文档介绍：4共表达dll4,p16基因真核表达载体构建及对白血病细胞K562的影响摘要目的:-dll4-16,并将构建的真核双表达载体通过Lipofectamine 2000转染慢性粒细胞白血病细胞株K562,观察目的基因在白血病细胞株K562中的表达情况,进而研究其对白血病细胞增殖情况的抑制作用,为进一步研究多种基因同时用于肿瘤治疗奠定一定基础。方法:-dll4-16,该载体包含野生型dll4cDNA 、p16cDNA基因,-dll4、-16。通过脂质体法将各组质粒转染入白血病细胞K562中,分为三阶段:一是转染48小时后,收集各组细胞,用RIPA裂解细胞,免疫印迹法检测外源性目的基因瞬时转染的表达水平;二是通过Cell Counting Kit-K-8),描绘各试验组细胞的生长曲线;三是转染48小时后,经70%预冷乙醇固定,PI染色后,通过流式细胞仪检测各试验组细胞的周期分布情况。结果:-dll4 、--dll4-16,通过限制性核酸内切酶酶切方法鉴定插入基因的位点与预期相符。通过脂质体法将各组质粒成功转染入白血病细胞株K562中,转染后48小时,采用免疫印迹技术检测到野生型dll4、p16基因在白血病细胞K562中的表达水平:实验组(-dll4-16转染组)和对照组1(-16转染组)细胞均表达P16蛋白,其余三组均不表达;实验组(-dll4-16转染组)及对照组2(-dll4转染组)细胞均高度表达dll4蛋白,其余三组均中度表达。转染48小时后,通过PI染色处理,经流式细胞仪检测各组细胞的细胞周期分布情况,相比较于对照组,实验组细胞G0/G1期增多,差异有显著统计学意义(P<)。细胞生长曲线显示,细胞生长受到明显抑制,尤其是实验组细胞。结论:-dll4-16能在白血病细胞K562中共同表达目的基因,诱导细胞周期停滞于G0/G1期,抑制细胞生长,但不诱导细胞凋亡。关键词: dll4基因 p16基因 质粒构建基因转染共表达周期 5 Construction of binant Plasmid -dll4-16 and its Effect on Leukemic Cells K562 Abstract Objective:This article constructs a vector containing human suppressor gene dll4 and p16 INK4a , uses the Lipofectamine 2000 to dry the dll4 and p16INK4a into the Leukemic cell line K562,and observing its expression and function in Leukemic further study many kinds of genes to use in the tumor treatment laying the foundation. Methods:binant plasmid -dll4-16 was designed and constructed for simultaneous expression of human suppressor genes dll4 and p16 INK4a in mammalian cell line. After transfected into Leukemic cells K562 with lipfectamine TM2000,divides into three stages: First, the extension dyes 48 hours later, collects each group of cells, poses the cells with RIPA, the expression of dll4 and p16INK4agenes was detected by Western-immunobltting ; Second, The cell proliferation was assessed K-8; Third, the changes in cell cycle was eva