摘要
目的评价二氮嗪预处理对大鼠海马神经细胞缺氧复氧时c—Jun氨基末端激酶（JNK）表达的影响。方法SD大鼠100只，出生〈24h，体重5～6g，断头取海马组织，制备海马神经细胞悬液，海马神经细胞接种于96孔板或直径35mm培养皿中，培养9～10d后，随机分为4组，每组36孔或8皿：对照组（C组）、缺氧复氧组（A／R组）、二氮嗪30μmol／L组（D1组）和二氮嗪100μmol／L组（D2组）。C组不行任何处理；A／R组置于特制的无菌密闭容器，容器中通入95％N2-5％CO2混合气体，流速0．2L／min，4h后放回原培养箱继续培养24h；D1组和D2组在培养液中加入二氮嗪，终浓度分别为30、100μmol／L，孵育1h后用全量培养液洗脱，1次／d，连续3d，随后处理同A／R组。于培养24h时行免疫组化染色，观察神经细胞病理学结果，四甲基偶氮唑蓝比色法测定海马神经细胞活力，Westernblot法测定海马神经细胞Bcl-2、Bax和JNK的表达。结果与c组比较，其余3组海马神经细胞Bcl-2、Bax和JNK表达均上调，活力降低（P〈0．05或0．01）；与A／R组比较，D。组海马神经细胞Bcl-2表达上调，Bax和JNK表达下调，活力升高（P〈0．05或0．01），D．组上述指标差异无统计学意义（P〉0．05）。结论二氮嗪预处理可能通过下调JNK的表达，抑制JNK信号通路，维持Bcl-2与Bax的平衡，减轻大鼠海马神经细胞缺氧复氧损伤。
Objective To investigate the effects of diazoxide.preconditioning on the expression of C-Jun N-terminal kinase （JNK） in primary cultured rat hippocampal neurons after anoxia-reoxygenation （A/R）. Methods One hundred SD rats （ 〈 24 h after birth） weighing 5-6 g were decapitated. Their hippocampi were removed and cut into chunks. Hippocampal neurons were enzymatically isolated. After being cultured for 9-10 d, the hippocampal neurons were randomly divided into 4 groups： group Ⅰ control （group C） ; group Ⅱ A/R; group Ⅲ ,Ⅳ preconditioning with diazoxide 30 and 100 μmol/L （group D1 , D2 ） . In group A/R, D1 and D2 the neurons were exposed to 95 % N2-5 % CO2 for 4 h followed by 24 h reoxygenation. In group D1 and D2 the neurons were pretreated with diazoxide 30 and 100 μmol/L respectively for 1 h before A/R for 3 consecutive days. The viability of neurons was measured by MTF assay. The expression of Bcl-2, Bax and JNK proteins was detected by Western blot analysis. Results The viability of neurons was significant lower in group A/R, D1 and D2 than in control group and was significantly higher in group D1 than in group A/R. The expression of Bcl-2, Bax and JNK proteins was significantly up-regulated in group A/R, D1 and D2 as compared with control group. The expression of Bcl-2 was significantly higher while the Bax and JNK protein expression was significantly lower in group D2 than in group A/R. Conclusion Preconditioning with diazoxide 100 μmol/L can ameliorate A/R injury to hippocampal neurons by down-regulating Bax expression and up-regulating Bcl-2 expression through inhibition of JNK signaling transduction pathway.
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2009年第4期368-371,共4页
Chinese Journal of Anesthesiology
