成体小鼠骨髓中神经干细胞的分离与培养

Isolation and culture of neural stem cellsfrom bone marrow of adult mouse

在无菌条件下抽取成体小鼠骨髓,在含体积分数1%B27,20ng/mLEGF和10ng/mLbFGF的F12-DMEM(1∶1)培养基中,于37℃、50mL/LCO2饱和湿度下常规培养,对培养的细胞进行nestin免疫荧光染色检测,并将pEGFP-N2基因在80V、80μs条件下采用电击法转化导入神经干细胞中。结果表明,培养7d后,有大部分细胞聚集成团,并大量增殖;Nestin免疫荧光染色后呈阳性;导入pEGFP-N2基因的神经干细胞培养后在荧光显微镜下可观察到绿色荧光。表明从成体小鼠获得的骨髓,在该试验培养条件下有神经干细胞生成,并且外源标记基因pEGFP-N2在其细胞中实现了表达。

The bone marrow was obtained from the femur of the adult mouse under the aseptic condition,cultured under 37 ℃ and 50 mL/L CO_2 saturated humidity in the F12-DMEM (1∶1) medium,which contains 1% B27,20 ng/mL EGF and 10 ng/ML bFGF.Immunofluorescence stain with Nestin of the separated cells was tested,and pEGFP-N_2 was induced in NSCs under condition of 80 V,80 μs by electrotransformation.As a result,the cells aggregate and greatly proliferate in number after one week of culture,immunofluorescence stain with nestin of the isolated cells shows positive result,the NSCs induced with pEGFP-N_2 are observed green fluorescence under the fluorescencemicroscope.The result indicates that the bone marrow obtained from the adult mouse in present culture condition contains NSCs,in which exogenetic gene pEGFP-N_2 is expressed.This method can be a new way of obtaining NSCs and the foundation of inducing correlated gene to treat neurodegeneration diseases.