吗啡后处理诱导小鼠海马脑片氧糖剥夺/再灌注耐受和对cPKCβⅡ/γ膜转位的影响  

Effects ofcPKCβⅡ/γ membrane translocation ischemic/hypoxic tolerance induced by morphine postconditioning in hippocampal slices

作　　者：胡海立[1] 杨静[1] 纪方[1] 李天佐[1] 李俊发[2] 

机构地区：[1]首都医科大学附属北京同仁医院麻醉科,100730 [2]首都医科大学神经生物学系,疼痛生物医学研究所

出　　处：《中华医学杂志》2011年第28期2011-2014,共4页National Medical Journal of China

基　　金：国家自然科学基金（30672023）;北京市教育委员会科技发展计划项目（KM200910025018）

摘　　要：目的 探讨吗啡药物后处理诱导海马脑片氧糖剥夺/再灌注损伤耐受的时间窗、经典型蛋白激酶CⅡ/γ亚型（cPKCβⅡ/γ）是否参与吗啡药物后处理诱导脑缺血再灌注损伤耐受.方法 急性离体小鼠海马脑片,随机分组：正常对照组（Con组）、氧糖剥夺组（Ogd组）、氧糖剥夺吗啡后处理组（Mor组）.观察氧糖剥夺20 min后予吗啡3 μmol/L孵育5、10、20、30、60 min后复氧复糖3 h各组孵育液中乳酸脱氢酶（LDH）漏出率、脑片细胞存活率并检测脑片cPKCβⅡ/γ膜转位的变化.结果 与Con组（99.9±0.0）%比,Ogd组（184.1±12.9）%和Mor 60min组（144.5±7.9）%LDH漏出率均增加（P〈0.05） ;与Ogd组比,Mor 20 min组（136.8±6.0）%、30 min组（142.3±6.2）%、60 min组（144.2±7.3）%LDH漏出率降低（P〈0.05）.与Con组比,Ogd组和Mor组脑片细胞存活率降低（P〈0.05） ;与Ogd组比,Mor 20、30 min组脑片存活率增加 （P〈0.05）.与Con组比较,Ogd组和Mor 5～60 min组cPKCγ膜转位水平均增高 （P〈0.05）;与Ogd组比较,Mor 20、30、60 min组cPKCγ膜转位水平下降 （P〈0.05）.而cPKCβⅡ的膜转位变化差异无统计学意义（P〉0.05）.结论 吗啡后处理对氧糖剥夺海马脑片有保护作用,其中cPKCγ膜转位可能与参与吗啡后处理对氧糖剥夺小鼠海马脑片耐受的形成.Objective To determine whether or not morphine postconditioning can induce ischemic/hypoxic tolerance in neurons subjected to reperfusion injury after oxygen-glucose deprivation （OGD）. Methods Hippocampal slices of 400 μm thickness were prepared from healthy adult male BALB/c mice. The slices were incubated in oxygen-saturated ACSF without or with calcium, then were subjected to OGD for 20 min. After recovery, the samples were immersed in oxygenated artificial fluid for 2 hours in the presence or absence of morphine postconditioning at 3 μmol/L during the first 5-60 min. The assessment of slices injury was performed by a determination of the intensity of slice stain incubated with TTC （2% 2, 3, 5-triphenyltetrazolium chloride） and the leakage rate of LDH also evaluated. At the designated periods during incubation, some slices were immersed into liquid nitrogen for a later analysis of Western blot. The frozen slices were homogenized, sonicated and centrifuged to separate soluble and particulate proteins. 10% SDS-PAGE Western blot was used to identify the changes of membrane-specific translocation of cPKCβⅡ/γ. Results After reperfusion, the cell survival significantly decreased with the elongation of OGD （51.4%）. The release rate of LDH （184.05%） significantly increased simultaneously. In hippocampal slices postconditioned with morphine for 20-60 min, the release rate of LDH （136%, 142%, 144%） significantly decreased as compared with the group OGD. In the hippocampal slices postconditioned with morphine for 10-30 min, the cell survival rate （64.9%, 69.9%, 63.5%） significantly increased as compared with reperfusion alone. cPKCγ of particulate fraction increased versus the control. And there was a corresponding decrease of cytosolic fraction. Morphine postconditioning significantly inhibited the cPKCγ isoform-specific membrane translocation. It declined from 136% in the group OGD to 123%, 118%, 114% in the group morphine 20-60 min. cPKCβⅡmembrane translocation had no change. Conclus

关 键 词：吗啡 脑 再灌注损伤 cPKCβⅡ/γ 

分 类 号：R614[医药卫生—麻醉学]