【摘要】 本研究在探讨β-联蛋白(β-catenin)序列特异的小发夹RNA(shRNA)干扰其表达后对K562细胞生长的影响。采用脂质体介导方法,将含编码β-联蛋白特异的shRNA的质粒转入K562细胞,经G418筛选提高细胞阳性率,用实时定量PCR和Western blot分别检测干扰后β-联蛋白转录水平及蛋白水平的表达变化,通过绘制生长曲线、MTT测定及集落培养等方法观察干扰后细胞生长能力的差别。结果发现:与对照组相比,转染后72小时干扰质粒可有效降低K562细胞β-联蛋白mRNA水平的表达(p<0.05),但短期培养对蛋白水平的表达未发现明显影响。经G418长期筛选后,对照组可见细胞阳性率逐渐提高,并可筛选到几近100%阳性的细胞克隆,而干扰组细胞则逐渐死亡。在G418存在下,干扰组与对照组K562细胞短期增殖曲线及MTT结果显示两组间无明显差异,但集落培养发现,干扰组细胞无论集落形成率(p<0.001)还是形成的集落大小均明显低于对照组,说明干扰质粒可影响细胞的集落形成能力。结论:β-联蛋白特异的shRNA干扰可以有效降低K562细胞中β-catenin基因的表达,降低细胞集落形成能力;K562细胞的生长依赖于β-联蛋白的存在,针对β-联蛋白的RNAi治疗或其它靶向治疗可能对CML治疗有效。更多还原

【Abstract】 In order to investigate the effect of shRNA targeted to β-catenin on the growth of K562 cells,plasmid containing β-catenin specific shRNA sequence was transfected into K562 cells by lipofectamine 2000,and G418 was added to screen the positive cells.Real-time PCR and Western blot were used to detect the expression of β-catenin.Cell growth curve,MTT and colony forming cell assays were used to evaluate the proliferation potential of cells.The results showed that the mRNA level of β-catenin was reduced significantly in K562 cells transfected into interfering plasmid as compared with control plasmid,while the protein level failed to demonstrate difference by the time of 72 hours after transfection.After long-term culture with G418,the count of positive cells enhanced in control group while no positive cells survived in the interfering group.Colony-forming cell assays revealed that the K562 cells in interfering group formed colonies with very small size and low forming rate,compared with the control group,though the growth curve and MTT failed to illustrate differences.It is concluded that the β-catenin-specific shRNA mediated by plasmid can effectively knockdown the expression of β-catenin gene and inhibit the colony-forming ability in K562 cells,it is a potential target for the therapy of CML,even in blast crisis.更多还原