摘要
目的 通过增强型绿色荧光蛋白（EGFP）与PTEN融合蛋白真核表达载体的构建和表达，观察PTEN基因对人脑胶质瘤细胞生长的影响。方法 （1）以RT—PCR方法扩增人的PTEN基因，经T—A亚克隆筛选后连入真核表达载体pEGFP—N1，构建融合蛋白表达载体。采用阳离子聚合物转染试剂，将重组质粒DNA瞬时转染至SHG-44细胞，检测融合蛋白的表达。（2）G418筛选出稳定转染的细胞（SHG-44-Z），并扩增培养，通过细胞形态学、生长曲线观察PTEN基因对细胞形态和增殖的影响，免疫组织化学法检测对胶质纤维酸性蛋白（GFAP）表达的影响。结果 （1）重组质粒阳性克隆的测序结果与GenBank报告序列一致。瞬时转染的SHG-44细胞于荧光显微镜下可见绿色荧光，流式细胞仪可检测到荧光表达量为17．8％，免疫细胞化学法检测到外源PTEN蛋白表达。证实pEGFP—PTEN融合蛋白表达载体构建成功，并在胶质瘤细胞得以正确表达。（2）转染组SHG-44-Z细胞株的生长增殖受到明显抑制，第7天细胞计数为未转染组细胞数的27．8％，且GFAP表达上调。结论携带绿色荧光蛋白的PTEN基因真核表达载体的构建，为进一步研究PTEN的作用机理及抑癌效应奠定了基础。
Objective To investigate the biological effects of PTEN gene on the malignant glioma cell line SHG-44. Firstly, A recombinant eukaryotic expression plasmid containing PTEN gene fused with EGFP （enhanced green fluorescence protein ） gene was constructed. Secondly, the expression of the recombinant vector in human glioma cells was detected. Methods （1） The human PTEN gene was amplified by RT-PCR and inserted into pEGFP-NI that was selected by T-A subclone, and the recombinant expression vector was obtained. After the recombinant plasmids were transfected into glioma SHG-44 cells by cation polymex, expression of fusion protein was tested. （2） The stable transfected cells were selected by G418 and amplified. Light microscopy and growth curve were used to measure the effects of PTEN expression on cell morphology and proliferation. Expression of GFAP （glial fibillary acidic protein ） was detected immunohistochemically. Results （ 1 ） The positive recombinant was sequenced and demonstrated to have the same sequence as that of PTEN gene in GenBank. It was proved that the eukaryotic expression vector pEGFP-PTEN have been constructed successfully and expressed in SHG-44 cells. （2） The SHG-44 cell growth was changed obviously. The expression of GFAP was increased. Conclusion The construction of PTEN eukaryotic expression vector containing green fluorescence protein gene will lay the basis for carrying out further studies on the function of PTEN gene.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2007年第4期245-248,共4页
Chinese Journal of Oncology
基金
国家自然科学基金资助项目（30670795）
广东省科技厅研究团队项目（015012）
国家科技部重大基础研究前期研究专项（2002ccc0400）
教育部高等学校博士学科点专项科研基金（20040559008）
关键词
抑癌基因PTEN
绿色荧光蛋白
基因克隆
胶质瘤
Tumor suppression gene PTEN
Green fluorescence protein gene
Gene clone
Glioma
