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目的克隆和分离儿童急性淋巴细胞白血病(ALL)差异表达基因,探讨小儿白血病的发病机制。方法 Ficoll密度梯度离心法分离单个核细胞,抽取mRNA,逆转录成cDNA;经RsaⅠ酶切,实验组cD-NA被分成两组,分别与两种不同的接头连接后,与对照组cDNA进行两次消减杂交和两次抑制性聚合酶链反应(PCR),提取、纯化PCR产物,与PT-Adv载体连接构建cDNA消减文库,利用PCR扩增、测序和同源性分析差异表达的序列。结果选取文库中60个克隆进行菌落PCR分析,可以检出大小为200～600bp的插入片段。从中随机挑选20个克隆进行测序、生物信息学分析,其中18个克隆与已知基因有高度序列同源性,符合率为98%～100%,共编码17种基因。这些基因大多涉及基因表达调控、DNA损伤修复、细胞周期、物质代谢等,均与细胞增殖、分化有关。此外发现2个新基因。结论本实验表明我们已成功构建小儿ALL相关消减cDNA文库,为小儿ALL发病机制的研究奠定了基础。
Objective To clone and isolate differentially expressed genes in children with acute lymphoblastic leukemia (ALL) and to explore the pathogenesis of childhood leukemia. Methods The mononuclear cells were isolated by Ficoll density gradient centrifugation, mRNA was extracted and reverse transcribed into cDNA. After digestion with Rsa Ⅰ, the cD-NA of experimental group was divided into two groups. After being ligated with two different linkers, Sub-subtractive hybridization and two inhibitory polymerase chain reaction (PCR) were used to extract and purify the PCR products. The cDNA subtractive library was constructed by ligating with PT-Adv vector. The differentially expressed sequences were analyzed by PCR amplification, sequencing and homology analysis. Results Sixty colonies from the library were selected for colony PCR analysis, and the size of 200-600bp insertions could be detected. Twenty randomly selected clones were sequenced and analyzed by bioinformatics. Among them, 18 clones had high sequence homology with known genes, with the coincidence rate of 98% -100%, encoding a total of 17 genes. Most of these genes involved in gene expression and regulation, DNA damage repair, cell cycle, material metabolism, are related to cell proliferation and differentiation. In addition, two new genes were found. Conclusion This experiment shows that we have successfully constructed pediatric ALL-related subtractive cDNA library, which laid the foundation for the pathogenesis of ALL in children.