發(fā)布時間：2018-01-03 21:06

  本文關(guān)鍵詞：縮宮素對大鼠十二指腸肌間神經(jīng)叢AH神經(jīng)元電生理特性的影響及其機制　出處：《山東大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 縮宮素 AH神經(jīng)元 BK通道 IP_3 PLC

【摘要】：目標(biāo)：經(jīng)典的觀點認(rèn)為縮宮素(oxytocin,OT)是下丘腦垂體分泌的九肽激素,其主要生理作用是促進(jìn)子宮平滑肌收縮并調(diào)節(jié)泌乳反射。近年來,越來越多的研究發(fā)現(xiàn)OT具有調(diào)節(jié)消化道運動和分泌的功能,可能是一種新的胃腸神經(jīng)肽。我們最新的研究發(fā)現(xiàn)OT可通過腸神經(jīng)叢抑制大鼠十二指腸縱行肌的自發(fā)收縮,但其機制尚不清楚。肌間神經(jīng)元根據(jù)電生理特性可分為AH和S神經(jīng)元兩種類型,AH神經(jīng)元為內(nèi)在感覺神經(jīng)元(intrinsic primary afferent neurons,IPANs),而S神經(jīng)元為中間和運動神經(jīng)元。在預(yù)實驗中,我們發(fā)現(xiàn)OT可使全部的AH神經(jīng)元(25/25)和部分S神經(jīng)元(10/30)膜電位發(fā)生超極化,因此我們推測OT抑制十二指腸的運動可能與其調(diào)節(jié)AH神經(jīng)元的活動有關(guān)。本課題的主要目的是研究OT對AH神經(jīng)元電生理特性的影響及其機制。 方法：將大鼠頸部脫臼處死,快速取出約1cm長的一段十二指腸,沿腸系膜剖開,粘膜面向上展開固定在盛有柯氏液的硅膠盤中。在解剖鏡下用顯微器械將粘膜、粘膜下層和環(huán)形肌依次剝離,剩余部分即為縱行肌-肌間神經(jīng)叢標(biāo)本(longitudinal muscle myenteric plexus,LMMP).將LMMP標(biāo)本置于含10mg/ml木瓜蛋白酶的柯氏液中37℃消化50min,將組織剪碎后再置于含1mg/ml膠原酶II的DMEM培養(yǎng)基中37℃消化55min,用玻璃吸管吹打約30次,1000r/min離心6min,棄去上清,用1m1含10%胎牛血清的DMEM將沉淀懸浮,37℃培養(yǎng),16h-24h后通過膜片鉗全細(xì)胞記錄模式分析OT對其膜電位和細(xì)胞膜上離子通道的影響；通過鈣成像方法監(jiān)測加入OT前后肌間神經(jīng)元胞漿中Ca2+濃度的改變；通過ELISA的實驗方法檢測LMMP標(biāo)本中IP3含量及OT釋放量；通過免疫熒光雙標(biāo)觀察OT受體(OTR)、BK通道α亞基、胞漿中OT及IPANs的標(biāo)記物calbindin28K在神經(jīng)元上的表達(dá)情況。 實驗結(jié)果： 1、OT對AH神經(jīng)元膜電位的影響 AH神經(jīng)元的靜息電位為-54mV±1mV (n=30),連續(xù)給藥15s后,濃度在10-7M-10-5M的OT均使膜電位發(fā)生超極化,幅度分別為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)。將AH神經(jīng)元用OTR阻斷劑atosiban(10-6M)孵育1min之后再給予OT,原本OT誘發(fā)的超極化反應(yīng)基本消失。 2、OT對AH神經(jīng)元動作電位參數(shù)的影響 連續(xù)給予OT(10-6M)60s之后,AH神經(jīng)元的1/2動作電位時程(action potential half width, AP1/2)由3.2ms±0.4ms降至2.9ms±0.3ms(P=0.04, n=12),閾刺激(threshold)由69mV±10mV增至130mV±21mV(P=0.02,n=12),去極化速率(action potential maximal depolarization, APdv/dt)及動作電位幅度(action potential amplitude, APamp)均不發(fā)生改變。3、OT對AH神經(jīng)元外向離子流的影響 在膜片鉗電壓鉗模式下,設(shè)置一個100ms的step,將鉗制電位從-80mV升至+50mV,觀察激發(fā)出的外向離子流。連續(xù)給藥60s后,三種濃度的OT均使這種外向離子流增加,增加的幅度分別為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)。若將細(xì)胞先用BK通道(large conductance calcium-activated potassium channels)的阻斷劑IbTX孵育1min再加入OT,原本OT引起的外向離子流增加的現(xiàn)象基本消失。但I(xiàn)K通道(intermediate conductance calcium-activated potassium channels)的阻斷劑clotrimazole和SK通道(small conductance calcium-activated potassium channels)的阻斷劑apamln均無此作用。 為確定BK通道是否在AH神經(jīng)元上有功能性表達(dá),進(jìn)一步觀察BK通道的激動劑NS1619對外向離子流的影響,發(fā)現(xiàn)當(dāng)膜電位牽制在+50mV時,NS1619可使其增大748pA±141pA(P=0.002, n=8),該作用可完全被IbTX阻斷。4、OT對肌間神經(jīng)元中鈣離子濃度的影響 OT可使12/30肌間神經(jīng)元胞漿中鈣離子濃度([Ca2+]i)升高。連續(xù)給藥50s后,[Ca2+]；升高到最大值,且分別在l00s(10-7M),150s(10-6M)和250s(10-5M)恢復(fù)到初始水平。三種濃度的OT作用50s分別使胞漿中鈣離子的相對濃度由基準(zhǔn)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)。 