摘要
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 explore the molecular mechanism of differentiation of human erythro-leukemia K562 cells induced by matrine. Methods The improved mRNA differential display reverse technique-polymerase chain reaction (DDRT-PCR) was used to screen the differentially expressed genes related to K562 cells between before and after being treated with matrine. The differentially expressed cDNA fragments were purified, cloned, sequenced, and Blast analyzed. RT-PCR was used to examine the expression levels of the differentially expressed cDNA. Northern blot was adopted to confirm the transcript length and distribution of the cDNA fragments with unknown differentiation, then their structures and functions were analyzed by comparing their base sequences with multiple public bioinformatic resources. Results Significant difference in gene expression was observed in K562 cells before and after being treated with matrine. Seventeen bands of differential genes were isolated, cloned, and sequenced. Four of them were analyzed in Genbank and confirmed by RT-PCR. The Blast result showed that three of them were the known genes, while one of them may be an unknown gene named as KH gene temporally. The Northern blot result showed that KH gene distributed in normal human brain tissue, whose transcript size was 1.35 kb. Conclusion Matrine plays an important role in regulating the differentiation-related gene expression. And K562 cell differentiation induced by matrine is a complex process in which many genes are involved. The KH gene may be a differentiation-related unknown candidate gene with changes of expression amount in K562 cells being treated with matrine that probably relates to cell canceration.
出处
《中草药》
CAS
CSCD
北大核心
2005年第2期224-228,共5页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(39670881
30171150)
关键词
苦参碱
K562细胞
诱导分化
差异表达基因
matrine
K562 cells
induced differentiation
differentially expressed genes
