缩宫素对大鼠十二指肠肌间神经丛AH神经元电生理特性的影响及其机制

发布时间：2018-01-03 21:06

  本文关键词：缩宫素对大鼠十二指肠肌间神经丛AH神经元电生理特性的影响及其机制　出处：《山东大学》2012年博士论文　论文类型：学位论文

  更多相关文章： 缩宫素 AH神经元 BK通道 IP_3 PLC

【摘要】：目标：经典的观点认为缩宫素(oxytocin,OT)是下丘脑垂体分泌的九肽激素,其主要生理作用是促进子宫平滑肌收缩并调节泌乳反射。近年来,越来越多的研究发现OT具有调节消化道运动和分泌的功能,可能是一种新的胃肠神经肽。我们最新的研究发现OT可通过肠神经丛抑制大鼠十二指肠纵行肌的自发收缩,但其机制尚不清楚。肌间神经元根据电生理特性可分为AH和S神经元两种类型,AH神经元为内在感觉神经元(intrinsic primary afferent neurons,IPANs),而S神经元为中间和运动神经元。在预实验中,我们发现OT可使全部的AH神经元(25/25)和部分S神经元(10/30)膜电位发生超极化,因此我们推测OT抑制十二指肠的运动可能与其调节AH神经元的活动有关。本课题的主要目的是研究OT对AH神经元电生理特性的影响及其机制。 方法：将大鼠颈部脱臼处死,快速取出约1cm长的一段十二指肠,沿肠系膜剖开,粘膜面向上展开固定在盛有柯氏液的硅胶盘中。在解剖镜下用显微器械将粘膜、粘膜下层和环形肌依次剥离,剩余部分即为纵行肌-肌间神经丛标本(longitudinal muscle myenteric plexus,LMMP).将LMMP标本置于含10mg/ml木瓜蛋白酶的柯氏液中37℃消化50min,将组织剪碎后再置于含1mg/ml胶原酶II的DMEM培养基中37℃消化55min,用玻璃吸管吹打约30次,1000r/min离心6min,弃去上清,用1m1含10%胎牛血清的DMEM将沉淀悬浮,37℃培养,16h-24h后通过膜片钳全细胞记录模式分析OT对其膜电位和细胞膜上离子通道的影响；通过钙成像方法监测加入OT前后肌间神经元胞浆中Ca2+浓度的改变；通过ELISA的实验方法检测LMMP标本中IP3含量及OT释放量；通过免疫荧光双标观察OT受体(OTR)、BK通道α亚基、胞浆中OT及IPANs的标记物calbindin28K在神经元上的表达情况。 实验结果： 1、OT对AH神经元膜电位的影响 AH神经元的静息电位为-54mV±1mV (n=30),连续给药15s后,浓度在10-7M-10-5M的OT均使膜电位发生超极化,幅度分别为4.4mV±0.5mV(P0.05,n=9：OT10-7M),12.7mV±0.7mV(P＜0.05,n=8：OT10-6M)和8.9mV±0.8mV(P0.05,n=8;OT10-5M)。将AH神经元用OTR阻断剂atosiban(10-6M)孵育1min之后再给予OT,原本OT诱发的超极化反应基本消失。 2、OT对AH神经元动作电位参数的影响 连续给予OT(10-6M)60s之后,AH神经元的1/2动作电位时程(action potential half width, AP1/2)由3.2ms±0.4ms降至2.9ms±0.3ms(P=0.04, n=12),阈刺激(threshold)由69mV±10mV增至130mV±21mV(P=0.02,n=12),去极化速率(action potential maximal depolarization, APdv/dt)及动作电位幅度(action potential amplitude, APamp)均不发生改变。3、OT对AH神经元外向离子流的影响 在膜片钳电压钳模式下,设置一个100ms的step,将钳制电位从-80mV升至+50mV,观察激发出的外向离子流。连续给药60s后,三种浓度的OT均使这种外向离子流增加,增加的幅度分别为552pA±72pA(P0.05,n=8;OT10-7M),1035pA±122pA(P0.05,n=8：OT10-6M)和1116pA±100pA(P0.05,n=8：OT10-5M)。若将细胞先用BK通道(large conductance calcium-activated potassium channels)的阻断剂IbTX孵育1min再加入OT,原本OT引起的外向离子流增加的现象基本消失。但IK通道(intermediate conductance calcium-activated potassium channels)的阻断剂clotrimazole和SK通道(small conductance calcium-activated potassium channels)的阻断剂apamln均无此作用。 为确定BK通道是否在AH神经元上有功能性表达,进一步观察BK通道的激动剂NS1619对外向离子流的影响,发现当膜电位牵制在+50mV时,NS1619可使其增大748pA±141pA(P=0.002, n=8),该作用可完全被IbTX阻断。4、OT对肌间神经元中钙离子浓度的影响 OT可使12/30肌间神经元胞浆中钙离子浓度([Ca2+]i)升高。连续给药50s后,[Ca2+]；升高到最大值,且分别在l00s(10-7M),150s(10-6M)和250s(10-5M)恢复到初始水平。三种浓度的OT作用50s分别使胞浆中钙离子的相对浓度由基准值1升高至1.3±0.2(P0.05,10-7M；n=8),1.3±0.2(P0.05,10-6M；n=11)和1.6±0.3(P0.05,10-5M；n=11)。 为进一步确定胞内增加的Ca2+的来源,我们将AH神经元分别用钙库清空剂thapsigargin(10-6M)和细胞膜上非特异性的钙离子通道阻断剂CdCl2(5×10-5M)孵育10min,再加入OT,外向离子流的增加值分别由678pA±78pA降至182pA±34pA(P0.001,n=8;DMSO+OT vs.thapsigargin+OT),由1042pA±96pA降至440pA±94pA(P0.001,n=10;vehicle+OT vs. CdCl2+0T). 5、OT致AH神经元BK通道开放的信号通路 将AH神经元分别用IP。受体的阻断剂2-APB(10-4M)和PLC的阻断剂U73122(10-5M)孵育10min,均可部分逆转OT引起外向离子流增加。LMMP标本在含OT(10-6M)的浴液中孵育1min,其中的IP3含量由16.0ng-ml±1.1ng/ml增加至22.0ng/ml±1.4ng/ml(P=0.001,n=16)。用atosiban(10-6M)预处理之后再加入OT,IP3含量为16.8ng/ml±0.7ng/ml(P=0.003,n=16;OT vs. atosiban+OT) 6.Atosiban和KCl对LMMP标本OT分泌量的影响 将LMMP标本(20μ g/300μ1)在柯氏液中孵育3min,OT分泌量为33.2ng/ml±2.6ng/ml(n=7),向柯氏液中加入atosiban(10-6M)或KCl (2×10-6M)OT分泌量分别增至47.1ng/ml±3.5ng/ml(P=0.01,b=7:atosiban treatment vs. control)和45.4ng/ml±2.5ng/ml((P=0.02,n=7:KCl treatment vs. control)。