慢病毒介导神经营养因子3基因修饰大鼠嗅鞘细胞的实验研究

Experimental study on lentivirus mediated neurotrophic factor 3 gene modification of rats olfactory ensheathing cells

目的:探究神经营养因子3(NT-3)基因修饰的嗅鞘细胞(OECs)分泌的NT-3在体内外的活性。方法:取SD大鼠胎鼠嗅球组织,进行原代培养、纯化和鉴定,将纯化后的OECs分为对照组(control)、空病毒感染组(Lenti-NC)以及Lenti-NT-3感染组(Lenti-NT-3)3组,分别在4~28 d,每隔4 d应用酶联免疫分析方法(ELISA)测细胞培养上清中NT-3的浓度。并将NT-3基因修饰的OECs(NT-3-OECs)注入SD大鼠脑部,2周后进行脑组织石蜡切片p75神经生长因子受体(p75 NGFR)、胶质纤维酸性蛋白(GFAP)免疫荧光检测,运用ELISA方法在4~16 d,每隔4 d测各组大鼠脑组织中NT-3的浓度。结果:纯化后OECs纯度为(92.03±2.17)%;慢病毒对OECs的感染率为(70.69±10.29)%;各时间点Lenti-NT-3组上清中NT-3含量均显著高于Control组与Lenti-NC组(P<0.05);NT-3-OECs移植入大鼠脑内2周后,可见p75 NGFR、GFAP表达阳性的OECs;各时间点Lenti-NT-3组脑组织中NT-3含量均显著高于Control组与Lenti-NC组(P<0.05)。结论:运用慢病毒载体构建的NT-3-OECs可在体内外高表达具有生物学活性的NT-3。

Objective:To investigate the biological activity of NT-3 secreted by olfactory ensheathing cells(OECs)modified by neurotrophic factor 3(NT-3)gene in vivo and in vitro.Methods:The olfactory bulb tissues of SpragueDawley rat fetuses were taken for primary culture,purification and identification.The purified OECs were divided into three groups:control group(Control),empty virus infection group(Lenti-NC)and Lenti-NT-3 infection group(LentiNT-3).From 4 to 28 days,Enzyme-linked immunosorbnent assay(ELISA)was used to measure the concentration of NT-3 in the cell culture supernatant every 4 days;NT-3 genetically modified OECs(NT-3-OECs)were injected into the brain of SD rats.After 2 weeks,the paraffin brain tissue sections were tested for p75 nerve growth factor receptor(p75 NGFR)and glial fibrillary acidic protein(GFAP)immunofluorescence.From 4 to 16 days,the ELISA method was used to measure the concentration of NT-3 in the brain tissue of the rats in each group every 4 days.Results:The purity of OECs after purification was(92.03±2.17)%,the infection rate of lentivirus to OECs was(70.69±10.29)%,the NT-3 content in the supernatant of Lenti-NT-3 group was significantly higher than Control group and Lenti-NC group at each time point(P<0.05).At 2 weeks after NT-3-OECs was transplanted into the rat brain,p75 NGFR-and GFAPpositive OECs were seen,NT-3 content in the brain tissue of the Lenti-NT-3 group was significantly higher than that of the Control group and Lenti-NC group at each time point(P<0.05).Conclusion:NT-3-OECs constructed by lentiviral vectors can highly express NT-3 with biological activity in vivo and in vitro.