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

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

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Bdnf和Gdnf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Bdnf和Gdnf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.