论文部分内容阅读
目的构建双质粒表达载体pIRES-p14ARF-p53,并研究其对骨肉瘤细胞增殖生长的抑制作用。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 (0.5 kb)亚克隆至pIRES载体中,通过聚合酶链反应(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,流式细胞仪测定瘤细胞DNA含量和细胞周期;逆转录(RT)-PCR和Western bolt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。噻唑蓝(MTT)比色法与细胞生长曲线观察细胞增殖情况。结果成功构建出双质粒表达载体plRES-p14ARF-p53。转染后瘤细胞形态由转染前密集排列的多角形、星形转变为排列稀疏的长梭形细胞,瘤细胞生长速度较前缓慢,群体倍增时间比转染前增加了2.3倍,且易出现脱壁并见散在瘤细胞凋亡;流式细胞仪检测发现转染后的瘤细胞多停滞于G,期;RT-PCR与Western blot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,转染MG-63后24、48、72、96 h瘤细胞生长抑制率分别为:33.43%、69.37%、66.19%、75.26%,差异有统计学意义(P<0.01)。结论μ野生型p53和p14ARF协同抑制骨肉瘤细胞的增殖促进瘤细胞的凋亡,为进一步研究p14ARF与p53在骨肉瘤基因治疗的体内实验奠定了基础。
Objective To construct a double plasmid expression vector pIRES-p14ARF-p53 and study its inhibitory effect on proliferation and proliferation of osteosarcoma cells. Methods The p14 cDNA (0.5 kb) amplified from cultured normal human hepatocyte line L02 cells was subcloned into pIRES vector using gene engineering technology. The expression of p14 was detected by polymerase chain reaction (PCR), restriction endonuclease digestion The recombinant plasmid pIRES-p14ARF-p53 was identified. The liposome-mediated transfection into osteosarcoma MG-63 cells, positive clones were screened, the DNA content and cell cycle of tumor cells were determined by flow cytometry. After transfection (RT) -PCR and Western bolt, Of tumor cells p53, p14ARF protein expression of qualitative and semi-quantitative detection. MTT assay and cell growth curve were used to observe cell proliferation. Results The double plasmid expression vector plRES-p14ARF-p53 was successfully constructed. After transfection, the morphology of the tumor cells was changed from densely arranged polygons and stars to star-shaped sparsely-shaped spindle cells. The growth of tumor cells was slower and the population doubling time was increased by 2.3 times compared with that before transfection The results of flow cytometry showed that the number of tumor cells in the transfected cells was mostly arrested in G stage. The mRNA and protein levels of p14ARF and p53 in target cells were detected by RT-PCR and Western blot, respectively The expression of MG-63 at 24, 48, 72 and 96 h after transfection was 33.43%, 69.37%, 66.19% and 75.26%, respectively. The difference was statistically significant (P <0.01). Conclusion μ wild-type p53 and p14ARF synergistically inhibit the proliferation of osteosarcoma cells and promote the apoptosis of tumor cells, which will lay the foundation for the further study of p14ARF and p53 in gene therapy of osteosarcoma in vivo.