同时向柯氏液中加入atosiban和KCl,OT分泌量增至62.6ng/ml±5.0ng/ml(P=0.002,n=7,KCl+atosiban vs.KCl;P0.005,n=7,KCl+atosiban vs. atosiban). 7、OTR和BK通道在LMMP标本上的定位OTR在部分肌间神经元细胞膜上表达,其中63/71阳性表达细胞呈圆形或椭圆形,近似于Dogiel type Ⅱ/AH神经元的形状。BK通道α亚基与OTR阳性表达细胞完全重合。 8、OT和calbindin28K在LMMP上的表达OT和AH神经元的标记物calbindin28K均在部分LMMP的神经元上表达。统计发现,73.1%(122/167)的OT阳性细胞上有calbindin28K表达,而82.4%(122/148)的AH神经元上有OT表达。 结论：OT作用于大鼠肌间神经丛AH神经元上的OT受体,激活细胞中的OTR-PLC-IP3-Ca2+信号通路,使细胞膜上的BK通道开放,K+外流引起膜电位发生超极化；OT可能是肠神经系统中一种起负反馈调节作用的自分泌神经肽。
[Abstract]:Objective : The classical point of view is that oxytocin ( OT ) is a 9 - peptide hormone secreted by the pituitary gland . Its main physiological function is to promote the contraction of uterine smooth muscle and regulate the lactation reflex . In recent years , more and more studies have found that OT has the function of regulating the movement and secretion of the digestive tract . In recent years , we have found that OT can be divided into two types of AH and S neurons . The main purpose of this study is to study the effect and mechanism of OT on the electrophysiological properties of AH neurons . Methods : The rats were sacrificed at the neck of the neck , and a length of duodenum was taken out rapidly . The mucous membrane was taken out along the mesentery . The mucous membrane facing upward was fixed in the silica gel disc containing the Koehler ' s solution . The mucosa , submucosa and annular muscle were peeled off sequentially under the dissecting microscope , and the remaining part was longitudinal muscle - myenteric plexus specimen ( LMMP ) . The effects of OT on membrane potential and ion channel of membrane were determined by ELISA . The concentration of IP3 and the amount of OT were analyzed by ELISA . The expression of calbindin 28K on neurons was observed by ELISA . The expression of calbindin 28K in the cells of OT and IPANs was observed by ELISA . Experimental results : Effect of OT on Membrane Potential of AH Neurons The resting potential of AH neurons was -54mV 卤 1mV ( n = 30 ) . After 15 s of continuous administration , the membrane potential was hyperpolarized at 10 - 7 M - 10 - 5M OT , respectively , and the amplitudes were 4.4 mV 卤 0.5mV ( P0.05 , n = 9 : OT10 - 7 M ) , 12.7mV 卤 0.7 mV ( P < 0.05 , n = 8 : OT10 - 6M ) and 8.9mV 卤 0.8mV ( P0.05 , n = 8 ; OT10 - 5M ) . The AH neurons were incubated with OTR blocking agent atosiban ( 10 - 6M ) for 1 min and OT was given . The hyperpolarization induced by OT disappeared . Effect of OT on Action Potential Parameters of AH Neurons After continuous administration of OT ( 10 - 6M ) 60s , the action potential half width ( AP1 / 2 ) of AH neurons decreased from 3.2 ms 卤 0.4 ms to 2.9 ms 卤 0.3 ms ( P = 0.04 , n = 12 ) . The threshold stimulation ( threshold ) increased from 69mV 卤 10mV to 130mV 卤 21mV ( P = 0.02 , n = 12 ) , and the action potential amplitude ( APdv / dt ) and action potential amplitude ( APamp ) did not change . In the mode of patch clamp voltage clamp , a 100ms step was set to increase the clamping potential from -80 mV to + 50mV , and the excited