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目的研究苦参碱诱导人红白血病K562细胞分化的分子机制。方法应用改良的mRNA差异显示逆转技术(DDRT-PCR)筛选苦参碱诱导K562细胞分化相关的差异表达基因;对差异cDNA片段纯化、克隆、测序及Blast分析,采用逆转录-聚合酶链式反应(RT-PCR)方法验证。进一步对其中的未知差异cDNA片段采用North-ern杂交确定其转录本的大小及组织分布特性,进而充分利用多种生物信息学资源对差异片段进行结构和功能的预测。结果苦参碱诱导K562细胞前后存在明显的基因表达差异,筛选获得了17条差异条带。其中4个差异显著片段经RT-PCR验证后,在Genbank中分析,有3个为已知基因,1个可能为未知基因,暂命名为KH基因。Northern杂交证实KH基因转录本的大小为1.35kb,分布于正常人体脑组织中。结论苦参碱具有诱导K562细胞基因表达谱发生变化的作用,由苦参碱诱导的K562细胞分化是一个多基因参与的过程;KH基因可能是苦参碱作用K562细胞后表达量发生改变的未知基因,可能与细胞癌变有关。
Objective To study the molecular mechanism of matrine-induced differentiation of human erythroleukemia K562 cells. Methods The differentially expressed genes related to matrine-induced K562 cell differentiation were screened by DDRT-PCR. The cDNA fragments were purified, cloned, sequenced and Blast analyzed by reverse transcription-polymerase chain reaction (RT-PCR) method validation. The unknown cDNA fragments were further identified by North-ern hybridization to determine the size and tissue distribution of the transcripts, and the full utilization of multiple bioinformatics resources to predict the structure and function of different fragments. Results Matrine induced a significant difference in gene expression before and after K562 cells, and 17 differential bands were screened. Among them, 4 significant fragments were verified by RT-PCR and analyzed in Genbank. There were 3 known genes and 1 unknown gene, which were named as KH gene temporarily. Northern blot confirmed that KH gene transcript size of 1.35kb, distributed in normal human brain tissue. Conclusion Matrine can induce the change of gene expression profile of K562 cells. Matrine-induced differentiation of K562 cells is a multi-gene involved process. KH gene may be unknown after matrine-induced K562 cells change Genes may be related to cancerous cells.