摘要
目的 探讨神经干细胞(NSCs)与神经营养因子-3(NT-3)基因修饰的嗅鞘细胞(OECs)联合移植对大鼠创伤性颅脑损伤(TBI)后神经功能的影响及机制。方法 将100只Sprague Dawley雄性大鼠按照随机数字表法分为假手术组、模型组、NSCs组、OECs组、联合治疗组,每组20只。模型组、NSCs组、OECs组、联合治疗组大鼠采用改良Feeney法构建TBI模型。造模1 d后,将不同的试剂注入各组大鼠损伤灶进行细胞移植,模型组大鼠注入10μL生理盐水,OECs组大鼠注入10μL NT-3基因修饰的OECs悬液,NSCs组大鼠注入10μL NSCs悬液,联合治疗组大鼠注入5μL NT-3基因修饰的OECs细胞悬液和5μL NSCs悬液,注射完成后继续常规饲养。分别于移植后1、7、14、28 d,各组随机取10只大鼠,采用改良神经功能缺损评分(mNSS)评估各组大鼠的神经功能缺损程度。移植后7 d,采用酶联免疫吸附试验法检测大鼠脑组织中白细胞介素(IL)-6、IL-1β及神经生长因子(NGF)水平。移植后28 d,采用苏木精-伊红染色法观察大鼠脑组织病理学改变,采用免疫组织化学染色法检测大鼠脑组织中NGF表达。结果 移植后1、7、14、28 d,模型组、NSCs组、OECs组、联合治疗组大鼠mNSS评分显著高于假手术组(P<0.05);移植后7、14 d,NSCs组、OECs组、联合治疗组大鼠mNSS评分显著低于模型组(P<0.05);移植后7、14 d,NSCs组、OECs组、联合治疗组大鼠mNSS评分比较差异无统计学意义(P>0.05);移植后28 d,NSCs组、OECs组、联合治疗组大鼠mNSS评分显著低于模型组,联合治疗组大鼠mNSS评分显著低于NSCs组和OECs组(P<0.05);移植后28 d,NSCs组与OECs组大鼠mNSS评分比较差异无统计学意义(P>0.05)。移植后28 d,假手术组大鼠脑组织结构完整,核仁明显,细胞排列有序;模型组大鼠损伤灶周围脑组织形态不规则,细胞肿胀,排列紊乱,可见坏死细胞及细胞核固缩,存在炎症细胞浸润;相较于模型组,NSCs组及OECs组大鼠脑组织细胞肿胀,排列紊乱,细胞核固缩、炎症浸润现象稍有改善;相较于模型组、NSCs组及OECs组,联合治疗组大鼠脑组织细胞排列相对规整,细胞肿胀减轻,核仁清晰。模型组大鼠脑组织中IL-6、IL-1β表达水平显著高于假手术组(P<0.05),联合治疗组大鼠IL-6、IL-1β表达水平显著低于模型组、NSCs组、OECs组(P<0.05),NSCs组、OECs组大鼠脑组织中IL-6、IL-1β水平显著低于模型组(P<0.05)。NSCs组、OECs组、联合治疗组大鼠脑组织中NGF阳性表达细胞数及NGF水平显著高于假手术组、模型组(P<0.05);假手术组与模型组大鼠脑组织中NGF阳性表达细胞数和NGF水平比较差异无统计学意义(P>0.05);联合治疗组大鼠脑组织中NGF阳性表达细胞数及NGF水平显著高于NSCs组、OECs组(P<0.05);NSCs组与OECs组大鼠脑组织中NGF阳性表达细胞数及NGF水平比较差异无统计学意义(P>0.05)。结论 NSCs和NT-3基因修饰的OECs联合移植治疗可显著减轻TBI大鼠神经功能缺损程度,提高NGF表达水平,减轻炎症反应,从而起到神经保护作用。
Objective To explore the effect and mechanism of combined transplantation of neural stem cells(NSCs)and olfactory ensheathing cells(OECs)modified by neurotrophin-3(NT-3)gene on neurological function after traumatic brain injury(TBI)in rats.Methods A total of 100 male Sprague Dawley rats were randomly divided into sham operation group,model group,NSCs group,OECs group and combined treatment group using a random number table method,with 20 rats in each group.The rats in the model group,NSCs group,OECs group and combined treatment group were used to construct TBI models by using the improved Feeney method.After 1 d of modeling,different reagents were injected into the damaged lesion of rats in each group for cell transplantation,the rats in the model group injected with 10μL physiological saline,the rats in the OECs group injected with 10μL OECs modified by NT-3 gene suspension,the rats in the NSCs group rats injected with 10μL NSCs suspension,the rats in the combined treatment group injected with 5μL OECs modified by NT-3 gene cell suspension and 5μL NSCs suspension,routine feeding was performed after injection.On 1,7,14,28 d after transplantation,10 rats were randomly selected from each group and the degree of neurological impairment was evaluated by the modified neurological deficiency scale(mNSS).On 7 d after transplantation,the levels of interleukin(IL)-6,IL-1βand nerve growth factor(NGF)in brain tissue of rat were detected by enzyme-linked immunosorbent assay.On 28 d after transplantation,the histopathology changes in brain tissue of rats were detected by hematoxylin-eosin staining,and the expression of nerve growth factor(NGF)in brain tissue was detected by immunohistochemical staining.Results On 1,7,14,28 d after transplantation,the mNSS scores of rats in the model group,NSCs group,OECs group and combined treatment group were significantly higher than those in the sham operation group(P<0.05);on 7,14 d after transplantation,the mNSS scores of rats in the NSCs group,OECs group and combined treatment group were significantly lower than those in the model group(P<0.05);on 7 and 14 d after