转染p16、p53对白血病细胞株K562及HL-60的抑制作用  

Inhibitory Effect of p16,p53 Transfection on Leukemic Cell Lines K562 and HL-60

作　　者：陈琦[1] 沈杰芳[1] 芮红兵[1] 苏津自[2] 卓光生[1] 康日辉[1] 林珺芳[1] 

机构地区：[1]福建医科大学附属第一医院血液内科,福建福州350005 [2]福建医科大学附属第一医院心内科,福建福州350005

出　　处：《中国实验血液学杂志》2010年第2期305-310,共6页Journal of Experimental Hematology

基　　金：福建医科大学科学研究发展基金资助(编号FJGXY04026)

摘　　要：本研究构建p53,p16基因真核表达载体,用脂质体法转染入白血病细胞,观察其在白血病细胞中的表达和对白血病细胞的作用。设计并构建包含野生型p53cDNA与p16cDNA的真核双表达载体pBudCE4.1-53-16,用脂质体法转染白血病细胞,用间接免疫细胞化学法、Western blot法检测鉴定外源性p53及p16的表达情况。通过CCK-8,流式细胞仪检测细胞的生长曲线、细胞周期、细胞凋亡。结果发现:p53p16基因双表达真核载体构建成功,体外转染入白血病细胞后能检测到外源性P53、P16蛋白在白血病细胞中表达。转染48小时后,试验组与对照组比较,细胞周期阻滞(p<0.001);试验组和对照组比较,细胞增殖下降,细胞凋亡增加(p<0.001)。结论:重组质粒pBudCE4.1-53-16构建成功,经体外转染白细胞细胞后2个目的基因能够在细胞中同时表达,并引起细胞周期阻滞于G0期,细胞凋亡增加。This study was purposed to construct a vector conta^ng human suppressor gene p53 and p16, and to investigate their expression and effect on K562 and HL-60 cells, pBudCE4.1-53-16 is a vector designed for simultaneous expression of human suppressor gene p53 and p16 in mammalian cell line. After transfection into K562 cells with lipo- fectamineTM 21300, the expression of p53 and p16 genes was detected by Western blot and immunocytochemical method. The growth curve, apoptosis, cell cycle were assayed by CCK-8 and flow cytometry. The results showed that the recom- binant plasimid pBudCE4.1-53-16 was constructed successfully and were verified by PCR and restriction analysis. The expression of P53 and P16 protein could be detected after transfecton into leukemia cells （ K562 and HL-60） for 48 hours. As compared with control group, the cell proliferation in experimental group was inhibited, the cells were arrested in GO phase and apoptotic cells increased（p 〈0. 001 ）. It is concluded that the recombinant plasmid pBudCE4.1-53-16 has been established, p16 and p53 in the recombinant plasmid pBudCE4.1-53-16 synchroniously expresse in leukemic cells after transfection in vitro for 2 days and results in reduced proliferation, GO arrest and apoptosis increase.

关 键 词：P53基因 P16基因 pBudCE4.1 基因转染 凋亡 

分 类 号：R733.7[医药卫生—肿瘤] Q78[医药卫生—临床医学]