CAJ | 학술논문

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55 ku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.
목적 연구삼여저양예괄응(HPC)적경전형단백격매CβⅡ(cPKCβⅡ)상호작용적뇌쇠단백반응조절단백-2(CRMP-2)대결혈뇌시부유보호작용.방법 성년웅성BALB/c소서수궤분위상양(Nor)、HPC、상양가수술(Nor+sham)、HPC가수술(HPC+sham)、상양결혈(Nor+I)화HPC결혈(HPC+I)등6조(매조n=6).응용소서정체HPC화뇌중동맥경사(MCAO)치뇌국부결혈모형,결합면역침정、쌍향응효전영화질보등기술,분리화감정여cPKCβⅡ상호작용단백;이용취병희선알응효전영(SDS-PAGE)화단백인적(Western blot)기술,분석CRMP-2린산화화단백강해수평재뇌HPC화결혈중적변화.결과 여Nor조비,HPC서뇌피층조직내유10충여cPKCβⅡ상호작용단백적표체발생료명현변화,기중CRMP-2재막상관단백조분중적표체량승고,이재포질단백조분중적표체량강저.재뇌결혈모형중,여Nor+Sham조상비,Nor+I조소서뇌피층결혈핵심구(Ic)CRMP-2린산화수평명현강저(P＜0.05,n=6);여Nor+I조상비,HPC+I조소서뇌피층Ic구내CRMP-2린산화수평명현증고(P＜0.05,n=6).뇌결혈가도치CRMP-2발생수해병반수착대량55 ku수해편단(BDP)적출현,단여Nor+I조상비,HPC+I조소서뇌피층결혈반영구(P)내CRMP-2수해편단감소,수해솔명현강저(P＜0.05,n=6).결론 CRMP-2삼여료HPC완해소서뇌결혈Ic구내CRMP-2린산화수평적강저화감소P구내CRMP-2수해편단종이감경결혈뇌조직적손상.
Objective To investigate whether conventional protein kinase C (cPKC ) βⅡ-interacting collapsin response mediator protein-2 (CRMP-2) provides neuroprotection against cerebral ischemic (I) injuries. Methods Male BALB/c mice were randomly divided into normoxic control (Nor) , HPC, Nor + Sham, HPC + Sham, Nor + I and HPC + I groups (n = 6 per group). Using our HPC and MCAO mouse models, we applied immunoprecipita-tion, two-dimensional electrophoresis and mass spectrometry to characterize cPKCβⅡ-interacting proteins and combined with SDS-PAGE and Western blot to quantitatively analyze CRMP-2 phosphorylation and degradation levels in the brain of mice after HPC and MCAO. Results The expression level of 10 cPKCβⅡ-interacting proteins changed obviously in cerebral cortex of HPC mice when compared with Nor group. One of these proteins, CRMP-2 protein level increased in particulate fraction and decreased in cytosolic fraction of cerebral cortex of HPC mice. CRMP-2 phosphorylation level in ischemic core (Ic) of cerebral cortex decreased significantly ( P < 0. 05 , n = 6) as compared with that of Nor + sham group, but CRMP-2 phosphorylation level in HPC +I group increased significantly as compared with that of Nor +I group ( P < 0. 05, n = 6). In ischemic cortex, CRMP-2 degradation (proteolysis) was observed as the appearance of 55 ku breakdown products (BDP). However, the CRMP-2 degradation level, BDPs products decreased significantly in penumbra ( P) of ischemic cortex from HPC +I group when we compared with that of Nor +I group (P < 0. 05, n = 6 ). Conclusion CRMP-2 is involved in attenuating the decrease of CRMP-2 phosphorylation in ischemic core and in inhibiting its degradation in penumbra of cerebral cortex of mice thereby to lessen the ischemic injuries.