甲基化抑制剂5-aza-2′-deoxycytidine对K562细胞SHP-1基因表达及细胞增殖与凋亡的影响  

Effect of A Methylation Inhibitor 5-aza-2'-deoxycytidine on SHP-1 Gene Expression,Proliferation and Apoptosis in K562 Cells

作　　者：罗建民[1] 李燕[2] 杨琳[1] 刘小军[1] 温树鹏[1] 王福旭[1] 张敬宇[1] 张学军[1] 董作仁[1] 

机构地区：[1]河北医科大学第二医院血液科,河北省血液病重点实验室,河北石家庄050000 [2]河北省人民医院血液科,河北石家庄050000

出　　处：《中国实验血液学杂志》2009年第2期309-314,共6页Journal of Experimental Hematology

基　　金：河北省自然科学基金资助项目(编号C2005000744);河北省科技厅指导项目(编号052761615)

摘　　要：本研究探讨5-氮杂胞苷(5-aza-2′-deoxycytidine,5-aza-CdR)对K562细胞中抑癌基因SHP-1的转录调控作用及对K562细胞增殖凋亡的生物学影响,为寻找肿瘤治疗新靶点提供实验依据。采用MSP方法检测SHP-1基因甲基化状态,MTT法检测不同剂量(0.5,1,2μmol/L)5-aza-CdR作用K562细胞1,3,5天时对K562细胞存活率的影响,流式细胞术(FCM)分析5-aza-CdR作用1,3,5天时细胞周期及凋亡率的变化,实时定量PCR(FQ-PCR)和Westernblot方法检测5-aza-CdR处理前后SHP-1mRNA及蛋白表达水平、p-STAT5蛋白表达变化。结果表明,K562细胞中存在SHP-1基因的甲基化,5-aza-CdR作用使K562细胞中SHP-1mRNA和蛋白重新表达,而p-JAK2蛋白表达随之呈下降趋势;5-aza-CdR明显抑制K562细胞增殖,并与药物浓度及作用时间呈正相关。JAK2特异性抑制剂AG490明显抑制K562细胞增殖;5-aza-CdR可使K562细胞凋亡率增加,且作用也呈时间依赖性,用药1,3,5天细胞凋亡率分别为9.3%,24.2%,37.7%。2μmol/L的5-aza-CdR处理24小时后,G0/G1期细胞逐渐增多,G2/M期细胞逐渐减少,细胞阻滞在G0/G1期。5-aza-CdR作用1,3,5天时G2/M期细胞比例分别为30.7%,23.4%,19.3%。结论:特异性甲基化转移酶抑制剂5-aza-CdR能使K562细胞中沉默的SHP-1基因重新表达,并可能通过JAK/STAT路径活化抑制白血病细胞增殖并诱导其凋亡。The aim of this study was to investigate the regulation of 5-aza-CdR on transcription of SHP-1 gene and effects on the proliferation and apoptosis of K562 ceils. Methylation-specific PCR（MSP） was used to detect CpG island methylation in SHP-1 promoter. MTT and flow cytometry were used to detect the growth and apoptosis of K562 cells after treatment with different comcentration of 5-aza-CdR. The expressions of SHP-1 mRNA and protein were determined by FQ-PCR and Western blot. The expression of p-JAK2 was assayed by Western blot. The result showed that methylation of SHP-1 gene promoter was detected in K562 cells, and the SHP-1 mRNA and protein were expressed again in K562 cells after treatment with 5-aza-CdR, meanwhile the expression of phosphorylated P-JAK2 was down-regulated; 5-aza-CdR significantly inhibited the cell growth in dose and time dependent manners. AG490 inhibited the cell proliferation. 5-aza-CdR increased the apoptosis rate of K562 cells also in dose- and time-depedent manners. The apoptosis rates of I（562 cells treated with 5-aza-CdR for 1, 3 and 5 days were 9.3%, 24.2% and 37.7% respectively. After treatment with 2μmol/L 5-aza-CdR for 24 hours, cells in G0/G1 phase increased gradually, cells in G2/M phase decreased gradually, cells were arrested in G0/G1 phase. The cell ratios in G2/M phase at 1, 3 and 5 days after treatment with 5-aza-CdR were 30.7%, 23.45 and 19.3% respectively. It is concluded that the 5-aza-CdR, inhibitor of specific methylation transferase, can re-express the silent SHP-1 gene in K.562 ceils, inhibits the proliferation of leukemia cells and induces cell apoptosis by activating JAK/STAT pathway.

关 键 词：甲基化抑制剂 5-氮杂脱氧胞苷 K562细胞 SHP-1基因 白血病 细胞增殖 

分 类 号：R733.71[医药卫生—肿瘤] R979.1[医药卫生—临床医学]