Ephrin-B2基因敲除小鼠脑损伤后星形胶质细胞活化增强的研究

Reactive astrocytes increase after traumatic brain injury in Ephrin-B2 knock-out mice

目的 观察小鼠脑损伤后Ephrin-B2配体在脑内的表达规律及Ephrin-B2信号传导在损伤修复过程中的作用.方法 培育Ephrin-B2基因敲除（KO）小鼠,将KO和C57BL/6野生型（WT）小鼠各116只建立可控性皮层损伤模型,且随机分为4组,于损伤后3、7、14、21 d4个时间点采集标本,进行以下检测：免疫荧光组织化学染色检测脑损伤后Ephrin-B2和胶质纤维酸性蛋白（GFAP）的表达;Western blot检测损伤周围Ephrin-B2、磷酸化Ephrin-B2（pEphrin-B2）和GFAP的表达变化;实时荧光定量聚合酶链反应（FQ-PCR）检测损伤周围及损伤同侧海马区域GFAP mRNA水平;尼氏染色检测损伤后21 d时损伤空洞体积.平衡木行走试验进行运动协调及平衡功能评分.体外划痕试验检测KO星形胶质细胞长入损伤区域的数量.结果 免疫组织化学染色和Western blot检测显示,WT小鼠脑损伤后21d内,Ephrin-B2和pEphrin-B2蛋白在损伤周围表达明显增加,主要表达于活化的星形胶质细胞膜和突起上.在脑损伤后早期（3 d）,KO小鼠活化星形胶质细胞的数量较WT小鼠明显增加,GFAP的表达上调;FQ-PCR检测显示KO小鼠损伤周围及同侧海马区域GFAP mRNA水平均较WT小鼠呈明显增高.尼氏染色显示KO小鼠在脑损伤后21d时形成的损伤空洞较小.划痕试验显示KO组星形胶质细胞增殖数量较WT组显著增多.行为学评分示KO小鼠在脑损伤后神经功能恢复较好.结论 Ephrin-B2反向信号传导在脑外伤后调控星形胶质细胞活化中起关键作用.

Objective To explore expression of Ephrin-B2 and its role of signal transduction on repair after traumatic brain injury （TBI） in mice.Methods Ephrin-B2 knock-out （KO） mice were bred.Controlled cortical impact （CCI） injury model was induced with each 116 KO and Wild-Type （WT） mice,and the animals were divided randomly into four groups.At 4 time points,including 3,7,14 and 21 d,their brain specimens were collected.Immunofluorescence was employed to detect alternations of Ephrin-B2 and glial fibrillary acidic protein （GFAP） after TBI.Western blotting were employed to determine alternations in Ephrin-B2,pEphrin-B2 and GFAP expression and GFAP mRNA level in injured cortex and ipsilateral hippocampus was tested by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） ; the volume of necrotic cavity was measured with Nissl Staining on 21 days; Beam walking test was conducted to detect the coordination and balance of motor.In scratch wound assay,the number of astrocytes entering the wound area was determined.Results Immunofuorescence and Western blotting showed Ephrin-B2,in WT mice,was strongly upregulated on the membrane and neurites of reactive astrocytes at the lesion epicenter during 21 days post-injury.On early stage after TBI,the number of reactive astrocytes in KO mice was more than WT mice,so was GFAP.The same mRNA change was detected for GFAP at the lesion epicenter and ipsilateral hippocampus.Nissl staining showed KO mice had smaller lesion volume at 21 days after TBI.Astrocytic behavior in a scratch wound assay revealed cell density was significantly greater in KO groups compared with WT groups.Better recovery of motor functions was found after injury in KO mice.Conclusion Ephrin-B2 ligand is a key regulator for reactive astrocytes after TBI by reverse signal.