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背景:在应激反应中,下丘脑-垂体-肾上腺皮质轴的激活对下丘脑促肾上腺皮质激素释放激素表达的增加起着关键的作用。然而关于通过何种途径调控下丘脑神经元表达促肾上腺皮质激素释放激素的神经内分泌活性,促肾上腺皮质激素释放激素能否激活下丘脑神经元,尚不十分清楚。目的:通过外源性促肾上腺皮质激素释放激素刺激培养下丘脑神经元,以观察细胞内环磷酸腺苷和胞浆内钙离子浓度的变化。设计:观察对比实验。单位:解放军第三军医大学大坪医院野战外科研究所,首都医科大学附属天坛医院神经外科。材料:实验于1999-12/2002-03在解放军第三军医大学大坪医院完成。取孕17d胎鼠下丘脑分散培养下丘脑神经元。方法:用外源性促肾上腺皮质激素释放激素刺激培养下丘脑神经元,促肾上腺皮质激素释放激素1受体特异性拮抗剂CP-154526预先处理下丘脑神经元。将培养的细胞分组:①促肾上腺皮质激素释放激素(10-12,10-10,10-8,10-6mol/L)刺激组。②预先用CP-154526(500μmol/L)处理再用促肾上腺皮质激素释放激素(10-12,10-10,10-8,10-6mol/L)刺激组。③分别设置相应的正常对照组,正常对照组用等渗盐水刺激。用PTI荧光成像系统测量和分析外源性促肾上腺皮质激素释放激素对培养下丘脑神经元胞浆内游离钙浓度变化,用放射免疫方法测定神经元内环磷酸腺苷含量。主要观察指标:①下丘脑神经元胞浆内游离钙浓度。②下丘脑神经元内环磷酸腺苷含量。结果:正常对照组的下丘脑神经元胞浆内游离钙浓度和环磷酸腺苷含量较低,外源性促肾上腺皮质激素释放激素刺激后,胞浆内游离钙浓度立即升高,神经元胞浆内环磷酸腺苷生成明显增加[(240±22),(153±11)nmol/L;(3.26±0.19),(0.44±0.02)pmol/dish,P<0.01];CP-154526可明显抑制促肾上腺皮质激素释放激素(10-6mol/L)刺激的下丘脑神经元胞浆内游离钙浓度和环磷酸腺苷含量的生成[钙离子浓度:(240±22),(171±16)nmol/L;环磷酸腺苷含量:(3.26±0.19),(2.33±0.21)pmol/dish,P<0.01]。结论:促肾上腺皮质激素释放激素可通过与其1受体结合直接作用于下丘脑神经元,使神经元胞浆内游离钙浓度和环磷酸腺苷含量明显增加,在调节下丘脑神经元激活过程中下丘脑合成和分泌的促肾上腺皮质激素释放激素可能起了重要作用。
BACKGROUND: The activation of the hypothalamic-pituitary-adrenocortical axis in stress response plays a key role in the increased expression of corticotropin-releasing hormone in the hypothalamus. However, it remains unclear as to whether corticotrophin-releasing hormone can activate hypothalamic neurons by regulating the neuroendocrine activity of corticotropin-releasing hormone in hypothalamic neurons. OBJECTIVE: To culture hypothalamic neurons by exogenous corticotropin-releasing hormone (HGH) to observe the changes of intracellular cAMP and intracellular calcium concentration. Design: observe the contrast experiment. Unit: Institute of Field Surgical Surgery, Daping Hospital, Third Military Medical University of Chinese PLA, Department of Neurosurgery, Tiantan Hospital, Capital Medical University. Materials: The experiment was performed at Daping Hospital of the Third Military Medical University of Chinese PLA from December 1999 to March 2002. Hypothalamic neurons were disseminated in hypothalamus of fetus and fetus at 17 days. METHODS: Hypothalamic neurons were stimulated with exogenous corticotropin-releasing hormone and hypothalamic neurons were pre-treated with CP-154526, a corticotropin-releasing hormone receptor-specific antagonist. The cultured cells were divided into groups: ① corticotropin-releasing hormone (10-12,10-10,10-8,10-6mol / L) stimulation group. ② pre-treated with CP-154526 (500μmol / L) and then corticotropin-releasing hormone (10-12,10-10,10-8,10-6 mol / L) stimulation group. ③ respectively set the corresponding normal control group, the normal control group with isotonic saline stimulation. PTI fluorescence imaging system was used to measure and analyze the changes of intracellular free calcium concentrations in cultured cortex of cultured hypothalamus with exogenous corticotropin-releasing hormone. The contents of intracellular cyclic AMPs in neurons were measured by radioimmunoassay. MAIN OUTCOME MEASURES: ①Hypothalamic neuronal cytoplasm free calcium concentration. ② hypothalamic neurons in the content of cyclic adenosine monophosphate. Results: In normal control group, the content of intracellular free calcium and cyclic adenosine monophosphate in hypothalamic neurons was low. After stimulated by exogenous corticotropin releasing hormone, the intracellular free calcium concentration increased immediately. The formation of adenosine monophosphate in the plasma was significantly increased ([(240 ± 22) vs (153 ± 11) nmol / L, (3.26 ± 0.19), (0.44 ± 0.02) pmol / Inhibition of corticotropin-releasing hormone (10-6 mol / L) -induced hypothalamic neuron intracellular free calcium concentration and cyclic adenosine monophosphate content [calcium ion concentration: (240 ± 22), (171 ± 16) nmol / L; cAMP content: (3.26 ± 0.19), (2.33 ± 0.21) pmol / dish, P <0.01]. CONCLUSION: Corticotropin-releasing hormone can act directly on neurons in the hypothalamus by binding to its 1 receptor, thereby increasing the intracellular free calcium concentration and cyclic adenosine monophosphate content in neurons. In regulating the activation of hypothalamic neurons Hypothalamic synthesis and secretion of corticotropin releasing hormone may play an important role.