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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 studies have shown that ADp14ARF transfection of p53-positive tumor cell lines, we can find obvious cell proliferation blocked phenomenon. Transfection of p53-negative tumor cell lines, although also showed inhibition of tumor cell proliferation, but to a lesser extent the former. Transfection of p14ARF and p53 at the same time can increase the expression of p53 and enhance the accumulation of p53, which can further promote the apoptosis of tumor cells. OBJECTIVE: To construct a double plasmid expression vector pIRES-p14ARF-p53 by gene engineering and observe its effect on flesh and blood Inhibition of tumor cell proliferation and growth. Design: Randomized controlled observation. SETTING: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Experiments were performed on public experimental platform of Immunology Department of School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology from January 2005 to October 2006. Human osteosarcoma MG-63 cells are provided by the Cell Department of Immunology Department, Tongji Medical College, Huazhong University of Science and Technology. Plasmids pIRES-p53 and pIRES vectors containing the full-length p53 gene were purchased from Wuhan Celestron Bio. Methods: The p14 cDNA (0.5kb) amplified from cultured normal human hepatocyte line L02 cells was subcloned into pIRES vector using genetic engineering technique. The recombinant plasmid pIRES- p14ARF-p53. The cells were divided into 3 groups: control group (MG-63 cells), empty vector control group (stably transfected with pIRES-neo Cells), p14ARF-p53 group (stably transfected with pIRES-p14ARF-p53 cells). ① using flow cytometry before and after transfection tumor DNA content and cell cycle. ② RT-PCR and Westernbolt were used to detect the expression of p53 and p14ARF protein in the stable transfected tumor cells. ③ Cell proliferation was observed by MTT assay and cell growth curve. MAIN OUTCOME MEASURES: ① DNA content and cell cycle of osteosarcoma cells. ② tumor cells p53, p14ARF protein expression. ③ cell proliferation. Results: Double plasmid vector pIRES-p14ARF-p53 was successfully constructed. ① DNA content of osteosarcoma cells and cell cycle: Flow cytometry showed that the transfected tumor cells were mostly arrested in G1 phase. ② The results of protein expression test: RT-PCR and Western blot confirmed that p14ARF and p53 gene were independently expressed in target cells at mRNA and protein levels respectively. ③ Cell growth: The growth inhibition rate of MG-63 cells were 33.43%, 69.37%, 66.19% and 75.26% respectively at 24, 48, 72 and 96h after transfection, which was significantly different from the control group (P <0.01). Conclusion: Wild-type p53 and p14ARF can synergistically inhibit osteosarcoma cell proliferation and promote tumor cell apoptosis.