损伤脑组织提取液对体外神经干细胞存活和分化的影响  

Effect of brain traumatic tissue extract on the survival and differentiation of neural stem cells

作　　者：李梅 邓春萍 董化江[2] 路美[1] 

机构地区：[1]中国人民武装警察部队后勤学院附属医院脑科中心,天津300162 [2]天津大学精密仪器与光电子工程学院,300072

出　　处：《国际生物医学工程杂志》2017年第5期339-345,共7页International Journal of Biomedical Engineering

摘　　要：目的 模拟颅脑创伤（TBI）后的化学微环境,探讨该微环境对神经干细胞（NSCs）存活和分化的影响.方法 获取TBI大鼠损伤区脑组织的匀浆液,用于模拟TBI后的化学微环境.分离、提取并培养大鼠原代NSCs,采用免疫荧光染色鉴定其表型.将实验分为对照组、正常脑组织提取液组（BTE组）和创伤脑组织提取液组（TBITE组）.动态观察各组细胞的生长情况和形态变化,24 h后行Western Blot检测凋亡相关蛋白Bax、Bcl-2、Caspase-3及Cleaved caspase-3的表达水平;3d后采用噻唑蓝实验和Western Blot检测NSCs的增殖情况;7d后行免疫荧光染色检测NSCs向神经元分化的水平.结果 Western Blot结果显示,与对照组相比,BTE组无明显细胞凋亡变化,而TBITE组NSCs凋亡显著增多,Bax（F=18.06,P＜0.01）和Cleaved caspase-3（F=23.86,P＜0.01）表达升高,Bcl-2表达降低（F=22.95,P＜0.01）,差异均具有统计学意义.噻唑蓝实验结果显示,与BTE组相比,TBITE组NSCs增殖水平降低（F=41.99,P＜0.01）,差异具有统计学意义.免疫荧光染色结果显示,与对照组相比,BTE组神经元分化率升高;而与BTE组相比,TBITE组神经元分化率降低（F=66.93,P＜0.01）,差异均具有统计学意义.结论 TBI后的损伤微环境可显著抑制NSCs的存活和分化能力,从而为明确TBI后内源性神经的再生机制提供了理论基础.Objective To simulate the chemical microenvironment after traumatic brain injury （TBI） and to investigate the effect of this microenvironment on the survival and differentiation of neural stem cells （NSCs）.Methods The brain tissue homogenate of TBI rat model was harvested to simulate the chemical microenvironment after TBI.The primary NSCs of rat model were isolated and extraction,and then identified the phenotype characteristics with immunofluorescence staining.The experiments were divided into control group,normal brain tissue extract group （BTE group） and traumatic brain injury tissue extract group （TBITE group）.The cell growth and morphological changes of each group were observed dynamically.The expression of apoptosis related protein,which includes Bax,Bcl-2,caspase-3 and cleaved caspase-3,were detected by Western Blot 24 h after experiments.The proliferation of NSCs was detected by MTT assay and Western Blot after 3 days.The differentiation level of NSCs to neurons was detected by immunofluorescence staining after 7 days.Results The results of Western Blot showed that compared with the control group,there was no significant change of apoptosis in the BTE group,while the apoptosis in the BTE group was significantly increased,showed a increase of expression levels of Bax （F=18.06,P〈0.01） and Cleaved caspase-3 （F=23.86,P〈0.01）,and a decrease of that of Bcl-2 （F=22.95,P〈0.01）.The results of MTT assay showed that compared with the BTE group,the proliferation of NSCs in the TBITE group was decreased （F=41.99,P〈0.01）.The immunofluorescence staining showed that compared with the control group,the neuronal differentiation rate was increased in the BTE group.Further,compared with the BTE group,the neuronal differentiation rate in the TBITE group was decreased （F=66.93,P〈0.01）.Conclusion The injury microenvironment after TBI can significantly inhibit the survival and differentiation of NSCs,which provides a theoretical basis for clarifying the mechanism of endogenous nerve

关 键 词：颅脑创伤 化学微环境 神经干细胞 脑组织提取液 

分 类 号：R651.15[医药卫生—外科学]