神经营养素-3基因转染小胶质细胞及其表达

TANSFECTION OF NEUROTROPHIN-3 GENE TO MICROGLIA AND ITS EXPRESSION

目的用神经营养素-3(NT-3)基因转染小胶质细胞并检测其表达情况。方法小胶质细胞分离纯化后,分为非转染组、空转染组及转染组,非转染组小胶质细胞不经质粒转染,空转染组及转染组分别将空质粒pcDNA3.1及真核重组质粒pcDNA3.1/NT-3转染小胶质细胞。然后经过G418筛选后,采用RT-PCR方法检测3组小胶质细胞中的NT-3mRNA水平,酶联免疫吸咐法(ELISA法)检测各组细胞培养液的吸光度值及NT-3蛋白的浓度。结果 RT-PCR检测显示,转染组可得到一250bp的基因片段。ELISA法检测显示,转染组细胞培养液的吸光度值明显高于非转染组及空转染组,差异有显著性(F=57.23,t=14.28、12.93,P<0.05)。转染组细胞培养液NT-3蛋白浓度亦明显高于非转染组及空转染组,差异有显著性(F=49.56,t=15.32、11.47,P<0.05)。结论 NT-3基因成功转染小胶质细胞,并获得高效表达。

Objective To transfect neurotrophin-3（NT-3）gene into microglia and test its expression. Methods Microglia were separated,purified and randomized to three groups as non-transfection group,empty-transfection group,transfection group.In non-transfection group,the microglia were not transfected by plasmid,and in empty-transfection group and transfection group,pcDNA3.1and pcDNA3.1/NT-3 were transfected to microglia,respectively.After G418 screening,the level of NT-3mRNA in microglia in the three groups was detected using RT-PCR,and the absorbance and NT-3protein concentration in each group were measured employing enzyme-linked immunoassay sensitive assay（ELISA）. Results RT-PCR showed a 250 bp segment obtained in the transfection group.ELISA showed that the absorbance in the cell culture fluid in transfection group was higher than that in both non-transfection and empty-transfection groups（F=57.23;t=14.28,12.93;P〈0.05）,and NT-3protein concentration was also higher（F=49.56;t=15.32,11.47;P〈0.05）. Conclusion NT-3gene was successfully transfected to microglia,and obtained efficient expression.