transplantation,there was no significant difference in mNSS scores among the NSCs group,OECs group and combined treatment group(P>0.05);on 28 d after transplantation,the mNSS scores of rats in the NSCs group,OECs group and combined treatment group were significantly lower than those in the model group,while the mNSS score of rats in the combined treatment group was significantly lower than that in the NSCs group and OECs group(P<0.05);there was no significant difference in mNSS scores of rats between the NSCs group and the OECs group on 28 d after transplantation(P>0.05).On 28 d after transplantation,the brain tissue structure of rats in the sham operation group was intact,with obvious nucleoli and orderly cell arrangement;the brain tissue around the lesion of rats in the model group had irregular morphology,swollen cells,disordered arrangement,visible necrotic cells and nuclear pyknosis,and infiltration of inflammatory cells;compared with the model group,the brain tissue around the lesion of rats in the NSCs group and OECs group showed slight improvement in cell swelling,disordered arrangement,nuclear pyknosis,and inflammatory infiltration;compared with the model group,NSCs group and OECs group,the brain tissue around the lesion of rats in the combined treatment group showed relatively regular cell arrangement,reduced cell swelling,and clear nucleoli.The expression levels of IL-6 and IL-1βin the brain tissue of rats in the model group were significantly higher than those in the sham operation group(P<0.05);the expression levels of IL-6 and IL-1βin the brain tissue of rats in the combined treatment group were significantly lower than those in the model group,NSCs group and OECs group(P<0.05);and the expressions of IL-6 and IL-1βin the brain tissue of rats in the NSCs group and OECs group were significantly lower than those of the model group(P<0.05).The number of NGF positive expression cells and NGF level in the brain tissue of rats in the NSCs group,OECs group and combined treatment group were significantly higher than those in the sham operation group and model group(P<0.05).There was no significant difference in the number of NGF positive expression cells and NGF level in the brain tissue of rats between the sham operation group and the model group(P>0.05).The number of NGF positive expression cells and NGF level in the brain tissue of rats in the combined treatment group were significantly higher than those in the NSCs group and OECs group(P<0.05).There was no significant difference in the number of NGF positive expression cells and NGF levels in the brain tissue of rats between the NSCs group and OECs group(P>0.05).Conclusion The combined transplantation of NSCs and OECs modified by NT-3 in TBI rats can significantly reduce the degree of neurological deficits,increase NGF expression levels,alleviate inflammatory reactions,and thus play a neuroprotective role.
作者
张召宇
李梦欣
李慧堂
李海明
金保哲
ZHANG Zhaoyu;LI Mengxin;LI Huitang;LI Haiming;JIN Baozhe(Department of Neurosurgery,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China;Henan Provincial Key Laboratory of Neural Repair,Weihui 453100,Henan Province,China)
出处
《新乡医学院学报》
CAS
2023年第6期501-506,共6页
Journal of Xinxiang Medical University
基金
2018年河南省医学科技攻关计划省部共建项目(编号:SBGJ2018057)。
关键词
神经生长因子
神经干细胞
嗅鞘细胞
创伤性脑损伤
nerve growth factor
neural stem cells
olfactory ensheathing cells
traumatic brain injury
作者简介
张召宇(1994-),男,河南南阳人,硕士研究生在读,住院医师,研究方向:神经损伤功能修复与保护;通信作者:金保哲(1970-),男,河南南阳人,博士,教授,主任医师,硕士研究生导师,研究方向:神经损伤功能修复与保护,E-mail:13938765496@163.com。
