单启动子双表达载体pIRES-p14ARF-p53的构建及对骨肉瘤细胞增殖的抑制(英文)

Construction of single promoter double expression vector pIRES-p14ARF-p53 and its inhibitory effect on proliferation of osteogenic sarcoma cells

背景:以往的研究表明,ADp14ARF转染p53阳性的肿瘤细胞系,可以发现明显的细胞增殖受阻现象。而转染p53阴性的肿瘤细胞系,尽管也可见肿瘤细胞增殖受阻,但在程度上明显轻于前者。同时转染p14ARF和p53两种基因,既增强p53表达又加强p53积累,能否更易促进肿瘤细胞凋亡?目的:利用基因工程技术构建双质粒表达载体pIRES-p14ARF-p53,并观察其对骨肉瘤细胞增殖生长的抑制作用。设计:随机对照观察。单位:华中科技大学同济医学院附属协和医院骨科。材料:实验于2005-01/2006-10于华中科技大学同济医学院基础医学院免疫教研室公共实验平台完成。人骨肉瘤MG-63细胞有华中科技大学同济医学院免疫教研室细胞室提供。含p53全长基因序列的质粒pIRES-p53和pIRES载体均购自武汉晶赛生物公司。方法:利用基因工程技术,将从培养的正常人肝细胞系L02细胞中扩增出的p14cDNA(0.5kb)亚克隆至pIRES载体中,通过PCR、限制性内切酶酶切鉴定重组质粒pIRES-p14ARF-p53。通过脂质体介导转染入骨肉瘤MG-63细胞中,并筛选出阳性克隆,将细胞分为3组:空白对照组(MG-63细胞),空载体对照组(稳定转染pIRES-neo细胞),p14ARF-p53组(稳定转染pIRES-p14ARF-p53细胞)。①采用流式细胞仪测定转染前后瘤细胞DNA含量和细胞周期。②逆转录PCR(RT-PCR)和Westernbolt对稳定转染后的瘤细胞p53、p14ARF蛋白的表达进行定性和半定量检测。③采用噻唑蓝比色法与细胞生长曲线观察细胞增殖情况。主要观察指标:①骨肉瘤细胞DNA含量和细胞周期。②瘤细胞p53、p14ARF蛋白的表达。③细胞增殖情况。结果:成功构建出双质粒表达载体pIRES-p14ARF-p53。①骨肉瘤细胞DNA含量和细胞周期:流式细胞仪检测发现转染后的瘤细胞多停滞于G1期。②蛋白表达检测结果:RT-PCR与Westernblot检测证实p14ARF、p53基因在靶细胞mRNA和蛋白水平分别有独立表达。③细胞生长情况:转染MG-63后24,48,72,96h瘤细胞生长抑制率分别为33.43%、69.37%、66.19%、75.26%,与空载体对照组差异显著(P<0.01)。结论:野生型p53和p14ARF可协同抑制骨肉瘤细胞的增殖促进瘤细胞的凋亡。

BACKGROUND： Previous researches indicate that ADp14ARF transfecting positive tumor cell line of p53 can inhibit the proliferation; in addition, the inhibitory effect is superior to transfection negative tumor cell line of p53. Whether simultaneous transfection of p14ARF and p53 genes can increase expression and accumulation of p53 and accelerate apoptosis of tumor cells needs further studies.OBJECTIVE： To construct double plasmid expression vector pIRES-p14ARF-p53 by using gene engineering so as to observe the inhibitory effect on proliferation of osteogenic sarcoma cells.DESIGN ： Randomized controlled observation.SETTING： Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology,MATERIALS： The experiment was carried out in the Public Laboratory Platform, Immune Researching Room, Basic Medical College, Tongji Medical College, Huazhong University of Science and Technology from January 2005 to October 2006. Human osteogenic sarcoma MG-63 cells were provided by Cell Laboratory, Immune Researching Room, Tongji Medical College, Huazhong University of Science and Technology. pIRES-p53 plasmid and pIRES vector containing p53 total-length gene order were provided by Wuhan Jingsai Biology Company.METHODS ： Based on gene engineering, p14DNA （0.5 kb） was amplified from cultured L02 cells of normal human hepatic cells into pIRES vector. Recombinant plasmid pIRES-p14ARF-p53 was determined with polymerase chain reaction （PCR） and restriction enzyme and transfected into human osteogenic sarcoma MG-63 cells through mediation of liposome to screen positive clones. Otherwise, cells were divided into throe groups, including blank control group （MG-63 cells）, blank vector control group （stably transfecting pIRES-neo cells） and p14ARF-p53 group （stably transfecting pIRES-p14ARF-p53 cells）. （1） DNA content and cycle of tumor cells were measured by using flow cytometry before and after transfection. （2） Reverse transcription polymerase chain reaction （RT-PCR） and Western blot were used to detect quantitative and semi-quantitative expression of p53 and p14ARF protein in tumor cells after stable transfection. （3） Thiazole blue chromatometry and growth curve were used to observe proliferation. MATN OUTCOME MEASURES： （1） DNA content and cycle of osteogenic sarcoma cells; （2）expressions of p53 and p14ARF protein in tumor cells; （3） proliferation.RESULTS： Double plasmid expression vector pIRES-p14ARF-p53 was constructed successfully. （1） DNA content and cycle of osteogenic sarcoma cells： Flow cytometry demonstrated that tumor cells mainly stayed in G1 phase after transfection. （2） Protein expression： RT-PCR and Western blot indicated that p14ARF and p53 gene independently expressed in target cell mRNA and protein, respectively. （3） Cell growth： At 24, 48, 72 and 96 hours after MG-63 transfection, inhibitory rates of tumor cells were 33.43%, 69.37%, 66.19% and 75.26%, respectively, which was significant difference as compared with blank vector control group （P 〈 0.01）.