利用稳定表达神经营养因子的神经干细胞治疗脊髓损伤的实验研究

Treatment of Spinal Cord Injury Using Neural Stem Cells with Stable Neurotrophin Expression

目的利用转基因技术将大鼠NT-3基因转入神经干细胞,获得稳定表达NT-3的细胞株,注入脊髓损伤大鼠模型的损伤部位进行治疗,观察外源细胞对脊髓的修复情况。本研究为脊髓损伤的干细胞治疗提供新的实验依据,为采用转基因技术提高脊髓损伤治疗效果奠定理论基础。方法①取孕14天大鼠胚胎皮层细胞,磁珠分选Nestin阳性(神经干细胞特异性标记)细胞群进行体外细胞培养至形成细胞克隆。将大鼠NT-3高表达载体转入神经干细胞,G418筛选出稳定表达NT-3的细胞继续培养。Western Blot检测证实得到的细胞可以稳定表达NT-3。②40只成年雌性Wistar大鼠麻醉后撞击锤损伤脊髓成脊髓损伤后九天,实验组大鼠每只注入5μL细胞(稳定表达NT-3)悬液(10000个/μL),对照1组动物每只注入5μL细胞培养基,对照2组动物每只注入5μL未经转染处理的细胞悬液(10000个/μL),皮肤缝合后放回动物房常规饲养。③移植后一周起,采用斜板试验和BBB(Basso-Beattie-Bresnahan)评分观察大鼠后肢运动功能恢复情况。结果①神经干细胞可以稳定高表达(NT-3);②高表达NT-3的神经干细胞移植后可以改善实验性脊髓损伤大鼠模型各项检测指标,对脊髓损伤具有潜在的治疗价值。结论 NT-3转染神经干细胞后对于实验动物的脊髓损伤治疗效果显著提高,是提高临床脊髓损伤疗效的潜在可行途径。

Objective To create stable transfection of rat neurotrophin （NT）-3 gene into neural stem cells （NSCs）, which were injected into the site of spinal cord injury （SCI） in rats for repairing SCI through these exogenous cells. Methods （1）The embryonic cortical cells were harvested from rats on gestational day 14 and Nestin positive cells were isolated. The transgenic vector of rat NT-3 was transfected into NSCs and these cells were further screened by Geneticin 418 （G418）. Western blot was used to confirm the stable expression of NT-3. （2）After anesthesia, the spinal cords of forty female Wistar rats were impacted to induce SCI. Nine days later, the rats in experimental group were injected with 5uL NT-3 cell suspension （10 000 cells/ uL）, the rats in control group 1 were injected with 5 uL medium and the rats in control group 2 were injected with 5 uL untransfected cell suspension （10 000 cells/ uL）. After suturing, these animals received conventional feeding in an animal house. （3）One week after transplantation, tilt tests and Basso-Beattie-Bresnahan （BBB） scores were used to observe the repair of motor function of rat hind limbs. Results （1）NSCs could stably overexpress NT-3. （2）The transplantation of NT-3 overexpressing NSCs could improve the recovery indices of experimental SCI rats. Conclusions NSCs stably expressing NT-3 can significantly improve the treatment outcome of SCI in experimental animals, providing a potentially feasible pathway for clinical treatment of SCI.