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中华脑科疾病与康复杂志(电子版) ›› 2019, Vol. 09 ›› Issue (02) : 101 -105. doi: 10.3877/cma.j.issn.2095-123X.2019.02.009

所属专题: 文献

基础研究

诱导型神经干细胞移植对颅脑创伤后神经营养因子分泌的影响
高谋1, 徐如祥2,(), 王文佳3, 董勤4, 丁柏匀2, 姚慧2, 杨志军2   
  1. 1. 100048 北京,解放军总医院第六医学中心神经外科
    2. 100700 北京,解放军总医院第七医学中心神经外科
    3. 572013 三亚,解放军总医院海南医院耳鼻咽喉头颈外科
    4. 100038 北京,首都医科大学附属复兴医院神经内科
  • 收稿日期:2019-02-09 出版日期:2019-04-15
  • 通信作者: 徐如祥
  • 基金资助:
    国家自然科学基金项目(81671189); 全军"十三五"军事医学创新工程项目(16CXZ001)

Neurotrophic effects of induced neural stem cell grafts following traumatic brain injury

Mou Gao1, Ruxiang Xu2,(), Wenjia Wang3, Qin Dong4, Boyun Ding2, Hui Yao2, Zhijun Yang2   

  1. 1. Department of Neurosurgery, The Sixth Medical Center, The PLA General Hospital, Beijing 100048, China
    2. Department of Neurosurgery, The Seventh Medical Center, The PLA General Hospital, Beijing 100700, China
    3. Department of ENT-HN, Hainan Hospital of PLA General Hospital, Sanya 572013, China
    4. Department of Neurology, Fu Xing Hospital, Capital Medical University, Beijing 100038, China
  • Received:2019-02-09 Published:2019-04-15
  • Corresponding author: Ruxiang Xu
  • About author:
    Corresponding author: Xu Ruxiang, Email:
引用本文:

高谋, 徐如祥, 王文佳, 董勤, 丁柏匀, 姚慧, 杨志军. 诱导型神经干细胞移植对颅脑创伤后神经营养因子分泌的影响[J/OL]. 中华脑科疾病与康复杂志(电子版), 2019, 09(02): 101-105.

Mou Gao, Ruxiang Xu, Wenjia Wang, Qin Dong, Boyun Ding, Hui Yao, Zhijun Yang. Neurotrophic effects of induced neural stem cell grafts following traumatic brain injury[J/OL]. Chinese Journal of Brain Diseases and Rehabilitation(Electronic Edition), 2019, 09(02): 101-105.

目的

研究诱导型神经干细胞(iNSCs)移植对颅脑创伤(TBI)后神经营养因子分泌的影响。

方法

采用自由落体脑打击装置制备雄性成年C57BL/6小鼠TBI模型,将神经功能缺损评分(NSS)4~8分者纳入TBI组,按照随机数字表法分为iNSCs移植组(12只)和磷酸盐缓冲液(PBS)处理组(9只)。同时设置假手术(sham)组(9只)。于TBI后12 h,用脑立体定向仪将含1×106个iNSCs单细胞悬液或等体积PBS分别移植到TBI小鼠脑内,于移植后7 d处死动物。分别取脑组织mRNA(3只/组)行逆转录定量实时聚合酶链反应(RT-qPCR)和蛋白(6只/组)行酶联免疫吸附测定(ELISA),检测脑源性神经营养因子(BDNF)和胶质细胞源性神经营养因子(GDNF)表达量;取iNSCs移植组(3只)动物脑组织行免疫荧光染色,观察iNSCs移植物分泌BDNF和GDNF。

结果

RT-qPCR示:相比sham组,TBI小鼠脑组织中BdnfGdnf基因转录水平明显降低;相比PBS处理组,iNSCs移植组TBI小鼠脑组织中BdnfGdnf基因转录水平明显升高,差异具有统计学意义(P<0.05)。ELISA示:相比sham组,TBI小鼠脑组织中BDNF和GDNF蛋白表达水平明显降低;相比PBS处理组,iNSCs移植组TBI小鼠脑组织中BDNF和GDNF蛋白表达水平明显升高,差异具有统计学意义(P<0.05)。免疫荧光染色示iNSCs移植组TBI小鼠脑内可见绿色荧光蛋白标记的iNSCs移植物迁移到达脑损伤区并表达BDNF和GDNF。

结论

经脑立体定向移植iNSCs可在TBI后脑组织中合成分泌神经营养因子BDNF和GDNF,发挥神经营养作用。

Objective

To study the neurotrophic effects of grafted induced neural stem cells (iNSCs) following traumatic brain injury (TBI).

Methods

Healthy adult male C57BL/6 mouse TBI models were established using a standardized weight-drop device. Mice having an neurological severity scores (NSS) of 4-8 were randomly assigned to two groups: the iNSC group (n=12) and the phosphate buffered solution (PBS) group (n=9). Sham-operated mice underwent the same procedures, but not head trauma (n=9). At 12 h post-injury, iNSCs (1×106 cells) or PBS were separately injected into the brains of TBI mice via stereotactic method. On day 7 after TBI, animals were sacrificed for molecular-biological and morphological analysis. RT-qPCR and ELISA were utilized to determine the levels of BDNF and GDNF expression. To explore the neurotrophic effects of iNSCs, we utilized immunofluorescent staining to detect the expression of BDNF and GDNF in iNSC grafts.

Results

RT-qPCR assay revealed that the expression levels of Bdnf and Gdnf were significantly lower in the TBI group than in the sham group (P<0.05). Furthermore, the levels of Bdnf and Gdnf expression were significantly higher in the iNSC group than in the PBS group (P<0.05). ELISA indicated that the expression levels of BDNF and GDNF were significantly lower in the TBI group than in the sham group (P<0.05). Furthermore, the levels of BDNF and GDNF expression were significantly higher in the iNSC group than in the PBS group (P<0.05). Immunofluorescent staining showed that GFP-labelling iNSCs migrated to the damaged area and expressed BDNF and GDNF.

Conclusion

Stereotactic transplantation of iNSCs can express BDNF and GDNF to play neurotrophic effects following TBI.

表1 颅脑创伤7 d后的各组小鼠脑组织中BdnfGdnf mRNA表达倍数(Mean±SD)
表2 颅脑创伤7 d后的各组小鼠脑组织中BDNF和GDNF蛋白表达水平(Mean±SD,pg/mg)
图1 颅脑创伤7 d后的iNSCs移植组小鼠脑组织免疫荧光染色检测(×400)
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