人重组NT-3基因体外转染胚胎干细胞及其表达

目的 探讨神经营养素-3(NT-3)基因体外转染小鼠胚胎干细胞(ES)的可能性及其表达情况.方法 用脂质体介导的方法,将重组真核表达载体PCDNA-3.1(+).NT-3瞬时转染ES.用细胞免疫组化及RT-PCR检测转染细胞NT-3蛋白及mRNA表达.ELISA检测细胞分泌上清液中NT-3蛋白表达.结果 细胞免疫组化结果显示,转染NT-3基因的ES细胞胞浆呈红色染色;RT-PCR得到200 bp的基因片段;ELISA检测细胞分泌上清液中NT-3蛋白表达呈阳性,与对照组有显著差别(P＜0.05).结论 重组真核表达载体PCDNA-3.1(+).NT-3可成功转染ES,获得稳定表达NT-3的ES细胞株.

Transfection of recombinant human NT-3 gene into mouse embryonic stem cell and its expression in vitro

Objective To investigate the possibility of in vitro expression of human neurotrophin-3 (NT-3) gene in mouse embryonic stem (ES) cells. Methods The reconlbinant eukaryotic expression vector,PCDNA-3.1(+)-NT-3 was transiently transfected into ES cells by means of liposome mediated method. NT-3 protein and mRNA were tested by immunohistochemical method and RT-PCR,respectively.NT-3 protein secretion in the supernatant of cell culture was detected by ELISA.Results By immunohistochemistry, ES cells were red stained in cytoplasma, showing that the transfected NT-3 plasmid was expressed in the ES cells.RT-PCR got a 200 bp segment in the transfected cells,and the NT-3 protein secretion in the supernatrdnt of transfected cell culture detected by ELISA was dramatically higher than that of control group(P＜0.05). Conclusions Successful transfection of recombinant PCDNA-3.1(+)-NT-3 into the ES cells that leads to stable expression of NT-3 gene can be a potential effective gene delivery approach in the treatnlent of Parkinson's disease.