大鼠胚胎神经干细胞分离培养及基因感染研究

Isolation,Culture and Infection of Neural Stem Cells from Fetal Rat

目的建立基因工程化神经干细胞(neural stemcells,NSCs)以便为治疗感音神经性聋的研究创造条件。方法分离、培养胎鼠海马组织神经干细胞,并用巢蛋白(nestin)免疫荧光组织化学染色鉴定;诱导神经干细胞分化,用神经元特异性烯醇化酶(neuron specific enolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫荧光组织化学染色鉴定分化细胞。通过细菌内同源重组方法构建携带有报告基因绿色荧光蛋白(green fluorescent protein,GFP)的重组腺病毒(Ad-GFP),并将其感染神经干细胞。结果①获得了大量未分化、呈巢状悬浮生长的神经干细胞团,并能分化为神经元和神经胶质细胞,且经传代培养10代后仍具干细胞特性;②97%以上的神经干细胞能被腺病毒感染;③被腺病毒感染的神经干细胞分化为神经元和神经胶质细胞后仍可有效表达GFP报告基因。结论本实验成功构建了携带GFP基因的重组腺病毒。从胚鼠海马组织分离、培养的细胞具有自我更新能力和多潜能分化能力,为中枢神经系统的干细胞,经基因工程化及传代后,仍具干细胞特性,可以作为细胞移植治疗感音神经性聋试验研究的供体细胞。

Objective To establish the genetically engineered neural stem cells（ NSCs） to create bases for the experimental study on treating sensorineural deafness. Methods After isolated from fetal rat hippocampus and identified with nestin immunocytochemical fluorescent staining , NSCs were induced to differentiate. The differentiated cells were detected respectively with neuron specific enolase（NSE） and glial fibrillary acidic protein（GFAP） immunocytochemical staining. The recombinant adenovirus with green fluorescence protein（Ad - GFP） was constructed by homologous recombination in bacteria and used to infect the obtained NSCs. Results ①Nest - like clusters of NSCs were obtained in the suspension and the cells could be differentiated into neurons and astrocytes, which retaining the main characteristics of NSCs after 10 passages of culture②Ahove 97% of NSCs were infected by Ad - GFP. （3）After infected by Ad - GFP, NSCs were induced to differentiate. The differentiated cells could efficiently express the GFP. Conclusion The Ad - GFP has been constructed successfully. The NSCs possess multipotency and self- renewable ability is believed to be NSCs of the central nervous system. Since they retain their characteristics and differentiation ability after passages and genetic engineering, they can be used as donor cells to experimental research on treating sensorineural deafness with transplantation of cells.