發(fā)布時(shí)間：2018-02-22 07:39

  本文關(guān)鍵詞： 托吡酯 泌汗障礙 外泌汗腺 乙酰膽堿酯酶 膽堿乙酰轉(zhuǎn)移酶 毒蕈堿M3受體 GABA能神經(jīng)元　出處：《第四軍醫(yī)大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 托吡酯(topiramate,TPM)是一種新的抗癲vN藥,其化學(xué)結(jié)構(gòu)為2,3:4,5-雙-O-(1-甲基亞乙基)-β-右旋-氨基磺酸吡喃果糖,在多種癲vN發(fā)作類型治療中有效,包括局灶性癲vN發(fā)作、原發(fā)性強(qiáng)直陣攣發(fā)作、Lennox-Gastaut綜合征等[1-2]。其作用機(jī)制有:1.可阻斷神經(jīng)元持續(xù)去極化導(dǎo)致的反復(fù)電位發(fā)放,此作用與使用TPM后的時(shí)間密切相關(guān),表明TPM可以阻斷鈉通道[3];2.可以增加γ-氨基丁酸(GABA)激活GABAA受體的頻率,加強(qiáng)氯離子內(nèi)流,增強(qiáng)抑制性中樞神經(jīng)遞質(zhì)的作用[4,5];3.降低谷氨酸AMPA受體的活性[6,7],抑制谷氨酸和天冬氨酸的釋放[8],降低興奮性中樞神經(jīng)遞質(zhì)的作用;4.抑制碳酸酐酶,特別是其同工酶CA II和CA IV [9];5.對K+通道電流的調(diào)節(jié)作用[10];6.抑制神經(jīng)元L-型電壓激活鈣離子通道[11]。 最近陸續(xù)報(bào)道TPM在治療兒童癲vN時(shí)出現(xiàn)少汗或無汗為主要表現(xiàn)的對熱不耐受現(xiàn)象[12-17],該現(xiàn)象是可逆的,當(dāng)停用TPM時(shí),少汗表現(xiàn)消失。泌汗障礙發(fā)生率一般在10%-20%左右,但有研究發(fā)現(xiàn)未出現(xiàn)臨床癥狀者,其發(fā)汗試驗(yàn)提示有出汗量下降,下降人數(shù)超過測試總?cè)藬?shù)的50%。這個(gè)現(xiàn)象提示TPM相關(guān)的泌汗障礙存在普遍性。 目前TPM引起泌汗障礙的機(jī)制尚不清楚。根本的原因是汗液的調(diào)節(jié)機(jī)制尚處于研究中。有研究提示泌汗障礙與TPM對碳酸酐酶亞型抑制有關(guān),該抑制作用減低了汗液原液的分泌,減少汗液的形成從而導(dǎo)致泌汗率下降。但作為一個(gè)酶類,其調(diào)節(jié)存在一個(gè)過程,機(jī)體并不能快速的調(diào)節(jié)此過程,因此這種假設(shè)還有待證實(shí)。在小鼠汗腺分泌細(xì)胞的管腔側(cè)和基底側(cè)均存在AQP5表達(dá),有實(shí)驗(yàn)發(fā)現(xiàn)該蛋白參與了汗液的分泌過程。但也有研究提出該蛋白與泌汗沒有聯(lián)系。因此,泌汗障礙的原因有待進(jìn)一步研究。 泌汗通路起始于丘腦下部視前區(qū),經(jīng)腦干側(cè)索中央下行,到達(dá)脊髓的中間外側(cè)柱,位于中間外側(cè)柱的節(jié)前神經(jīng)元發(fā)出纖維到達(dá)同側(cè)的交感神經(jīng)節(jié),換元后發(fā)出節(jié)后纖維支配汗腺器官。人類在高溫或運(yùn)動(dòng)時(shí)的熱驅(qū)散主要依靠小汗腺的蒸發(fā)而帶來的散熱作用。因此,汗液的產(chǎn)生對熱耐受程度產(chǎn)生重要的影響,出汗可以防止運(yùn)動(dòng)中出現(xiàn)過熱現(xiàn)象。汗腺由交感節(jié)后神經(jīng)元軸突支配,不同于一般的交感神經(jīng)節(jié)后遞質(zhì),汗液的分泌以乙酰膽堿為主要神經(jīng)遞質(zhì)。嚙齒類動(dòng)物的汗腺主要位于足墊和足指(趾),小汗腺的出汗可以由擬膽堿劑、腎上腺素等注射或熱刺激運(yùn)動(dòng)刺激激發(fā)。TPM是如何引起在兒童的泌汗障礙的,是否與對膽堿能系統(tǒng)的作用有關(guān)系?實(shí)驗(yàn)以此著手展開研究。 本研究首先采用硅膠印跡模型觀察了TPM在小鼠引起的泌汗功能情況。其次在此模型基礎(chǔ)上應(yīng)用HE染色法和電鏡下觀察TPM引起的泌汗障礙是否與汗腺結(jié)構(gòu)變化有關(guān)系;采用免疫組化染色觀察支配汗腺的一般神經(jīng)末梢、膽堿能神經(jīng)、腎上腺能神經(jīng)及膽堿能M3受體的表達(dá)變化。并結(jié)合蛋白印跡和免疫組織化學(xué)觀察膽堿酯酶在汗腺的表達(dá),進(jìn)行膽堿酯酶活性測定。此外對GABA神經(jīng)元參與脊髓和丘腦下部區(qū)域的交感活動(dòng)進(jìn)行了形態(tài)上的初步的探索。實(shí)驗(yàn)結(jié)果如下: (1)2周齡的幼鼠每天經(jīng)灌胃給予TPM(80mg/kg),連續(xù)給藥一個(gè)月后和停藥后一周,對小鼠進(jìn)行匹羅卡品刺激發(fā)汗試驗(yàn),發(fā)現(xiàn)TPM處理后小鼠與對照組小鼠相比其分泌汗腺數(shù)目下降17%,單位汗腺的泌汗量下降40%。光鏡結(jié)構(gòu)和汗腺的超微結(jié)構(gòu)未見明顯改變。 (2)與對照相比,TPM處理對汗腺腺周一般神經(jīng)支配PGP9.5、膽堿能神經(jīng)支配標(biāo)志物ChAT、腎上腺能標(biāo)志物TH神經(jīng)支配面積無明顯影響;對膽堿能毒蕈堿M3受體表達(dá)以及對星狀神經(jīng)節(jié)神經(jīng)遞質(zhì)表達(dá)無明顯影響。單個(gè)汗腺分泌部的面積無明顯變化。 (3)TPM處理后汗腺AChE免疫熒光及蛋白免疫印跡表達(dá)結(jié)果與對照相比無明顯差異,汗腺組織勻漿后提示TPM對AChE活性無明顯影響。 (4)交感節(jié)前神經(jīng)元存在ChAT和GAD65免疫熒光雙標(biāo)記。熱激活后,丘腦下部熱敏感神經(jīng)元與GABA能神經(jīng)元存在雙標(biāo)記。 從這些結(jié)果中可以初步得出以下結(jié)論: (1)TPM(80mgkg-1d-1)較長時(shí)期給藥在一定程度上降低了小鼠的泌汗功能,停藥后泌汗功能恢復(fù)。 (2)TPM導(dǎo)致的泌汗障礙沒有影響到小鼠汗腺的面積大小和形態(tài)、超微結(jié)構(gòu)。 (3)長期的TPM處理可降低小鼠外泌汗腺對匹羅卡品的分泌反應(yīng)性,TPM未影響到小鼠汗腺的交感神經(jīng)發(fā)育和支配、汗腺周圍的膽堿能毒蕈堿M3受體表達(dá),對汗腺部位的AChE表達(dá)及其活性也無明顯影響。 (4)GABA能神經(jīng)元可能參與節(jié)前膽堿能神經(jīng)元調(diào)節(jié)、GABA能神經(jīng)元參與丘腦下部對高溫的反應(yīng)。這種參與活動(dòng)可能與TPM泌汗障礙相關(guān)。
[Abstract]:Topiramate (topiramate, TPM) is a new antiepileptic drug vN, the chemical structure of 2,3:4,5- double -O- (1- methylethylidene) - beta - - amino acid fructophyranose, in a variety of vN types in the treatment of epilepsy seizures, including focal epilepsy vN seizures, primary clonic seizures the mechanism of [1-2]. Lennox-Gastaut syndrome: 1. blocked repeatedly issued potential long-lasting depolarization of neurons caused by this effect and the use of TPM is closely related to the time, showed that TPM could block the sodium channel [3]; 2. can increase the gamma aminobutyric acid (GABA) GABAA receptor activation frequency, strengthen chloride influx enhancement of [4,5] inhibitory neurotransmitters; 3. [6,7] decreased the activity of glutamate AMPA receptors, inhibit the release of glutamate and aspartate [8], reduce the excitability of the central neurotransmitter role; 4. inhibition of carbonic anhydrase, especially the CA II and CA IV [9] isozyme 5. regulation of K+ channel current [10]; 6. inhibition of neuron L- type voltage activation of calcium channel [11].
TPM has been recently reported on heat intolerance [12-17] Hypohidrosis or anhidrosis mainly in the treatment of children with epilepsy vN, this phenomenon is reversible, with the cessation of TPM, less was disappeared. Hypohidrosis occurred at around 10%-20%, but the study found no symptom, the sweat test indicate sweating decline, decline in the number of exceed the total number of test 50%. suggesting that TPM related Hypohidrosis exists.
