胰岛素、瘦素对大鼠下丘脑NPY神经元大电导钙激活钾通道的调节及机制

发布时间：2018-06-10 09:02

本文选题：胰岛素 + 瘦素　；参考：《华中科技大学》2008年硕士论文

【摘要】： 下丘脑是神经-内分泌两大机能系统的结合点,在维持机体生命活动和内环境稳态中起重要的作用。由脂肪细胞分泌的瘦素(leptin)与胰岛β细胞分泌的胰岛素(insulin)在下丘脑的神经-内分泌反馈环路中起到将外周信号传入下丘脑的作用。它们作用于下丘脑中枢,通过改变神经元的电兴奋性来调节某些特定的效应神经元,如神经肽Y (NPY)神经元的生理功能。大电导钙激活钾通道(large conductance calcium-activated potassium channels,BK通道)是众多K+通道中的一种,在中枢神经系统(CNS)中广泛分布于树突,轴突,突触末端,以及神经元胞体。在细胞发生动作电位期间,通道可以被膜的去极化和细胞内Ca2+水平的增高所激活,其直接效应是产生快后超极化电位。因此,通道的作用主要是使神经元动作电位的时程维持在正常范围内,使神经元放电频率减少,降低神经元的兴奋性,从而最终影响神经冲动的传导以及神经递质的释放。通道功能特性的改变,将对神经元的活动和脑的正常功能产生重要影响。然而,下丘脑内NPY神经元散在分布的特点使得很难对单个神经元电生理活动进行观察。为了研究这一类神经元的内在活动及其对insulin和leptin的电生理反应,我们通过建立将形态学特征与免疫细胞化学技术相结合的方法来鉴定NPY神经元,并运用全细胞膜片钳技术,研究insulin和leptin对BK通道的作用及其机制。第一部分Insulin对培养大鼠下丘脑NPY神经元BK通道的影响及机制目的:NPY神经元是下丘脑内调节摄食和能量代谢的主要效应细胞,大量研究已证实,insulin能够抑制NPY神经元的活性。BK通道在调节神经细胞兴奋性上发挥了重要作用。本实验通过研究insulin对NPY神经元BK通道的作用,探讨insulin调控这类神经元兴奋性的电生理机制。方法:(1)采用免疫细胞化学技术鉴定与摄食相关的NPY神经元;(2)结合形态学特征,采用全细胞膜片钳技术及及记录后的再次鉴定比对,研究insulin对这类神经元BK通道的调节作用及其可能的信号通路。结果:(1)NPY神经元具有明显形态学特征,典型的NPY神经元细胞体较小,成三角形或纺锤形,大部分具有1-3个细小的,不分枝的一级树突。(2)Insulin (0.1-30 nM)浓度依赖性地增加BK电流。3 nM insulin在膜电位为+20-+60 mV时可使BK电流的I-V曲线显著上移,并且能够使BK通道的稳态激活曲线左移。(3)Insulin对BK电流的增大作用能够被其受体阻断剂以及磷脂酰肌醇3激酶(PI3-k)阻断剂所抑制,而不受丝裂原活化蛋白激酶(MAPK)阻断剂的影响。结论:Insulin可浓度依赖性地增加BK通道电流,且这种作用是通过IR介导的PI3-k途径实现。第二部分Leptin对培养大鼠下丘脑NPY神经元BK通道的影响及机制目的:生理状态下,leptin发挥着与insulin相似的功能,CNS中leptin同样能够抑制NPY神经元的活性。本实验通过研究leptin对NPY神经元BK通道的作用,探讨leptin调控这类神经元兴奋性的电生理机制,观察leptin对NPY神经元BK通道的作用是否与insulin类似。方法:(1)采用免疫细胞化学技术鉴定NPY神经元;(2)结合形态学特征,采用全细胞膜片钳技术及及记录后的再次鉴定比对,研究leptin对这类神经元BK通道的调节作用及其信号通路。结果:(1)Leptin对NPY神经元BK通道的影响与insulin极其类似。Leptin(1.0-300 nM)浓度依赖性地增加NPY神经元BK电流。30 nM leptin在膜电位为+20 mV-+60 mV时可使BK电流的I-V曲线显著上移,而对通道的电压依赖的激活特性以及激活阈电位没有影响。30 nM leptin还能够使BK通道的稳态激活曲线左移,而对曲线斜率没有显著影响。(2)leptin受体(LEPR)-门神激酶2(JAK2)-PI3-k信号通路阻断剂能够抑制leptin引起的NPY神经元BK电流的增大,而MAPK阻断剂则不产生影响。结论:Leptin可浓度依赖性地增加BK通道电流,且这种作用是通过LEPR介导的PI3-k途径实现。第三部分Insulin及leptin对培养大鼠下丘脑NPY神经元BK通道的相互作用目的:生理环境中,insulin和leptin共同存在于下丘脑部位发挥生理作用,且大量研究表明这两类激素在下丘脑的信号转导通路之间存在着交叉对话。因此本实验观察insulin和leptin共同存在时对NPY神经元BK通道的作用,以及激素之间的相互影响。方法:(1)采用免疫细胞化学技术鉴定NPY神经元;(2)结合形态学特征,采用全细胞膜片钳技术及记录后的再次鉴定比对,研究insulin和leptin共同作用于NPY神经元BK通道的效果以及一种激素预孵育是否会对另一种激素的作用产生影响。结果:(1)外液中同时加入3 nM insulin和30 nM leptin显著增大NPY神经元BK电流幅值,且增大作用同样可以被PI3-k阻断剂抑制。然而两种激素共同作用的效果与3 nM insulin或30 nM leptin单独作用时的效果相近,并未出现叠加效应。(2)3 nM insulin预孵育神经元5 min后再观察30 nM leptin对BK电流的作用,发现insulin预孵育后的神经元对leptin失去反应性。同样,用30nM leptin预孵育的神经元对insulin也失去反应性。结论:同时加入insulin以及leptin所产生的效应与单独加入其中一种激素所产生的效应类似,用其中一种激素预孵育神经元则取消了另一种激素对BK电流的增加作用。
[Abstract]:Neuropeptide Y ( NPY ) neurons play an important role in maintaining life activity and homeostasis .

