维甲酸和Shh因子联合诱导神经干细胞分化为胆碱能神经元及其体内移植的实验研究

Experimental study on cholinergic neurons differentiated from neural stem cells through the united induction of RA and Shh and transplanted into rats

目的探讨维甲酸和Shh因子联合诱导大鼠神经干细胞州SCs）分化为胆碱能神经元的可行性。方法分离、培养和鉴定大鼠的NSCs，联合应用维甲酸和Shh因子诱导NSCs分化．应用胆碱能细胞特异抗体VAChT作组化鉴定，通过流式细胞仪计数诱导后胆碱能神经元的比例：并将其注射到大鼠脊髓，观察体内存活情况。结果维甲酸和Shh因子联合诱导NSCs可以产生较高比例的VAChT阳性细胞．阳性率达77．26％±0．99％，明显优于对照组：移植体内的细胞可以存活并具有一定迁移能力。结论应用维甲酸和Shh因子联合诱导NSCs能产生较高比例的胆碱能神经元，可以为修复脊髓损伤提供新的细胞来源。

Objective To provide new cell source for tissue engineering spinal cord injury （SCI） repair with cholinergic neurons differentiated from neural stem cells （NSCs） through the united induction of retinoic acid （RA） and Sonic hedgehog （Shh）. Methods Before the united induction of RA and Shh, NSCs were isolated, cultured and identified, Then they were differentiated into cholinergic neurons and the vesicular acetylcholine transporter （VAChT）, the specific antibody of cholinergic cells, was applied for immunohistochemical identification. Fluorescence-activated cell sorting （FACS） was used to count the ratio of cholinergic neurons after induction. Finally, cholinergic cells were injected into the spinal cord of rats for the observation of their survival in vivo. Results The united application of RA and Shh could induce VAChT-positive cells with a ratio which was as high as 77.26%＋0.99% and obviously superior to that in the control group; cells transplanted into the body could survive and were able to migrate in vivo. Conclusion KA and Shh applied in combination may induce NSCs to differentiate into cholinergic neurons with a comparatively high ratio, which can be used to provide new cell source for SCI repair and be helpful for the investigation of relevant therapeutic effects.