The mechanism of TPM induced Hypohidrosis is unclear. The basic reason is that the research is still in the adjustment mechanism in the sweat. Some studies suggest that Hypohidrosis and TPM on inhibition of carbonic anhydrase isoforms, the inhibitory effect of reduced secretion of perspiration, reduce the formation of resulting sweat sweat. But as the rate of decline a regulating enzymes, there is a process of adjusting the process of the body were not fast, so this hypothesis remains to be proven. The secretion of luminal side cells and basal side of the mouse sweat gland of AQP5 were detected, experiments have revealed that the protein involved in the secretion process of sweat. But there are also presented the urinary protein and no sweat contact. Therefore, the reason for Hypohidrosis needs further study.
The sweat pathway originates in the preoptic area of the hypothalamus, the central lateral funiculus of the brain stem down, reach the intermediolateral column of the spinal ganglion is located in the intermediolateral column of preganglionic neuron fibers from the ipsilateral reach, change the yuan after postganglionic fibers from sweat glands. Human dominated at high temperature or dissipate heat during exercise mainly rely on small sweat glands the evaporation caused by the cooling effect. Therefore, sweat production is an important influence on heat tolerance, sweating can prevent overheating in movement. The sweat glands innervated by sympathetic postganglionic neurons in sympathetic postganglionic axons, different neurotransmitters in general, sweat secretion by acetylcholine as the main neurotransmitter. The main rodent animal sweat glands located in the foot pad and fingers (toes), eccrine sweat by cholinergic agent, epinephrine injection or thermal stimulation of the motor stimuli how.TPM causes in children Whether the secretion of sweat is related to the effect on the cholinergic system? Experiments are conducted to start the study.
In this study, using silica gel blot model to detect the sweat function of TPM in mice. Then based on the model using HE staining and electron microscope observation of Hypohidrosis caused by TPM whether a relationship with the sweat gland structure changes; immunohistochemical staining observation of dominant nerve endings generally sweat glands, cholinergic nerve, adrenal gland adrenergic and cholinergic M3 receptor expression. Combined with Western blot and immunohistochemistry to observe the expression of cholinesterase in sweat, determination of cholinesterase activity. In addition a preliminary exploration on the morphology of GABA neurons in the spinal cord and the subthalamic region of sympathetic activity. The experimental results are as follows:
(1) 2 week old mice per day by intragastric administration of TPM (80mg/kg), administered continuously for a month later and after stopping for a week, the mice were pilocarpine stimulated sweat test, found that TPM treated mice compared with control mice the secretion of sweat glands secrete sweat number decreased by 17%, per unit of sweat gland no ultrastructural 40%. decreased light microscopic structure and sweat glands significantly changed.
(2) compared with the control group, TPM treatment of sweat gland innervation PGP9.5 weeks in general, cholinergic innervation marker ChAT, adrenergic marker TH innervation area had no obvious effect on cholinergic; expression of muscarinic M3 receptor expression and no significant effect on stellate ganglion neurotransmitters. Single sweat glands of the area no obvious change.
(3) the expression of AChE immunofluorescence and protein immunoblot in sweat glands after TPM treatment was not significantly different from that in contrast. After homogenization of sweat glands, TPM showed no significant effect on AChE activity.
(4) there is a double marker of ChAT and GAD65 immunofluorescence in the preganglionic neurons. After heat activation, there is a double mark in the hypothalamic thermosensitive neurons and the GABA neurons.
From these results, the following conclusions can be concluded:
(1) the administration of TPM (80mgkg-1d-1) to a certain extent reduced the function of sweat secretion in mice to a certain extent, and the function of secreting sweat was restored after the drug was stopped.
(2) the secretion of sweat caused by TPM did not affect the size and morphology of the sweat glands of mice and the ultrastructure.
(3) long term TPM treatment can reduce the secretion of reactive mouse eccrine sweat on pilocarpine, TPM did not affect the growth and sympathetic innervation of mouse sweat glands, sweat glands around the cholinergic expression of muscarinic receptors M3, AChE expression and activity of sweat gland has no significant impact.
(4) GABA neurons may participate in the regulation of cholinergic neurons before preganglionic. GABA neurons participate in the hypothalamic response to high temperature. This involvement may be related to TPM's sweating disorder.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R965;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 黃遠(yuǎn)桂,陳云春,杜芳,李銳,江文;托吡酯治療癲vN時(shí)出現(xiàn)的泌汗障礙的臨床分析[J];中國神經(jīng)精神疾病雜志;2004年05期

2 黃紹平,郭亞樂,和光祖,陳征起,何娟;托吡酯致癲vN患兒類暑熱癥42例臨床分析[J];中國實(shí)用兒科雜志;2004年10期

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本文編號(hào)：1523884