人PTEN基因表达载体的构建及其在U251细胞株中的表达

目的:克隆人野生型和突变型PTEN的cDNA,并构建其表达载体,探讨其表达质粒在人胶质瘤U251细胞中的表达情况。方法:分别从新鲜胎盘组织和结肠癌组织中提取总RNA,采用RT-PCR的方法扩增人野生型和突变型PTEN基因全长cDNA,分别克隆入pMD18-T载体上,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸序列分析,再分别将两段基因定向克隆入pcDNA3.1(+)载体中构建表达载体。结果:反转录PCR扩增后的产物,在约1.4kb处出现明亮的特异性条带,pcDNA-PTEN-WT细胞的生长曲线生长速度较另3种细胞明显减慢。结论:转染入U251细胞,发现突变型PTEN蛋白对细胞生长无明显抑制作用,而野生型PTEN蛋白对细胞生长有明显抑制作用。

Construction of PTEN gene expression vector and its expression in glioma cell lines U251

Objective To clone the cDNA of both wide and mutant type of the human tumor suppressor gene PTEN and construct its expression vector respectively, investigate the effects of PTEN on cell growth in human brain glioma U251 cells. Method Two DNA fragments were amplified from human placenta tissues and cancer of colon tissues by using RT-PCR and were cloned into pMD 18-T vector. After sequencing, the fragments were linked with expression vector pcDNA3.1(+) vector. Results Product of RT-PCR had a clear specific ladder bands at 1.4 kb. PcDNA-PTEN-WT cell growth curves was slower than other cells. Conclusion The two vectors were transfected into U251. The progress of cell cycle was obviously arrested in the U251 transfected with wild PTEN gene, but was not the thing.