人脑源性神经营养因子基因转染神经干细胞治疗大鼠视神经损伤

Treatment of optic nerve injury by transplanting hBDNF-GFP gene transfected neural stem cells

目的探讨转染人脑源性神经营养因子（brain—derived neurotrophic factor，hBDNF）-绿色荧光蛋白（GFP）基因神经干细胞体内移植后在视神经损伤大鼠视网膜内的存活、迁移和分化情况，以及其对视神经损伤后视网膜神经节细胞（retinal ganglion cells，RGCs）存活的影响。方法（1）取30只SD大鼠制作右眼视神经部分损伤模型，采用随机数字表法随机均分成两组，分别于玻璃体腔内移植GFP基因转染神经干细胞（GFP—NSCs）、hBDNF和GFP双基因转染神经干细胞（hBDNF—GFP—NSCs）。8周后处死大鼠，取右眼眼球做冰冻切片，然后分别用胸腺细胞表面糖蛋白（Thy1．1）、胶质纤维酸性蛋白（GFAP）、β-微管蛋白（β-tubulin）、视紫红质（rhodopsin）抗体进行免疫标记，观察移植细胞的迁移分化情况。（2）取30只SD大鼠制作右眼视神经部分损伤模型，随机均分为三组：损伤组、GFP组、BDNF组。损伤组大鼠玻璃体腔内注射等渗盐水，而GFP组、BDNF组则分别于玻璃体腔内移植GFP—NSCs和hBDNF—GFP—NSCs，每只眼球玻璃体腔内移植5×10^4个细胞。8周后处死大鼠，取右侧眼球视网膜铺片，行Thy1．1免疫荧光染色后，荧光显微镜下观察计数RGCs细胞数量。结果（1）移植后，两组细胞均可在视网膜内存活、迁移，并产生细胞分化，两组细胞均可以分化成胶质细胞、神经元。移植hBDNF—GFP—NSCs组少量细胞分化为节细胞样细胞（Thy1．1^＋），而GFP—NSCs组未发现有分化为Thy1．1阳性细胞。（2）视网膜铺片Thy1．1免疫荧光染色后计数发现，移植hBDNF—GFP—NSCs的视网膜神经节细胞为（1461±154）个／mm^2，明显多于移植GFP—NSCs组（1244±187）个／mm^2（P〈0．05）。结论移植后的hBDNF—GFP—NSCs可以分化为神经元和神经胶质细胞，少数可分化为视网膜神经节样细胞。hBDNF—GFP—NSCs移植可以增加RGCs的存活数量，有助于视神经损伤后的修复。

Objective To explore the survival, migration and differentiation of neural stem cells transfected by human brain-derived neurotrophic factor （hBDNF） and green fluorescent protein （GFP） genes and explore its impact on the survival of retinal ganglion cells （RGCs） after transplanted into the retina of rats with optic nerve injury. Methods （ 1 ） Thirty SD rats with right optic nerve injury were transplanted with GFP-transfected NSCs （GFP-NSCs） or hBDNF- and GFP-transfected NSCs （hBDNF- GFP-NSCs） into vitreous cavity respectively, eight weeks after which the rats were sacrificed and the right eyes removed for frozen section. Then, Thy1. 1, GFAP, β-tubulin, rhodopsin antibody were used as markers to observe the migration and differentiation of the transplanted cells. （2） Thirty SD rats models with partial right optic nerve injury were randomly divided into three groups：injury group, GFP group and BDNF group. Injury group was injected with 5 μl normal saline into vitreous cavity, while the other two groups were transplanted with GFP-NSCs or hBDNF-GFP-NSCs into the vitreous cavity, with 5 ×10^4 cells per eye. Eight weeks later, the rats were sacrificed and the right eyes enucleated for Thy1. 1 immune staining to count the number of RGCs under fluorescence microscope. Results （ 1 ） After transplantation, the transplanted cells could live, migrate and differentiate in both groups. The cells could differentiate into glial cells and neurons. In hBDNF-GFP-NSCs group, small amount of cells could differentiate into RGCs-like cells （Thy1. 1 ＋ ）, but none in GFP-NSCs group. （2） Thy1. 1 immune staining showed （1 461 ±154） RGCs/mm^2 in the retina of hBDNF-GFP-NSCs group, which was significantly more than （ 1 244 ±187 ） RGCs /mm^2 in GFP-NSCs group （ P 〈 0.05 ）. Conclusions After transplantation, hBDNF-GFP-NSCs can differentiate into glial cells, neurons and even RGCs-like cells. As well, transplantation of hBDNF-GFP-NSCs can increase the survival number of RGCs and help repair of the injured optic nerve.