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目的研究参与低氧预适应(HPC)的经典型蛋白激酶CβⅡ(cPKCβⅡ)相互作用的脑衰蛋白反应调节蛋白-2(CRMP-2)对缺血脑是否有保护作用。方法成年雄性BALB/c小鼠随机分为常氧(Nor)、HPC、常氧假手术(Nor+sham)、HPC假手术(HPC+sham)、常氧缺血(Nor+I)和HPC缺血(HPC+I)等6组(每组n=6)。应用小鼠整体HPC和脑中动脉梗死(MCAO)致脑局部缺血模型,结合免疫沉淀、双向凝胶电泳和质谱等技术,分离和鉴定与cPKCβⅡ相互作用蛋白;利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Western blot)技术,分析CRMP-2磷酸化和蛋白降解水平在脑HPC和缺血中的变化。结果与Nor组比,HPC鼠脑皮层组织内有10种与cPKCβⅡ相互作用蛋白的表达发生了明显变化,其中CRMP-2在膜相关蛋白组分中的表达量升高,而在胞质蛋白组分中的表达量降低。在脑缺血模型中,与Nor+Sham组相比,Nor+I组小鼠脑皮层缺血核心区(Ic)CRMP-2磷酸化水平明显降低(P<0.05,n=6);与Nor+I组相比,HPC+I组小鼠脑皮层Ic区内CRMP-2磷酸化水平明显增高(P<0.05,n=6)。脑缺血可导致CRMP-2发生水解并伴随着大量55ku水解片段(BDP)的出现,但与Nor+I组相比,HPC+I组小鼠脑皮层缺血半影区(P)内CRMP-2水解片段减少,水解率明显降低(P<0.05,n=6)。结论CRMP-2参与了HPC缓解小鼠脑缺血Ic区内CRMP-2磷酸化水平的降低和减少P区内CRMP-2水解片段从而减轻缺血脑组织的损伤。
Objective To investigate whether there is a protective effect of CRMP-2 on ischemic brain involved in the interaction of classical cPKCβⅡ with hypoxic preconditioning (HPC). Methods Adult male BALB / c mice were randomly divided into three groups: Nor, HPC, Nor + sham, HPC + sham, Nor + I and HPC Blood (HPC + I) and other 6 groups (n = 6 each group). The model of cerebral ischemia induced by whole cerebral artery occlusion (MCC) and middle cerebral artery occlusion (MCAO) in mice was established. The proteins interacting with cPKCβⅡ were isolated and identified by immunoprecipitation, two-dimensional gel electrophoresis and mass spectrometry. Polyacrylamide gel electrophoresis SDS-PAGE) and Western blotting were used to analyze the changes of CRMP-2 phosphorylation and protein degradation in cerebral HPC and ischemia. Results Compared with Nor group, the expression of 10 proteins interacting with cPKCβⅡ in HPC rat cortex changed significantly. The expression of CRMP-2 in membrane-associated protein components increased, while in cytoplasmic protein group The level of expression decreased. Compared with Nor + Sham group, the level of CRMP-2 phosphorylation in cortex ischemic core (Ic) of Nor + I group was significantly decreased (P <0.05, n = 6) Compared with I group, the level of CRMP-2 phosphorylation in cortex of Ic group was significantly increased in HPC + I group (P <0.05, n = 6). Cerebral ischemia resulted in the hydrolysis of CRMP-2 accompanied with the appearance of a large amount of 55ku hydrolytic fragment (BDP). Compared with Nor + I group, CRMP in cortex of ischemic penumbra of HPC + I group -2 hydrolysis fragments decreased, the hydrolysis rate was significantly reduced (P <0.05, n = 6). Conclusion CRMP-2 is involved in the reduction of CRP-2 phosphorylation and the decrease of CRMP-2 hydrolyzate in P area of ICC in mice with cerebral ischemia, and thus alleviate the damage of ischemic brain tissue.