氧调控性神经生长因子基因修饰神经干细胞移植治疗急性脊髓损伤  

The experimental research of hypoxia-regulated NGF modified neural stem cells in acute spinal cord injury

作　　者：陈梦吉 叶佳辉 应一博 陈敏[1] 窦海成[1] 倪文飞[1] 朱思品 Chen Mengji;Ye Jiahui;Ying Yibo;Chen Min;Dou Haicheng;Ni Wenfei;Zhu Sipin(Department of orthopedics,The Second Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第二医院骨科,325000

出　　处：《中华骨科杂志》2020年第10期669-679,共11页Chinese Journal of Orthopaedics

摘　　要：目的探讨氧调控性神经生长因子(nerve growth factor,NGF)基因修饰神经干细胞(neural stem cells,NSCs)移植治疗急性脊髓损伤(spinal cord injury,SCI)的可行性并观察脊髓损伤后的功能修复情况。方法以腺相关病毒(adeno-associated virus,AAV)为载体构建基因修饰神经干细胞,制备SCI动物模型3 d后将氧调控性神经生长因子基因修饰的神经干细胞移植到脊髓损伤部位作为AAV-5HRE-NGF-NSCs组(NGF组);另设GFP修饰的神经干细胞组(AAV-5HRE-GFP-NSCs组,GFP组);假手术组(空白组);SCI组(对照组)。在移植后第1、3、7、10、14、21、28、35、42、60 d共10个时间点采用Basso-Beattie-Bresnahan(BBB)运动功能评分,斜板试验和脚步印迹检测大鼠后肢运动功能的恢复情况。通过盒式磁带录像(video cassette recorder,VCR)图像及定量测定大鼠离地高度,错误脚步及后肢轮替动作检验大鼠的后肢支持力及灵活度。通过观察脊髓直观图粗测大鼠脊髓损伤程度。通过尼氏染色、HE染色和免疫荧光方法评价脊髓损伤区的神经元修复及形态学变化情况。通过CM-DiI追踪移植神经干细胞并用免疫荧光的方法分析干细胞的分化情况。结果基因修饰神经干细胞移植60 d后,NGF组大鼠的BBB,斜板测试和脚步印迹试验的功能评分均高于SCI组和GFP组,差异有统计学意义(P<0.05);通过VCR图像分析,NGF组大鼠的后肢支持力与活动灵活度优于SCI组和GFP组,差异有统计学意义(P<0.05);通过脊髓直观图分析,各组大鼠脊柱肉眼观对比图示NGF组脊柱未呈现明显萎缩和颜色加深,损伤程度低于SCI组和GFP组;通过尼氏染色、HE染色和免疫荧光方法检测,相比于SCI组与GFP组,NGF组在移植部位NeuN呈明显阳性,同时在形态学水平可见明显再生的神经结构,且SCI空洞面积减小,神经元和尼氏小体增多,差异具有统计学意义(P<0.05)。通过CM-DiI追踪神经干细胞,用NeuN标记神经元,用GFAP标记星形胶质细Objective To investigate the feasibility of transplantation of neural stem cells(NSCs)modified by hypoxia-regulated nerve growth factor(NGF)gene to treat acute spinal cord injury(SCI)and observe the functional repair after SCI.Methods Adeno-associated virus(AAV)was used as the vector to construct gene-modified NSCs.Three days after SCI attack on the animal model,the NSCs modified by hypoxia-regulated NGF were transplanted to the site of SCI as the NGF group.The GFP-modified neural stem cell group(GFP group),sham group,SCI group were set up.Hindlimb motor function was assessed by Basso-Beattie-Bresnahan(BBB)Locomotor Rating Scale,inclined plane tests and footprint analysis at 10 time points on day 1,3,7,10,14,21,28,35,42 and 60 after transplantation.The video cassette recorder(VCR)image and quantitative measurement of the height of the rat from the ground,the foot error and plantar steps were used to test the hindlimb support and flexibility of the rats.The degree of spinal cord injury in rats was roughly measured by observing the visual map of the spinal cord.The neuronal repair and morphological changes in SCI area were evaluated by Nissl staining,HE staining and immunofluorescence.CM-DiI was used to trace neural stem cells and to analyze the differentiation of NSCs by immunofluorescence.Results Two months after transplantation of genetically modified NSCs,the BBB,inclined plane tests and footprint Analytical scores of NGF group rats were higher than those of SCI group and GFP group(P<0.05);Through VCR image analysis,the hindlimb support and mobility of the rats in the NGF group were better than those in the SCI group and GFP group,and the difference was statistically significant(P<0.05).Visual analysis showed that the spinal cord of the rats in each group was visually compared to the NGF group,and the spine did not show significant atrophy and color deepening,and the degree of injury was lower than that of the SCI group and GFP group;Through Nissl staining,HE staining and immunofluorescence detection,obviously

关 键 词：神经生长因子 神经干细胞 脊髓损伤 动物 基因修饰 

分 类 号：R651.2[医药卫生—外科学]