outward ion flow was observed . After 60 s of continuous administration , three concentrations of OT increased the outward ion flow , the amplitude of which was 552pA 卤 72pA ( P0.05 , n = 8 ; OT10 - 7 M ) , 1035pA 卤 122 pA ( P0.05 , n = 8 : OT10 - 6M ) and 1116pA 卤 100pA ( P0.05 , n = 8 : OT10 - 5M ) . However , the blocking agent clotrimazole and SK channel blocker apamln of the blocking agent clotrimazole and SK channel ( small calcium - activated potassium channels ) of the IK channel ( intermediate calcium - activated potassium channels ) had no effect . In order to determine whether the BK channel has functional expression on AH neurons , the effect of BK channel agonist NS1619 on ion flow is further observed . It is found that NS1619 can increase 748pA 卤 141pA ( P = 0.002 , n = 8 ) when the membrane potential is tied to + 50mV . The effect of OT on the concentration of calcium ion in intermuscular neurons can be blocked completely . The concentration of Ca ~ ( 2 + ) in the cytoplasm of 12 / 30 muscle cells was increased . After 50 s of continuous administration , the relative concentrations of Ca ~ ( 2 + ) in cytoplasm were increased to the maximum , and the relative concentrations of Ca ~ ( 2 + ) in cytoplasm were increased to 1.3 卤 0.2 ( P0.05 , 10 - 7 M ; n = 8 ) , 1.3 卤 0.2 ( P0.05 , 10 - 6M , n = 11 ) and 1.6 卤 0.3 ( P0.05 , 10 - 5M ; n = 11 ) . In order to further determine the source of intracellular Ca 2 + , the AH neurons were incubated for 10 min with a calcium bank emptying agent thapsigargin ( 10 - 6M ) and a non - specific calcium channel blocker CdCl2 ( 5 脳 10 - 5M ) on the cell membrane . The addition of OT and outward ion flow was reduced from 678pA 卤 78pA to 182pA 卤 34pA ( P0.001 , n = 8 ; DMSO + OT vs . thapsigargin + OT ) , respectively , from 1042pA 卤 96pA to 440pA 卤 94pA ( P0.001 , n = 10 ; vehicle + OT vs . CdCl2 + 0T ) . 5 . Signal pathway open to the BK channel of the OT - induced AH neurons The concentration of IP3 was increased from 16.0 ng - ml 卤 1.1 ng / ml to 22.0ng / ml 卤 1.4 ng / ml ( P = 0.001 , n = 16 ) . After pretreatment with atosiban ( 10 - 6M ) , the content of IP3 was 16.8 ng / ml 卤 0.7 ng / ml ( P = 0.003 , n = 16 ; OT vs . atosiban + OT ) 6 . Effects of Atosiban and KCl on OT Secretion in LMMP Specimens The amount of OT was 33.2ng / ml 卤 2.6 ng / ml ( n = 7 ) . atosiban ( 10 - 6M ) or KCl ( 2 脳 10 - 6M ) OT secretion was increased to 47.1 ng / ml 卤 3.5 ng / ml ( P = 0.01 , b = 7 : atosiban treatment vs . control ) and 45.4 ng / ml 卤 2.5 ng / ml ( P = 0.02 , n = 7 : KCl treatment vs . control ) . At the same time , atosiban and KCl were added to the Koshi solution , the secretion of OT increased to 62.6 ng / ml 卤 5.0ng / ml ( P = 0.002 , n = 7 , KCl + atosiban vs . KCl ; P0.05 , n = 7 , KCl + atosiban vs . atosiban ) . 7 . The localization of OTR and BK channels on LMMP specimens was expressed on some of the intermuscular neuronal membranes , in which 63 / 71 positive expression cells were round or oval , approximate to the shape of Dogiel type 鈪，

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