CAJ | 학술논문

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5×104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/mm2,明显多于移植GFP-NSCs组(1 244±187)个/mm2(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.
목적 탐토전염인뇌원성신경영양인자(brain-derived neurotrophic factor,hBDNF)-록색형광단백(GFP)기인신경간세포체내이식후재시신경손상대서시망막내적존활、천이화분화정황,이급기대시신경손상후시망막신경절세포(retinal ganglion cells,RGCs)존활적영향.방법 (1)취30지SD대서제작우안시신경부분손상모형,채용수궤수자표법수궤균분성량조,분별우파리체강내이식GFP기인전염신경간세포(GFP-NSCs)、hBDNF화GFP쌍기인전염신경간세포(hBDNF-GFP-NSCs).8주후처사대서,취우안안구주빙동절편,연후분별용흉선세포표면당단백(Thy1.1)、효질섬유산성단백(GFAP)、β-미관단백(β-tubulin)、시자홍질(rhodopsin)항체진행면역표기,관찰이식세포적천이분화정황.(2)취30지SD대서제작우안시신경부분손상모형,수궤균분위삼조:손상조、GFP조、BDNF조.손상조대서파리체강내주사등삼염수,이GFP조、BDNF조칙분별우파리체강내이식GFP-NSCs화hBDNF-GFP-NSCs,매지안구파리체강내이식5×104개세포.8주후처사대서,취우측안구시망막포편,행Thy1.1면역형광염색후,형광현미경하관찰계수RGCs세포수량.결과 (1)이식후,량조세포균가재시망막내존활、천이,병산생세포분화,량조세포균가이분화성효질세포、신경원.이식hBDNF-GFP-NSCs조소량세포분화위절세포양세포(Thy1.1+),이GFP-NSCs조미발현유분화위Thy1.1양성세포.(2)시망막포편Thy1.1면역형광염색후계수발현,이식hBDNF-GFP-NSCs적시망막신경절세포위(1461±154)개/mm2,명현다우이식GFP-NSCs조(1 244±187)개/mm2(P<0.05).결론 이식후적hBDNF-GFP-NSCs가이분화위신경원화신경효질세포,소수가분화위시망막신경절양세포.hBDNF-GFP-NSCs이식가이증가RGCs적존활수량,유조우시신경손상후적수복.
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 × 104 cells per eye. Eight weeks later, the rats were sacrificed and the right eyes enucleated for Thyl. 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/mm2 in the retina of hBDNF-GFP-NSCs group, which was significantly more than (1 244 ± i 87 ) RGCs/mm2 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, trans-plantation of hBDNF-GFP-NSCs can increase the survival number of RGCs and help repair of the injured optic nerve.