大鼠脊髓损伤后β淀粉样蛋白的表达及γ分泌酶抑制剂对其作用机制的影响  

Effects of expression of amyloid β protein and γ-secretase inhibitor on mechanisms of spinal cord injury in rats

作　　者：郭磊[1,2] 侯景明[3] 钟剑峰[2] 刘佳[2] 孟繁星[2] 陈杨葭 冯杰扬 焦皎[2] 孙天胜[1,2] 

机构地区：[1]解放军医学院,北京100853 [2]北京军区总医院全军创伤骨科研究所,北京100700 [3]第三军医大学附属西南医院,重庆400038

出　　处：《解放军医学院学报》2016年第4期371-376,共6页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金项目(81301679)~~

摘　　要：目的探讨β淀粉样蛋白(amyloidβ,Aβ)在大鼠脊髓损伤(spinal cord injury,SCI)后的表达变化及γ分泌酶抑制剂DAPT对其作用机制的影响。方法选用雌性SD大鼠117只,随机分成3组:假手术组(Sham组,31只)、脊髓损伤组(SCI组,43只)、干预组(DAPT组,43只),采用Allen’s打击法制作大鼠脊髓损伤模型,DAPT组于造模前及术后给予DAPT药物溶液胃管灌注,并于术后6 h、1 d、3 d、7 d、14 d、21 d、28 d取血液、脑脊液及损伤段脊髓组织,分别行酶联免疫吸附试验(ELISA)检测血清和脑脊液Aβ水平,免疫印迹法(Western blot)检测脊髓组织淀粉样前体蛋白(amyloid precursor protein,APP)、β淀粉样蛋白、轴突生长抑制因子受体(neurite growth inhibitor receptor 1,Ng R1)、Rho相关激酶2(Rho related kinase 2,Rock2)蛋白水平,免疫组化分析观察Aβ表达并计数阳性细胞,采用BBB评分评估大鼠后肢运动功能恢复情况。结果 SCI组大鼠Aβ水平明显高于Sham组(P<0.05),血清和脑脊液中Aβ水平在SCI后6 h开始升高,3 d时达到高峰,7 d时降至正常水平,应用DAPT能够明显降低SCI后Aβ水平(P<0.05);脊髓损伤后3 d SCI组Aβ、Ng R1、Rock2蛋白水平较Sham组明显升高(P<0.05),DAPT组Aβ、Ng R1、Rock2蛋白水平较SCI组明显降低(P<0.05);免疫组化分析可见损伤区脊髓Aβ阳性细胞明显增多,DAPT组Aβ阳性细胞数明显少于SCI组(P<0.05);DAPT组大鼠BBB评分明显高于SCI组(P<0.05);SCI大鼠血清和脑脊液Aβ水平与运动功能BBB评分呈负相关(P<0.05)。结论大鼠SCI后Aβ表达上调,通过应用γ分泌酶抑制剂DAPT减少Aβ表达能够促进运动功能恢复,其机制可能是通过下调Ng R1蛋白表达、抑制Rho/Rock通路激活,从而减少白质区髓鞘结构损伤。Objective To explore expression changes of amyloid β（Aβ） protein after spinal cord injury in rats and the effect of γ-secretase inhibitor DAPT on mechanisms of spinal cord injury in rats. Methods Totally 117 adult SD rats were randomly divided into three groups： sham group（n=31）, spinal cord injury（SCI） group（n=43） and SCI＋DAPT（DAPT） group（n=43）. Models of spinal cord injury were established by modified Allen’s assay, and rats in DAPT group were treated with γ secretase inhibitor DAPT by gastric tube perfusion before and after SCI procedure. Rats were sacrificed at 6 h, 1 d, 3 d, 7 d, 14 d, 21 d, 28 d after injury. Protein levels of Aβ in serum and cerebro-spinal fluid（CSF） were determined by ELISA assay, and content of amyloid precursor protein β（APP）, Aβ, Axon growth inhibitory factor receptor（Ng R1）, Rho related kinase（Rock2） were determined by Western blot. Aβ positive cells in injured spinal cord were observed with immunohistochemical analysis, and hind limb motor function of rats was evaluated weekly by BBB（Basso Beattie and Bresnahan） scores. Results Protein levels of Aβ in serum and CSF in SCI group elevated from 6 h（P 〈0.05） and reached the peak at 3 d post- injury with a time-dependent manner, which was significantly higher than sham group（P 〈0.05）. Administration of DAPT led to a significant decrease of Aβ protein levels in serum and CSF（P 〈0.05）. Compared with sham group, the protein level of Aβ, Ng R1 and Rock2 in SCI group at 3 d after spinal cord injury increased significantly（P 〈0.05）, while it decreased significantly in DAPT group when compared with SCI group（P 〈0.05）. Immunohistochemical analysis showed that the amount of Aβ positive cells in injured spinal cord increased significantly, and it was significantly less in DAPT group than SCI group（P 〈0.05）, and the BBB score in DAPT group was significantly higher than SCI group（P 〈0.05）. There existed a negative correlation between Aβ levels in

关 键 词：脊髓损伤 Β淀粉样蛋白 轴突生长抑制因子受体 Rho相关激酶 大鼠 

分 类 号：R651.2[医药卫生—外科学]