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胰島素、瘦素對(duì)大鼠下丘腦NPY神經(jīng)元大電導(dǎo)鈣激活鉀通道的調(diào)節(jié)及機(jī)制

發(fā)布時(shí)間:2018-06-10 09:02

  本文選題:胰島素 + 瘦素; 參考:《華中科技大學(xué)》2008年碩士論文


【摘要】: 下丘腦是神經(jīng)-內(nèi)分泌兩大機(jī)能系統(tǒng)的結(jié)合點(diǎn),在維持機(jī)體生命活動(dòng)和內(nèi)環(huán)境穩(wěn)態(tài)中起重要的作用。由脂肪細(xì)胞分泌的瘦素(leptin)與胰島β細(xì)胞分泌的胰島素(insulin)在下丘腦的神經(jīng)-內(nèi)分泌反饋環(huán)路中起到將外周信號(hào)傳入下丘腦的作用。它們作用于下丘腦中樞,通過(guò)改變神經(jīng)元的電興奮性來(lái)調(diào)節(jié)某些特定的效應(yīng)神經(jīng)元,如神經(jīng)肽Y (NPY)神經(jīng)元的生理功能。大電導(dǎo)鈣激活鉀通道(large conductance calcium-activated potassium channels,BK通道)是眾多K+通道中的一種,在中樞神經(jīng)系統(tǒng)(CNS)中廣泛分布于樹(shù)突,軸突,突觸末端,以及神經(jīng)元胞體。在細(xì)胞發(fā)生動(dòng)作電位期間,通道可以被膜的去極化和細(xì)胞內(nèi)Ca2+水平的增高所激活,其直接效應(yīng)是產(chǎn)生快后超極化電位。因此,通道的作用主要是使神經(jīng)元?jiǎng)幼麟娢坏臅r(shí)程維持在正常范圍內(nèi),使神經(jīng)元放電頻率減少,降低神經(jīng)元的興奮性,從而最終影響神經(jīng)沖動(dòng)的傳導(dǎo)以及神經(jīng)遞質(zhì)的釋放。通道功能特性的改變,將對(duì)神經(jīng)元的活動(dòng)和腦的正常功能產(chǎn)生重要影響。然而,下丘腦內(nèi)NPY神經(jīng)元散在分布的特點(diǎn)使得很難對(duì)單個(gè)神經(jīng)元電生理活動(dòng)進(jìn)行觀察。為了研究這一類(lèi)神經(jīng)元的內(nèi)在活動(dòng)及其對(duì)insulin和leptin的電生理反應(yīng),我們通過(guò)建立將形態(tài)學(xué)特征與免疫細(xì)胞化學(xué)技術(shù)相結(jié)合的方法來(lái)鑒定NPY神經(jīng)元,并運(yùn)用全細(xì)胞膜片鉗技術(shù),研究insulin和leptin對(duì)BK通道的作用及其機(jī)制。 第一部分Insulin對(duì)培養(yǎng)大鼠下丘腦NPY神經(jīng)元BK通道的影響及機(jī)制 目的:NPY神經(jīng)元是下丘腦內(nèi)調(diào)節(jié)攝食和能量代謝的主要效應(yīng)細(xì)胞,大量研究已證實(shí),insulin能夠抑制NPY神經(jīng)元的活性。BK通道在調(diào)節(jié)神經(jīng)細(xì)胞興奮性上發(fā)揮了重要作用。本實(shí)驗(yàn)通過(guò)研究insulin對(duì)NPY神經(jīng)元BK通道的作用,探討insulin調(diào)控這類(lèi)神經(jīng)元興奮性的電生理機(jī)制。 方法:(1)采用免疫細(xì)胞化學(xué)技術(shù)鑒定與攝食相關(guān)的NPY神經(jīng)元;(2)結(jié)合形態(tài)學(xué)特征,采用全細(xì)胞膜片鉗技術(shù)及及記錄后的再次鑒定比對(duì),研究insulin對(duì)這類(lèi)神經(jīng)元BK通道的調(diào)節(jié)作用及其可能的信號(hào)通路。 結(jié)果:(1)NPY神經(jīng)元具有明顯形態(tài)學(xué)特征,典型的NPY神經(jīng)元細(xì)胞體較小,成三角形或紡錘形,大部分具有1-3個(gè)細(xì)小的,不分枝的一級(jí)樹(shù)突。(2)Insulin (0.1-30 nM)濃度依賴性地增加BK電流。3 nM insulin在膜電位為+20-+60 mV時(shí)可使BK電流的I-V曲線顯著上移,并且能夠使BK通道的穩(wěn)態(tài)激活曲線左移。(3)Insulin對(duì)BK電流的增大作用能夠被其受體阻斷劑以及磷脂酰肌醇3激酶(PI3-k)阻斷劑所抑制,而不受絲裂原活化蛋白激酶(MAPK)阻斷劑的影響。 結(jié)論:Insulin可濃度依賴性地增加BK通道電流,且這種作用是通過(guò)IR介導(dǎo)的PI3-k途徑實(shí)現(xiàn)。 