miR-20b对重型创伤性脑损伤小鼠运动功能障碍的影响及其可能机制  

Effect of miR-20b on the motor dysfunction after traumatic brain injury in mice and the underlying mechanism

作　　者：赵程程 刘玉飞[2] 李赟[2] 何毅[2] 张协军[2] 李维平[2] Zhao Chengcheng;Liu Yufei;Li Yun;He Yi;Zhang Xiejun;Li Weiping(Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging,School of Biomedical Engineering,Shenzhen University Health Science Center,Shenzhen 518060,China;Department of Neurosurgery,First Affiliated Hospital of Shenzhen University,Shenzhen Second People′s Hospital,Shenzhen 518025,China)

机构地区：[1]深圳大学医学部生物医学工程学院,广东省生物医学信息检测与超声成像重点实验室,深圳518060 [2]深圳大学第一附属医院,深圳市第二人民医院神经外科,深圳518025

出　　处：《中华创伤杂志》2022年第3期260-267,共8页Chinese Journal of Trauma

基　　金：国家自然科学基金(81772685);广东省基础与应用基础研究基金(2019A1515011241);中国博士后科学基金(2021M702287)。

摘　　要：目的:探讨miR-20b对重型创伤性脑损伤(TBI)小鼠运动功能障碍的影响及其可能机制。方法:将60只C57BL/6J小鼠按随机数字表法分为假手术组、TBI组和TBI+miR-20b激动剂(Agomir-20b)组,每组20只。采用控制性脑皮质损伤法构建重型TBI模型。伤后TBI组尾静脉立刻注射50μmol/L Agomir阴性对照溶液200μl,TBI+Agomir-20b组尾静脉立刻注射50μmol/L Agomir-20b溶液200μl。伤后3,7 d,采用TUNEL法检测神经元凋亡比例;Western blot检测损伤周围区皮层凋亡相关蛋白[活化的半胱氨酸蛋白酶-3(caspase-3)、活化的多聚腺苷二磷酸核糖聚合酶(PARP)、B细胞淋巴瘤-2(Bcl-2)及Bcl-2相关X蛋白(Bax)]的表达水平;爬杆试验评估小鼠神经行为学变化;实时荧光定量PCR(RT-qPCR)检测损伤周围区皮层炎症因子[白细胞介素(IL)-1β、IL-6、IL-10、诱导型一氧化氮合酶(iNOS)、精氨酸酶(Arg)及巨噬细胞甘露糖受体-1(CD206)]mRNA表达水平。结果:TUNEL法显示,伤后3,7 d,假手术组小鼠损伤周围区皮层神经元凋亡比例较TBI组及TBI+Agomir-20b组显著减少(P均<0.01)。伤后3,7 d,TBI+Agomir-20b组小鼠损伤周围区皮层神经元凋亡比例较TBI组显著减少(P均<0.01)。Western blot显示,与假手术组相比,TBI组活化的caspase-3、活化的PARP及Bax蛋白表达水平在伤后3,7 d显著升高(P均<0.01),Bcl-2蛋白表达水平在伤后3,7 d显著降低(P均<0.01);而TBI+Agomir-20b组活化的caspase-3、活化的PARP及Bax蛋白表达水平在伤后3,7 d无显著改变(P均>0.05),Bcl-2蛋白表达水平在伤后3 d显著降低(P<0.01),伤后7 d无显著改变(P>0.05)。与TBI+Agomir-20b组相比,TBI组活化的caspase-3、活化的PARP及Bax蛋白在伤后3,7 d显著升高,Bcl-2蛋白表达水平显著降低(P均<0.05或0.01)。爬杆试验显示,伤后3,7 d,TBI组上台时间及步滑比例大于假手术组和TBI+Agomir-20b组(P均<0.01)。RT-qPCR显示,与TBI+Agomir-20b组相比,TBI组IL-1β、IL-6及iNOS mRNA表达水平在伤后3,7 d显著�Objective To investigate effect of miR-20b on the motor dysfunction after traumatic brain injury(TBI)in mice and the underlying mechanism.Methods Sixty C57BL/6J mice were divided into sham group,TBI group and TBI+miR-20b Agomir(Agomir-20b)group according to the random number table,with 20 mice per group.A model of severe TBI was induced by controlled cortical impact.After injury,the mice in TBI group were subjected to tail-vein injection of 200μl Agomir-negative control at dosage of 50μmol/L and the mice in TBI+Agomir-20b group were subjected to tail-vein injection of 200μl Agomir-20b at dosage of 50μmol/L.At days 3 and 7 postinjury,the rate of neuronal apoptosis in the pericontusional region was detected by TUNEL assay,expression levels of apoptosis-related proteins in the pericontusional region were detected by Western blot analysis,including cleaved caspase-3,cleaved poly adenosine diphosphate-ribose polymerases(PARP),B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax),motor function was evaluated by beam walking test,and expression levels of cytokine mRNAs in the pericontusional region were detected by real-time quantitative PCR(RT-qPCR),including interleukin(IL)-1β,IL-6,IL-10,inducible nitric oxide synthase(iNOS),arginase(Arg)and macrophage mannose receptor 1(CD206).Results In TUNEL assay,the rate of neuronal apoptosis in sham group was significantly lower than that in TBI group and TBI+Agomir-20b group at days 3 and 7 postinjury(all P<0.01),and there was a significantly lower rate of neuronal apoptosis in TBI+Agomir-20b group as compared with TBI group(all P<0.01).In Western blot analysis,significantly increased levels of cleaved caspase-3,cleaved PARP and Bax proteins and lowered level of Bcl-2 protein were observed in TBI group at days 3 and 7 postinjury as compared with sham group(all P<0.01);similar levels of cleaved caspase-3,cleaved PARP and Bax proteins were found in TBI+Agomir-20b group at days 3 and 7 postinjury as compared with sham group(all P>0.05),and level of Bcl-2 protein in TBI+

关 键 词：脑损伤 神经元 凋亡 

分 类 号：R651.15[医药卫生—外科学]