SHH方案诱导胚胎干细胞分化为神经干细胞移植促进小鼠损伤脊髓结构与功能的恢复

目的比较分析体外诱导小鼠胚胎干细胞分化为神经干细胞的方法并观察神经干细胞移植后对脊髓损伤小鼠的治疗作用。方法采用维甲酸(retinoic acid, RA)方案及音猬因子(sonihedgehog,SHH)方案进行体外诱导培养,然后用抗Nestin、NeuN、GFAP、O1抗体进行免疫细胞化学染色鉴定。脂质体转染法将LacZ基因导入小鼠胚胎干细胞,继而在SHH方案诱导后移植进入脊髓横断小鼠体内,分别于移植后1个月、2个月冰冻切片进行X-gal染色追踪ES细胞并对X-gal阳性细胞行免疫荧光组织化学染色鉴定细胞类型;BBB评分分析了小鼠后肢运动功能的恢复。结果RA与SHH方案得到的Nestin阳性细胞的比例分别为32.54%和68.51%。细胞移植后1个月、2个月均可在脊髓横断小鼠体内检测到大量的X-gal阳性细胞,它们与宿主的脊髓组织相整合。免疫荧光组织化学染色显示X-gal阳性细胞主要表达ChAT和MBP。细胞移植组BBB评分高于对照组。结论与RA相比,SHH是一种较为高效的诱导剂,经SHH诱导得到的神经干细胞移植到脊髓横断的小鼠体内至少可存活2个月,表达成熟用胆碱能神经元的标志及髓鞘碱性蛋白,并且可以促进脊髓损伤小鼠运动功能的恢复。

Transplanted neural stem cells derived from embryonic stem cells by SHH methods to promote recovery of structure and function in spinal cord transected mice

Objective To compare different methods inducing mouse embryonic stem (ES) cells differentiation into neural stem cells(NSCs) in vitro, further analyze the transplantation therapeutic effects of neural stem cells in the spinal cord transected mice. Methods Mouse ES cells were induced into NSCs in vitro by RA and SHH methods and identified morphologically and investigated immunocytochemically by Nestin ? NeuN ?GFAP?O1. To trace the transplanted cells, we constructed a ES cell line stably expressing LacZ gene which was transfected into ES cells by lipofect. Then the induced mouse ES cells carrying LacZ gene were transplanted into the spinal cord transected mice. The differentiation of NSCs were detected by immunofluorescence histochemistry, and the locomotor functions of the mice were evaluated by BBB score after one or two months. Results The percentages of the Nestin positive cells induced by RA and SHH methods were 32.54%?68.51% respectively. One and two months after transplantation, many X-gal positive cells were detected to be integrated into the host tissue, migrate about 1 cm , showing ChAT and MBP- positive,and the locomotor function of the injured mice improved after transplantation. Conclusion SHH induction method shows a higher efficiency,and the induced ES cells not only can keep alive at least two months,but also express marker of cholinergic neuron and MBP, further promote the functional recovery of the spinal cord transected mice.