腺病毒介导EGFP基因转染神经干细胞的实验研究

Experimental study on the transfection of neural stem cells with recombinant adenovirus-mediated enhanced green fluorescent protein gene

目的探讨腺病毒(adenovirus)介导的增强型绿色荧光蛋白(EGFP)基因在神经干细胞(NSCS)的表达规律及转染对神经干细胞增殖、分化的影响。方法用不同感染复数的携带EGFP基因的腺病毒(Ad/EGFP)转染NSCS,在荧光倒置相差显微镜下观察EGFP的表达情况,同时用台盼蓝染色、细胞球计数、免疫细胞化学方法分别检测对照组和转染组细胞的活力、增殖、分化情况。结果Ad/EGFP转染16 h后NSCS发出绿色荧光,在第7天左右荧光强度达高峰并可持续稳定表达20天左右,Nestin表达阳性。在分化后细胞的胞体与突起均可见绿色荧光,部分细胞β-Ⅲtubu lin、GFAP表达阳性。同时观察到对照组和转染组的NSCS在第7天、14天、21天时细胞活力、增殖、分化情况均无统计学差异(P>0.05)。结论Ad/EGFP可以高效转染新生大鼠海马NSCS,且不影响NSCS的存活、生长和分化。EGFP基因是神经干细胞的理想的报告基因。

Objective To observe the expression of enhanced green fluorescent protein （EGFP） in neural stem cells （NSCs ） with adenovirus vector - mediated enhanced GFP gene （Ad/EGFP） and the changes in the biological characters of NSCs in relation to the transfection. Methods NSCs from the hippocampus of neonate rat were isolated, cultured, passaged and identified. The NSCs were transfected with varied doses of EGFP - adenovirus vector to observe the EGFP expression under phase contrast microscope and fluorescentmicroscope. The vitality, multiplication and differentiation of the cells were examined by using trypan blue staining etc. , and the results were compared between the transfected and the control groups. Results The emission of green fluorescence in neurospheres started 16 hours after the transfection, intensified gradually, reached its peak on day 7 and maintained that high level for more than 20 days. The green fluorescence was also present in the cell body and fine dendrites of the neurons and glial cells in the transfected group. No significant differences were found in cell vitality, multiplication and differentiation between the transfectd and control groups on days 7, 14 and 21. Conclusion Ad/EGFP can efficiently transduce NSCs without causing changes in their biological characteristics, and therefore is an ideal method for genetic labeling of NSCs.