【摘要】 目的研究氯胺酮预先给药对缺氧复氧诱导大鼠小脑颗粒神经元(CGNs)凋亡的影响,及磷脂酰肌醇-3-激酶(P13K)/Akt通路在其中的作用。方法出生7～8 d的清洁级SD大鼠,雌雄不拘,体重15～20g；制备CGNs,培养的8d的CGNs随机分为5组,对照组(A组)、缺氧复氧组(B组)、氯胺酮预先给药组(C组)、PI3K/Akt通路的特异性抑制刺Ly294002-氯胺酮预先给药组(D组)、Ly294002组(E组)。B组将CGNs放入特制缺氧盒中,缺氧3h后复氧；C组缺氧前1h加入200μmol/L氯胺酮；D组加入氯胺酮前30 min加入20μmol/L Ly294002；E组加入20μmol/L Ly294002。复氧16h后进行下述指标的观察。二乙酸荧光素染色法测定CGNs存活率,Hoechst 33258核染色检测CGNs凋亡细胞核,琼脂糖凝胶电泳检测DNA片断化水平,蛋白免疫印迹法(Westem blot法)检测CGNs磷酸化A kt、磷酸化GSK3β及总Akt水平。结果缺氧复氧可诱导CGNs的凋亡,降低CGNs磷酸化Akt和磷酸化GSK3β水平(P＜0．05),氯胺酮预先给药可减轻缺氧复氧诱导的上述改变,氯胺酮对CGNs的这种保护作用可被Ly294002部分抑制。结论氯胺酮预先给药可减轻缺氧复氧诱导大鼠CGNs凋亡,其机制与激活PI3K/Akt通路有关。更多还原

【Abstract】 Objective To investigate the effect of ketamine pretreatment on hypoxia/reoxygenation （H/ R）-induced apoptosis in cultured rat cerebellar granule neurons （CGNs） and determine if phosphafidyl-inositol 3- kinase （PI3K）/Akt pathway is involved in the mechanism of protection.Methods CGNs were isolated from 7-8 day old SD rats of beth sexes weighing 15-20 g and cultured for 8 days and then divided into 5 groups:Ⅰcontrol group;ⅡH/R group CGNs were placed in a hypoxia box for 3 h and then reoxygenated;Ⅲketamine pretreatment group——CGNs were incubated with ketamine 200μmol·L^-1 for 1 h before H/R;ⅣLy294002-ketamine pretreatment group——CGNs were first incubated with Ly294002 （a specific P13K inhibitor） 20μmol·L^-1 for 30 min and then with ketamine 200μmol·L^-1 for another 1 h before H/R;V Ly 294002 pretreatment group——CGNs were incubated with Ly294002 20μmol·L^-1 for 30 min before H/R.After 16 h reoxygenation the neuronal survival was determined by fluoreseein diacetat （FDA） staining and apoptofic nuclei were detected by Hoechst 33258 nuclear staining.DNA fragments were detected by agarose gel electrophoresis and western blot analysis was used to detect the levels of phosphor-Akt,phosphor-GSK3βand total Akt.Results H/R significantly increased apoptosis in CGNs and decreased neuronal survival rate and the levels of phosphor-Akt and phosphor-GSK3βin CGNs as compared to the control group.Ketamine pretreatment protected CGNs against apoptosis induced by H/R and significantly increased the neuronal survival rate and the levels of phosphor-Akt and phosphor-GSK3βas compared with H/R group.Ly294002 attenuated the neuroprotective effect of ketamine pretreatment.Conclusion Ketamine can protect CGNs against apoptosis induced by H/R via P13K/Akt pathway.更多还原