组重复实验间的相关系数分别是,慢性髓细胞性白血病组为0.991,健康组为0.988。④半定量反转录-聚合酶链反应验证了芯片分析结果的可靠性。结论:通过比较慢性髓细胞性白血病患者与健康人单个核细胞的基因表达谱的差异,发现了大量的差异表达基因。这些数据将为寻找可用于慢性髓细胞性白血病患者疾病治疗的分子靶标提供有用的信息。
BACKGROUND: Chronic myelogenous leukemia is characterized by malignant clonal proliferation of hematopoietic stem cells. Chronic myelogenous leukemia patients without effective treatment inevitably evolve into a blast crisis, the prognosis is often very poor. Therefore, it is important to elucidate the mechanism of chronic myelogenous leukemia at the whole genome level. Objective: To observe the gene expression profile of bone marrow mononuclear cells in patients with chronic myelogenous leukemia using AppliedBiosystems expression chip system. Design: Observation and comparative analysis. Unit: Southern Medical University Institute of Genetic Engineering. PARTICIPANTS: Two bone marrow samples (one patient with chronic myelogenous leukemia and one healthy) were from Department of Hematology, Guangzhou General Hospital of Guangzhou Military Region. Methods: The experiment was performed at Institute of Genetic Engineering, Southern Medical University from October 2004 to September 2005. Mononuclear cells were isolated from bone marrow samples of one patient with chronic myelogenous leukemia and one healthy individual, respectively. Total RNA was extracted and mRNA was purified. The mRNA samples were labeled by reverse transcriptase in vitro amplification, and the labeled cRNA samples were hybridized with the chips. The ABI1700 chemiluminescence chip analyzer was used to analyze the gene expression profiles of bone marrow mononuclear cells. The repeatability of chip results was evaluated by comparing the data of different chips in the same sample. MAIN OUTCOME MEASURES: (1) Evaluation of total RNA and labeled cRNA quality. ② chip repeatability verification. ③ chip hybridization results. ④ semi-quantitative reverse transcription - polymerase chain reaction results. Results: ①Using statistical data analysis tools, a total of 6706 differential genes were found by comparing the gene expression differences between mononuclear cells from healthy people and patients with chronic myelogenous leukemia. Among them, there are 68 genes that are closely related to chronic myelogenous leukemia, up-regulated 17 and down-regulated 51. ② The majority of differentially expressed genes located in the C / EBPalpha signaling pathway and the CD40 receptor signaling pathway were reduced in their expression levels. (3) Through the relativity of the experimental results and the analysis of the consistency of the test, it is confirmed that the repeatability of the chip result is better. The correlation coefficients between the two groups were 0.991 in chronic myeloid leukemia group and 0.988 in healthy group. ④ Semi-quantitative reverse transcription-polymerase chain reaction confirmed the reliability of the chip analysis results. CONCLUSION: A large number of differentially expressed genes were found by comparing the gene expression profiles of mononuclear cells from healthy people with chronic myelogenous leukemia. These data will provide useful information for finding molecular targets for the treatment of diseases in patients with chronic myeloid leukemia.