Effect and Mechanism of Insulin on BK Channel of Hypothalamus NPY Neurons in Rat

Objective : NPY neurons are the main effector cells regulating food intake and energy metabolism in the hypothalamus , and a large number of studies have shown that insulin can inhibit the activity of NPY neurons . BK channels play an important role in regulating the excitation of neural cells .

Methods : ( 1 ) The NPY neurons related to food intake were identified by immunohistochemical technique ; ( 2 ) The regulatory effects of insulin on the BK channel of these neurons and possible signal pathways were studied by using the technique of whole cell patch clamp technique and the re - identification ratio after recording .

Results : ( 1 ) NPY neurons had obvious morphological characteristics , and the typical NPY neurons were small , triangular or fusiform , most of them had 1 - 3 small , unbranched primary dendrites . ( 2 ) Insulin ( 0.1 -30 nM ) increased BK current .

Conclusion : Insulin can increase BK channel current in a concentration - dependent manner , and this effect is achieved by IR - mediated PI3 - k pathway .

The Effect and Mechanism of the Second Part of Leptin on the BK Channel of NPY Neurons in the Hypothalamus of Rat

AIM : To investigate the effect of leptin on NPY neurons and to explore the electrophysiological mechanism of leptin on NPY neurons , and to observe the effect of leptin on the BK channel of NPY neurons , and to observe whether leptin is similar to insulin .

Methods : ( 1 ) NPY neurons were identified by immunohistochemical technique ; ( 2 ) The effect of leptin on the BK channel of these neurons and the signal pathway were studied by using the technique of whole cell patch clamp technique and the re - identification ratio after recording .

Results : ( 1 ) The effect of leptin on the BK channel of NPY neurons was similar to that of insulin . Leptin ( 1.0 - 300 nM ) increased the BK current of NPY neurons significantly .

Conclusion : Leptin can increase BK channel current in a concentration - dependent manner , and this effect is achieved by LEPR - mediated PI3 - k pathway .

Effect of Insulin and leptin on BK channel in hypothalamic NPY neurons in rats

Objective : In the physiological environment , insulin and leptin play a physiological role in the hypothalamic area , and a large number of studies have shown that there is a cross - dialogue between the two types of hormones in the signal transduction pathways of the hypothalamus . Therefore , the effect of insulin and leptin on the BK channel of NPY neurons and the interaction between hormones are observed .

Methods : ( 1 ) NPY neurons were identified by immunohistochemical technique . ( 2 ) The effect of insulin and leptin on the BK channel of NPY neurons and the effect of one hormone pre - incubation on the function of another hormone were studied .

RESULTS : ( 1 ) At the same time , 3 nM insulin and 30 nM leptin were added to the external solution to significantly increase the current amplitude of the NPY neurons , and the effect of the two hormones was similar to the effect of 3 nM insulin or 30 nM leptin alone . The effect of 30 nM leptin on BK current was observed after 5 min of 3 nM insulin pre - incubation .

Conclusion : At the same time , the effect of insulin and leptin is similar to that produced by one of the hormones alone . One of these hormone preincubation neurons cancels the increasing effect of another hormone on BK current .
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R33

【共引文献】