电击转染技术在神经干细胞中的应用研究

Application of electrotransformation techniques in neural stem cells

目的:探讨利用电击转染技术将pEGFP-N2基因导入神经干细胞的条件。方法:取SD胎鼠(14 d)的纹状体,将细胞于37℃,5%CO2的培养箱中培养,N estin免疫荧光染色检测,并将pEGFP-N2基因分别在100 V、80μs,200V、80μs,300V、80μs以及400V、80μs的4种不同电击转染条件下,导入神经干细胞。结果:N estin免疫荧光染色后呈阳性,确定为神经干细胞。在200V、80μs条件下,转染效率达80%以上,是4种条件下的最高转染效率。结论:在200V,80μs的电击转染条件下,基本无细胞死亡,达到80%以上转染效率,实现了4种不同条件中的最高转染效率。

Objective ： To investigate the condition of inducing pEGFP-N2 to neural stem cells. Methods：The striatums were obtained from the fetal mice which were pregenant for 14 days （El4）under the aseptic condition,cultured under 37℃ and 5% CO2 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-N2 was induced in NSCs under condition of 100 V/80μs, 200V/80μs, 300V/80μs and 400V/80μs respectively by electrotrans formation. Results ： Immunofluorescence stain with nestin of the isolated cells shows positive result, under condition of 200V. 80μs , 80 % NSCs are observed. Conclusion： under condition of 200V/80μs ,among the NSCs induced with pEGFP-N2 cultured for 2 days ,almost no cells are dead and 80% NSCs are observed in the condition of 200V.80μs by elecotrotransformation.