第二部分Leptin對(duì)培養(yǎng)大鼠下丘腦NPY神經(jīng)元BK通道的影響及機(jī)制 目的:生理狀態(tài)下,leptin發(fā)揮著與insulin相似的功能,CNS中l(wèi)eptin同樣能夠抑制NPY神經(jīng)元的活性。本實(shí)驗(yàn)通過(guò)研究leptin對(duì)NPY神經(jīng)元BK通道的作用,探討leptin調(diào)控這類(lèi)神經(jīng)元興奮性的電生理機(jī)制,觀察leptin對(duì)NPY神經(jīng)元BK通道的作用是否與insulin類(lèi)似。 方法:(1)采用免疫細(xì)胞化學(xué)技術(shù)鑒定NPY神經(jīng)元;(2)結(jié)合形態(tài)學(xué)特征,采用全細(xì)胞膜片鉗技術(shù)及及記錄后的再次鑒定比對(duì),研究leptin對(duì)這類(lèi)神經(jīng)元BK通道的調(diào)節(jié)作用及其信號(hào)通路。 結(jié)果:(1)Leptin對(duì)NPY神經(jīng)元BK通道的影響與insulin極其類(lèi)似。Leptin(1.0-300 nM)濃度依賴性地增加NPY神經(jīng)元BK電流。30 nM leptin在膜電位為+20 mV-+60 mV時(shí)可使BK電流的I-V曲線顯著上移,而對(duì)通道的電壓依賴的激活特性以及激活閾電位沒(méi)有影響。30 nM leptin還能夠使BK通道的穩(wěn)態(tài)激活曲線左移,而對(duì)曲線斜率沒(méi)有顯著影響。(2)leptin受體(LEPR)-門(mén)神激酶2(JAK2)-PI3-k信號(hào)通路阻斷劑能夠抑制leptin引起的NPY神經(jīng)元BK電流的增大,而MAPK阻斷劑則不產(chǎn)生影響。 結(jié)論:Leptin可濃度依賴性地增加BK通道電流,且這種作用是通過(guò)LEPR介導(dǎo)的PI3-k途徑實(shí)現(xiàn)。 第三部分Insulin及l(fā)eptin對(duì)培養(yǎng)大鼠下丘腦NPY神經(jīng)元BK通道的相互作用 目的:生理環(huán)境中,insulin和leptin共同存在于下丘腦部位發(fā)揮生理作用,且大量研究表明這兩類(lèi)激素在下丘腦的信號(hào)轉(zhuǎn)導(dǎo)通路之間存在著交叉對(duì)話。因此本實(shí)驗(yàn)觀察insulin和leptin共同存在時(shí)對(duì)NPY神經(jīng)元BK通道的作用,以及激素之間的相互影響。 方法:(1)采用免疫細(xì)胞化學(xué)技術(shù)鑒定NPY神經(jīng)元;(2)結(jié)合形態(tài)學(xué)特征,采用全細(xì)胞膜片鉗技術(shù)及記錄后的再次鑒定比對(duì),研究insulin和leptin共同作用于NPY神經(jīng)元BK通道的效果以及一種激素預(yù)孵育是否會(huì)對(duì)另一種激素的作用產(chǎn)生影響。 結(jié)果:(1)外液中同時(shí)加入3 nM insulin和30 nM leptin顯著增大NPY神經(jīng)元BK電流幅值,且增大作用同樣可以被PI3-k阻斷劑抑制。然而兩種激素共同作用的效果與3 nM insulin或30 nM leptin單獨(dú)作用時(shí)的效果相近,并未出現(xiàn)疊加效應(yīng)。(2)3 nM insulin預(yù)孵育神經(jīng)元5 min后再觀察30 nM leptin對(duì)BK電流的作用,發(fā)現(xiàn)insulin預(yù)孵育后的神經(jīng)元對(duì)leptin失去反應(yīng)性。同樣,用30nM leptin預(yù)孵育的神經(jīng)元對(duì)insulin也失去反應(yīng)性。 結(jié)論:同時(shí)加入insulin以及l(fā)eptin所產(chǎn)生的效應(yīng)與單獨(dú)加入其中一種激素所產(chǎn)生的效應(yīng)類(lèi)似,用其中一種激素預(yù)孵育神經(jīng)元?jiǎng)t取消了另一種激素對(duì)BK電流的增加作用。
[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 .
【學(xué)位授予單位】:華中科技大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2008
【分類(lèi)號(hào)】:R33

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào):2002673


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