颅脑创伤动物血清预处理诱导型神经干细胞移植对补体活化的影响  

Effects of stereotaxic transplantation of induced neural stem cells receiving traumatic brain injury mouse serum pre-treatment on complement activation

作　　者：高谋 徐如祥[2] 王文佳 董勤[4] 丁柏匀 姚慧 杨志军 Gao Mou;Xu Ruxiang;Wang Wenjia;Dong Qin;Ding Boyun;Yao Hui;Yang Zhijun(Department of Neurosurgery,The Sixth Medical Center,The PLA General Hospital,Beijing 100048,China;Affiliated Bayi Brain Hospital,The Seventh Medical Center,The PLA General Hospital,Beijing 100700,China;Department of ENT-HN,Hainan Hospital of PLA General Hospital,Sanya 572013,China;Department of Neurology,Fu Xing Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]解放军总医院第六医学中心神经外科,北京100048 [2]解放军总医院第七医学中心附属八一脑科医院,北京100700 [3]解放军总医院海南医院耳鼻咽喉头颈外科,三亚572013 [4]首都医科大学附属复兴医院神经内科,北京100038

出　　处：《中华神经创伤外科电子杂志》2019年第4期227-232,共6页Chinese Journal Of Neurotraumatic Surgery：Electronic Edition

基　　金：国家自然科学基金面上项目（81671189）;全军“十三五”军事医学创新工程项目（16CXZ001)

摘　　要：目的研究颅脑创伤(TBI)动物血清预处理诱导型神经干细胞(iNSCs)立体定向移植对TBI后补体活化的影响。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48只神经功能缺损评分(NSS)为4~8分的小鼠纳入TBI组,按照随机数字表法选取24只用于制备TBI小鼠血清和热灭活TBI小鼠血清,余下24只按照随机数字表法分为TBI小鼠血清预处理iNSCs(TBI-iNSCs)移植组(6只)、热灭活TBI小鼠血清预处理iNSCs(HITBI-iNSCs)移植组(6只)、磷酸盐缓冲液(PBS)预处理iNSCs(PBS-iNSCs)移植组(6只)和对照(Control)组(6只)。同时设置假手术(Sham)组(6只)。于TBI后12h,用脑立体定向仪分别将含1×106个TBI-iNSCs、HITBI-iNSCs、PBS-iNSCs单细胞悬液或等体积PBS移植到TBI小鼠脑内。于TBI后14d处死动物,取脑组织行Westernblot检测补体活化产物(C3d和C9)、补体调节因子Crry和细胞凋亡标记物activeCaspase-3等蛋白表达水平;取脑组织行免疫荧光染色和原位末端标记(TUNEL)染色,观察TBI-iNSCs、HITBI-iNSCs和PBS-iNSCs移植对TBI小鼠脑内NeuN抗体阳性和TUNEL阳性细胞数量的影响。结果Westernblot检测示:相比Sham组,PBS-iNSCs组、HITBI-iNSCs组、TBI-iNSCs组和Control组C3d、C9和activeCaspase-3表达水平明显增高;而相比Control组,PBS-iNSCs组、HITBI-iNSCs组和TBI-iNSCs组C3d、C9和activeCaspase-3表达水平明显降低;相比PBS-iNSCs组和HITBI-iNSCs组,TBI-iNSCs组C3d、C9和activeCaspase-3表达水平明显降低,组间差异具有统计学意义(P<0.05)。此外,相比Control组,PBS-iNSCs组、HITBI-iNSCs组、TBI-iNSCs组和Sham组Crry表达水平明显增高;相比Sham组,PBS-iNSCs组、HITBI-iNSCs组和TBI-iNSCs组Crry表达水平明显增高;相比PBS-iNSCs组和HITBI-iNSCs组,TBI-iNSCs组Crry表达水平明显增高,组间差异具有统计学意义(P<0.05)。免疫荧光染色和TUNEL染色示:PBS-iNSCs组和HITBI-iNSCs组NeuN抗体阳性和TUNEL阳性的细胞数量差异无统计学意义;然而,与PBS-iNObjective To study the effects of stereotaxic transplantation of induced neural stem cells (iNSCs) receiving traumatic brain injury (TBI) mouse serum pre-treatmenton complement activation following 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 enrolled in TBI group (n=48). Twenty-four TBI mice were randomly selected for preparation of TBI mouse serum and heat-inactivated TBI mouse serum, andthe other 24 mice were randomly divided into 4 groups: the TBI-iNSC group (n=6), the HITBI-iNSC group (n=6), the phosphate buffered solution (PBS)-iNSC group (n=6) and the Control group (n=6). Sham-operated mice underwent the same procedures, but not head trauma (n=6). At 12 h after TBI, TBI-iNSCs, HITBI-iNSCs, PBS-iNSCsor PBS were separately injected into the brains of TBI mice via stereotactic method. On day 14 after TBI, animals were sacrificed for molecular-biological andmorphological analysis. We performed western blot to analyze the expression of the C3d, C9, Crry and active Caspase-3 in brain tissues. Next, immunofluorescent stainingand terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining were utilized to determine the effects of TBI-iNSC, HITBI-iNSC and PBS-iNSC grafts on NeuN+ and TUNEL+ cells in the brains of TBI mice. Results Western blot analysis revealed that the expression of C3d, C9 and active Caspase-3 in the brains of the Sham group was significantly lower than that in PBS-iNSC, HITBI-iNSC, TBI-iNSC and Control groups (P<0.05). In contrast, the expression of C3d, C9 and active Caspase-3 in the brains of the Control group was significantly higher than that in PBS-iNSC, HITBI-iNSC and TBI-iNSC groups (P<0.05). Moreover, the levels of C3d, C9 and active Caspase-3 in the brain were substantially higher in the PBS-iNSC and HITBI-iNSC groups than in the TBI-iNSC group (P<0.05). Additionally, the expression of Crry in the brain of the Control group was significan

关 键 词：诱导型神经干细胞 颅脑创伤 Crry因子 补体调节分子 立体定向移植 

分 类 号：R651.15[医药卫生—外科学] R-332[医药卫生—临床医学]