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目的 观察 3 异丁基 1 甲基黄嘌呤 ( 3 Isobutyl 1 methylxanthine ,IBMX)对 5mmol·L-1KCl诱导的大鼠小脑颗粒神经元凋亡的拮抗作用并探讨其与 [cAMP]i和Ca2 +的关系。方法 建立KCl 5mmol·L-1诱导的大鼠小脑颗粒神经元凋亡模型 ,用荧光二酯素法分析神经元存活 ,Hoechest332 5 8核染色和DNA琼脂糖凝胶电泳分析凋亡 ,放射免疫法测定cAMP浓度。结果 IBMX呈浓度依赖性拮抗 5mmol·L-1KCl诱导的神经元死亡 ,并明显减少核形态异常的神经元 ,使DNA电泳梯带变浅或完全消失 ;IBMX( 1.0mmol·L-1)可持续升高神经元 [cAMP]i,福司可林 2 .0 μmol·L-1升高 [cAMP]i作用与IBMX类似 ,但不能模拟IBMX的作用 ;此外 ,IBMX的拮抗作用不被cAMP竞争性抑制剂Rp cAMP和PKA的特异阻断药H 89阻断 ;单用或联用钙调素依赖的蛋白激酶Ⅱ的特异抑制剂KN 93和磷酯酰肌醇 3位羟基激酶的特异性抑制剂LY2 940 0 2也不能阻断。结论IBMX抗低钾诱导的大鼠小脑颗粒神经元凋亡作用不依赖cAMP和Ca2 +。
Objective To observe the antagonism of 3 Isobutyl 1 methylxanthine (IBMX) on apoptosis of rat cerebellar granule neurons induced by 5 mmol·L-1 KCl and to explore its antagonism with [cAMP] i and Ca2 + relationship. Methods The apoptosis model of cerebellar granule neurons induced by KCl 5 mmol·L-1 in rats was established. The survival of neurons was analyzed by the fluorescent diester method, apoptosis was analyzed by Hoechest3328 nuclear staining and DNA agarose gel electrophoresis, and radioimmunoassay The cAMP concentration was measured. Results IBMX antagonized 5 mmol·L-1 KCl-induced neuronal death in a concentration-dependent manner and significantly reduced the number of neurons with abnormal nuclear morphology, which led to the shallowing or disappearance of DNA ladder. IBMX (1.0 mmol·L-1) Elevated neuron [cAMP] i, forskolin 2 .0 μmol·L-1 increased [cAMP] i similar to IBMX, but not simulate the role of IBMX; In addition, IBMX antagonistic effect is not cAMP competitive inhibition Agent Rp cAMP and PKA blockers H89 block; single or combined with calmodulin-dependent protein kinase Ⅱ specific inhibitor KN 93 and phosphatidylinositol 3-hydroxy kinase specific inhibitor LY2 940 0 2 can not be blocked. Conclusion IBMX anti-hypokalemia-induced apoptosis of rat cerebellar granule neurons is independent of cAMP and Ca2 +.