為進(jìn)一步確定胞內(nèi)增加的Ca2+的來源,我們將AH神經(jīng)元分別用鈣庫清空劑thapsigargin(10-6M)和細(xì)胞膜上非特異性的鈣離子通道阻斷劑CdCl2(5×10-5M)孵育10min,再加入OT,外向離子流的增加值分別由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神經(jīng)元BK通道開放的信號通路 將AH神經(jīng)元分別用IP。受體的阻斷劑2-APB(10-4M)和PLC的阻斷劑U73122(10-5M)孵育10min,均可部分逆轉(zhuǎn)OT引起外向離子流增加。LMMP標(biāo)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)預(yù)處理之后再加入OT,IP3含量為16.8ng/ml±0.7ng/ml(P=0.003,n=16;OT vs. atosiban+OT) 6.Atosiban和KCl對LMMP標(biāo)本OT分泌量的影響 將LMMP標(biāo)本(20μ g/300μ1)在柯氏液中孵育3min,OT分泌量為33.2ng/ml±2.6ng/ml(n=7),向柯氏液中加入atosiban(10-6M)或KCl (2×10-6M)OT分泌量分別增至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)。同時向柯氏液中加入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標(biāo)本上的定位OTR在部分肌間神經(jīng)元細(xì)胞膜上表達(dá),其中63/71陽性表達(dá)細(xì)胞呈圓形或橢圓形,近似于Dogiel type Ⅱ/AH神經(jīng)元的形狀。BK通道α亞基與OTR陽性表達(dá)細(xì)胞完全重合。 8、OT和calbindin28K在LMMP上的表達(dá)OT和AH神經(jīng)元的標(biāo)記物calbindin28K均在部分LMMP的神經(jīng)元上表達(dá)。統(tǒng)計發(fā)現(xiàn),73.1%(122/167)的OT陽性細(xì)胞上有calbindin28K表達(dá),而82.4%(122/148)的AH神經(jīng)元上有OT表達(dá)。 結(jié)論：OT作用于大鼠肌間神經(jīng)叢AH神經(jīng)元上的OT受體,激活細(xì)胞中的OTR-PLC-IP3-Ca2+信號通路,使細(xì)胞膜上的BK通道開放,K+外流引起膜電位發(fā)生超極化；OT可能是腸神經(jīng)系統(tǒng)中一種起負(fù)反饋調(diào)節(jié)作用的自分泌神經(jīng)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 鹵 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 鹵 0.5mV ( P0.05 , n = 9 : OT10 - 7 M ) , 12.7mV 鹵 0.7 mV ( P < 0.05 , n = 8 : OT10 - 6M ) and 8.9mV 鹵 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 鹵 0.4 ms to 2.9 ms 鹵 0.3 ms ( P = 0.04 , n = 12 ) . The threshold stimulation ( threshold ) increased from 69mV 鹵 10mV to 130mV 鹵 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 鹵 72pA ( P0.05 , n = 8 ; OT10 - 7 M ) , 1035pA 鹵 122 pA ( P0.05 , n = 8 : OT10 - 6M ) and 1116pA 鹵 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 鹵 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 鹵 0.2 ( P0.05 , 10 - 7 M ; n = 8 ) , 1.3 鹵 0.2 ( P0.05 , 10 - 6M , n = 11 ) and 1.6 鹵 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 鹵 78pA to 182pA 鹵 34pA ( P0.001 , n = 8 ; DMSO + OT vs . thapsigargin + OT ) , respectively , from 1042pA 鹵 96pA to 440pA 鹵 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 鹵 1.1 ng / ml to 22.0ng / ml 鹵 1.4 ng / ml ( P = 0.001 , n = 16 ) . After pretreatment with atosiban ( 10 - 6M ) , the content of IP3 was 16.8 ng / ml 鹵 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 鹵 2.6 ng / ml ( n = 7 ) . atosiban ( 10 - 6M ) or KCl ( 2 脳 10 - 6M ) OT secretion was increased to 47.1 ng / ml 鹵 3.5 ng / ml ( P = 0.01 , b = 7 : atosiban treatment vs . control ) and 45.4 ng / ml 鹵 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 鹵 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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