稳定表达野生型p53基因人骨肉瘤细胞株的建立与鉴定

Establishment and identification of human osteosarcoma cell line stably expressing wild-type p53 gene

目的建立稳定表达外源性野生型p53基因的人骨肉瘤细胞株(U-2 OS),为进一步研究野生型p53基因在骨肉瘤自杀基因治疗中的协同作用提供体外实验模型建立与鉴定的方法。方法利用分子生物学基因克隆技术将人类野生型p53基因cDNA片段插入到表达载体pIRES-EGFP中,得到重组质粒pIRES-EGFP-p53,并通过PCR、酶切电泳等方法进行质粒鉴定;然后用该重组质粒转化大肠杆菌BL21细胞,转化菌落经PCR扩增后,将提取的质粒DNA用脂质体介导的转染方法导入U-2 OS细胞中,经荧光显微镜下人工筛选得到稳定转染的骨肉瘤细胞系。结果成功地构建出包含有野生型p53 cDNA片段的重组质粒pIRES-EGFP-p53,通过PCR、酶切电泳等方法鉴定质粒大小和位点均符合实验设计;并将该重组质粒成功导入人骨肉瘤细胞株U-2 OS细胞中,经荧光显微镜下人工筛选得到稳定转染的骨肉瘤细胞系,命名为U-2-p53 OS。转染后的骨肉瘤细胞出现散在的凋亡现象。结论利用基因转染技术,建立了稳定表达外源性野生型p53基因的人骨肉瘤U-2-p53 OS细胞系,为进一步研究野生型p53基因在骨肉瘤自杀基因治疗中的协同作用奠定了基础。

Objective To establish an in vitro model of human osteosarcoma cell strain（U-2 OS）stably expressing the exogenetic wild-type p53 gene for further studying the role of wild-type p53 gene in osteosarcoma suicide gene therapy.Methods The molecular biology technology was used to insert the human wild-type p53 cDNA fragment into the expression vector pIRES-EGFP for obtaining the recombinant plasmid pIRES-EGFP-p53,and then it was identified by PCR,enzyme electrophoresis and other methods.Then the recombinant plasmid was transformed into E.coli BL21 cells.After the transformed colony was amplified by PCR,the plasmid DNA was transfected into U-2 OS cells by liposome-mediated transfection method.Under fluorescence microscopy,the stably transfected osteosarcoma cell line was manually screened.Results A recombinant plasmid pIRES-EGFP-p53 containing wild-type p53 cDNA fragment was successfully constructed.The results of PCR,enzyme electrophoresis and other methods to identify the size and position of plasmid,were consistent with the experimental design.After transfection,the human osteosarcoma cell line was named U-2-p53 OS.The transfected osteosarcoma cells showed weak apoptosis.Conclusion The establishment of human osteosarcoma U-2-p53 OS cell lines by gene transfer technology lay a foundation for the further study of wild-type p53 gene in osteosarcoma suicide gene therapy.