神经干细胞联合神经营养因子-3基因修饰嗅鞘细胞移植对大鼠脑损伤后的修复作用  

Effects of neural stem cells in combination with nerve neurotrophic factor 3 gene modified olfactory ensheathing cell co-transplants on functional recovery after traumatic brain injury in the adult rats

作　　者：闫耀宗 金保哲[1] 张新中[1] 李海明 周文科[1] 张岱男 Yan Yaozong;Jin Baozhe;Zhang Xinzhong;Li Haiming;Zhou Wenke;Zhang Dainan(Department of Neurosurgery,the First Affiliated Hospital of Xinxiang Medical University,Henan Key Laboratory of Neural Regeneration,Weihui 453100,China)

机构地区：[1]新乡医学院第一附属医院神经外科,河南省神经修复重点实验室,河南卫辉453100

出　　处：《中华实验外科杂志》2020年第1期71-74,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81701085);河南省卫计委科技攻关计划项目(SBGJ2018057);新乡医学院研究生科研创新支持计划资助项目(YJSCX201835Y);新乡医学院精神神经医学学科群开放课题(2016PN-KFKT-20)。

摘　　要：目的观察神经干细胞(NSCs)联合神经营养因子-3(NT-3)基因修饰嗅鞘细胞(OECs)移植对颅脑损伤(TBI)大鼠神经修复的作用。方法取新生乳鼠海马和嗅球原代培养NSCs及OECs并鉴定。用含NT-3基因的慢病毒载体转染OECs,构建NT-3基因修饰OECs(OECs-NT-3)。取50只体重(200±20)g SD成年雄性大鼠(山西医科大学实验动物中心)随机分为假手术组、模型组、OECs-NT-3移植组、NSCs移植组、OECs-NT-3联合NSCs移植组,分组后制作大鼠颅脑损伤模型,给予相应细胞移植处理。细胞移植后1、7、14、28 d行改良神经功能评分(mNSS),并各时间点取脑组织,制备石蜡切片行苏木精-伊红(HE)染色,原位缺口末端标记法(TUNEL)检测,溴脱氧尿嘧啶核苷(BrdU)、p75免疫组织化学染色。结果大鼠mNSS评分:移植28 d,联合组[(4.00±1.05)分]与模型组[(8.50±1.43)分]、OECs-NT-3组[(5.50±1.35)分]、NSCs组[(6.00±1.49)分]比较,差异有统计学意义(t=-7.997、-2.764、-3.464,P<0.05),而OECs-NT-3组与NSCs组比较,差异无统计学意义(t=-0.785,P>0.05)。HE染色示假手术组、模型组、OECs-NT-3组、NSCs组、联合组损伤灶细胞密度依次为(220±22)、(299±47)、(347±35)、(390±72)、(523±55)个/每高倍镜视野。联合组脑组织结构排列相对规整,损伤灶细胞密度较模型组、OECs-NT-3组、NSCs组显著提高,差异有统计学意义(t=7.246、5.996、3.373,P<0.05)。p75及BrdU免疫组织化学提示两种移植细胞能持续存活且广泛分布在损伤灶周围。移植7 d,假手术组、模型组、OECs-NT-3组、NSCs组、联合组细胞凋亡指数依次为(1.67±0.17)%、(47.49±7.92)%、(31.22±3.97)%、(34.48±4.69)%、(18.17±3.13)%。联合组与模型组、OECs-NT-3组、NSCs组比较,差异有统计学意义(t=5.963、4.470、5.012,P<0.05)。结论神经干细胞联合NT-3基因修饰嗅鞘细胞移植可减轻大鼠脑损伤后神经细胞凋亡,修补局部缺损,对神经功能的恢复具有显著促进作用。Objective To observe the effects of neural stem cells(NSCs)combined with neurotrophin-3 gene modified olfactory ensheathing cells(OECs-NT-3)co-transplants on functional recovery after traumatic brain injury in the adult rats.Methods NSCs and OECs were cultured from hippocampus and olfactory bulb of neonatal rats and then identified.OECs-NT-3 was constructed by lentiviral vector containing NT-3 gene.Male SD rats weighing(200±20)g were randomly divided into sham group,model group,OECs-NT-3 transplantation group,NSCs transplantation group and OECs-NT-3 combined NSCs transplantation group.The rat brain injury model was established.The corresponding cells were transplanted.At day 1,7,14 and 28 after transplantation,the modified neurological severity score(mNSS)was used to evaluate neurological function,and brain tissues were taken at each time point for paraffin sections.Hematoxylin-eosin staining was performed.Cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)method.Immunohistochemical staining of BrdU and p75 was done.Results At day 28 of transplantation,the mNSS scores in rats in the combined group(4.00±1.05)were significantly different from those in the model group(8.50±1.43),the OECs-NT-3 group(5.50±1.35)and the NSCs group(6.00±1.49)(t=-7.997,-2.764,-3.464,P<0.05),but there was no statistically significant difference between OECs-NT-3 group and NSCs group(t=-0.785,P>0.05).Hematoxylin and eosin(HE)staining revealed that the cell density in the sham group,model group,OECs-NT-3 group,NSCs group and the combined group was(220±22),(299±47),(347±35),(390±72)and(523±55)cells,respectively.As compared with the model group,OECs-NT-3 group and NSCs group,the cell density in the combined group was significantly increased(t=7.246,5.996,3.373,P<0.05),and the tissue structure was relatively regular.The p75 and BrdU immunohistochemistry showed that the two kinds of transplanted cells could survive continuously and were widely distributed around the lesion.At day 7 aft

关 键 词：神经干细胞 神经营养因子-3基因 基因修饰 嗅鞘细胞 联合移植 颅脑损伤 

分 类 号：R651[医药卫生—外科学]