摘要
目的探讨外源性Notch2胞内段基因过表达对慢性粒细胞白血病(Chronic myeloid leukemia,CML)细胞株K562增殖的影响及其机制。方法将携带Notch2胞内段(ICN2)基因的质粒转染K562细胞,MTT法检测细胞的增殖;流式细胞仪检测细胞的细胞周期分布;RT-PCR检测Notch2基因全长mRNA的转录,Western blot检测Notch2蛋白的表达;RT-PCR检测Notch通路下游靶基因Hes1、Hey1及细胞增殖、凋亡相关基因numb、Bcl-2、NF-κB和TGF-β1的mRNA转录水平。结果与未转染组相比,转染组K562细胞数量减少,增殖显著受抑(P<0.01);转染48 h后的K562细胞G1期细胞比例显著增多(P<0.01),S期细胞比例显著减少(P<0.01);Notch2基因mRNA及下游靶基因Hes1和Hey1 mRNA转录水平均明显增强(P<0.01),Notch2蛋白表达量增加(P<0.01);numb基因mRNA转录水平无变化;Bcl-2表达下调,NF-κB和TGF-β1表达上调。结论Notch2胞内段基因过表达可抑制K562细胞增殖,其机制可能是通过上调TGF-β1和NF-κB基因表达及下调Bcl-2基因表达,将细胞阻滞于G1期来实现的。
Objective To investigate the effect of overexpression of intracellular domain of Notch2 gene on proliferation of chronic myeloid leukemia （CML） K562 ceils as well as the relevant mechanism. Methods K562 ceils were transfected with plasmid carrying the intracellular domain of Notch2 gene （ICN2）, then determined for proliferation level by MTT method, and for cell cycle distribution by flow cytometry. The transcription of full-length mRNA of Notch2 gene was determined by RT-PCR, and the expression of Notch2 protein by Western blot. The transcription levels of mRNAs of downstream target genes Hesl and Heyl as well as cell proliferation and apoptosis-associated numb, Bcl-2, NF-KB and TGF-β1 were determined by RT-PCR. Results Compared with those of untransfected K562 cells, the count of transfected cells decreased, while the proliferation was inhibited significantly （P 〈 0. 01 ） ; the percentage of K562 cells at G, stage 48 h after transfection increased （P 〈 0. O1 ）, while that at S stage decreased （P 〈 O. 01 ） ; the transcription levels of Notch2, Hesl and Heyl mRNAs （P 〈 0. 01 ） as well as expression level of Notch2 protein （P 〈 0. 01） increased significantly, while the transcription level of numb mRNA showed no significant change; the expression of Bcl-2 was downregulated, while those of NF-KB and TGF-131 were up-regulated. Conclusion The overexpression of intracellular domain of Notch2 gene inhibited the proliferation of K562 cells by a potential mechanism of up-regulating the expressions of TGF-β1 and NF-kB genes, down-regulating that of Bcl-2 gene, and arresting the cells at G1 stage.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第11期1278-1281,1289,共5页
Chinese Journal of Biologicals
基金
重庆市自然科学基金(CSTC
2009BB5401)
