【摘要】：研究背景及目的:經(jīng)美國FDA批準(zhǔn),全身麻醉藥丙泊酚于1989年正式上市,是具有里程碑意義的事件。自此丙泊酚憑借其起效快、無蓄積、恢復(fù)迅速等優(yōu)勢,在臨床上廣泛應(yīng)用。目前世界范圍內(nèi)每年有上千萬例外科手術(shù)在丙泊酚麻醉下進(jìn)行。丙泊酚已成為臨床應(yīng)用最為廣泛且不可或缺的全身靜脈麻醉藥物。然而和其他全麻藥一樣,丙泊酚的麻醉作用機(jī)制至今仍然不清楚。揭示全麻機(jī)理是神經(jīng)科學(xué)和麻醉學(xué)的夢想。2005年《Science》雜志主編Donald Kennedy將全麻機(jī)理列為學(xué)術(shù)界今后100年面臨的125個(gè)世紀(jì)難題之一[1],攻破該堡壘亦將成為神經(jīng)科學(xué)和麻醉學(xué)領(lǐng)域里程碑式的飛躍。全麻引起的可逆性意識(shí)消失作為大腦的特殊非生理狀態(tài),與學(xué)習(xí)記憶、睡眠覺醒等大腦高級(jí)功能息息相關(guān)[2]。近年來越來越多的研究結(jié)果支持全麻藥物的作用機(jī)制并非以往認(rèn)為的——麻醉藥物對大腦整體的非特異性廣泛抑制,而是由各解剖位置、功能特點(diǎn)不同的神經(jīng)回路構(gòu)成的復(fù)雜網(wǎng)絡(luò)協(xié)同參與麻醉藥物引起的大腦功能可逆性改變[3-4],即麻醉藥物通過作用于特定大腦區(qū)域,阻斷不同腦區(qū)的功能聯(lián)系,打破信息在高級(jí)中樞的正、負(fù)反饋平衡而發(fā)揮麻醉作用。Ying的研究提示丘腦網(wǎng)狀核(thalamic reticular nucleus,TRN)在丙泊酚的全麻機(jī)理中發(fā)揮了重要作用[5]。然而,丙泊酚是否以及如何作用于TRN并產(chǎn)生麻醉作用仍然不清楚。TRN在大腦信息調(diào)控中的地位極其特殊。它由GABA能抑制性神經(jīng)元組成,調(diào)控丘腦-皮層環(huán)路,是網(wǎng)狀上行激動(dòng)系統(tǒng)的一部分。自2011年起,陸續(xù)有文獻(xiàn)報(bào)道通過光遺傳、電生理等技術(shù)發(fā)現(xiàn)TRN內(nèi)部存在不同的功能性子網(wǎng),即TRN各個(gè)亞核團(tuán)的GABA能神經(jīng)元投射至丘腦及皮層的不同區(qū)域并發(fā)揮不同的生理作用[6]。由于TRN特殊的解剖位置、生理功能,并且和睡眠、覺醒、意識(shí)等大腦功能密切相關(guān),啟發(fā)并促使我們探索它在麻醉這個(gè)無知覺、無意識(shí)、類似而有別于睡眠的特殊非生理狀態(tài)中所起的作用。本課題致力于探索TRN內(nèi)部亞核團(tuán)參與丙泊酚發(fā)揮麻醉作用的具體分子、細(xì)胞、網(wǎng)絡(luò)及行為機(jī)制,為丙泊酚中樞麻醉的網(wǎng)絡(luò)學(xué)說及相關(guān)核團(tuán)定位提供新的依據(jù)和方向。研究方法:(1)使用鵝膏蕈氨酸分別對TRN各亞核團(tuán)進(jìn)行化學(xué)性毀損,檢測核團(tuán)毀損后大鼠對丙泊酚麻醉的反應(yīng)及敏感性改變。(2)利用即刻表達(dá)基因C-Fos和GAD-67免疫熒光共標(biāo)檢測TRN頭端(anterior thalamic reticular nucleus,a TRN)和TRN尾端(posterior thalamic reticular nucleus,p TRN)的GABA能神經(jīng)元在清醒、丙泊酚麻醉、麻醉復(fù)蘇三個(gè)階段的功能狀態(tài)。(3)通過分子免疫學(xué)方法驗(yàn)證丙泊酚的主要分子靶點(diǎn)GABAa受體β3亞基(GABAa receptor-β3 subunit,GABAa R-β3)在TRN各亞核團(tuán)中的存在和分布。(4)在大鼠雙側(cè)a TRN處注射GABAa受體競爭性拮抗劑荷包牡丹堿(bicuculline,BIC)阻斷該受體,觀察其行為改變。待大鼠狀態(tài)平穩(wěn)后,檢測其對丙泊酚麻醉的反應(yīng)及敏感性改變。(5)制備針對GABAa R-β3的小干擾RNA(small interfering RNA,si RNA)病毒,下調(diào)a TRN處GABAa R-β3的表達(dá)。免疫印跡法驗(yàn)證病毒干擾效果。待病毒起效后檢測大鼠對丙泊酚麻醉的反應(yīng)及敏感性改變。研究結(jié)果:(1)TRN各亞核團(tuán)被化學(xué)性毀損后,僅a TRN毀損組大鼠對丙泊酚的敏感性有明顯且持續(xù)的提高(1)a TRN毀損組大鼠相較于對照組大鼠在接受丙泊酚靜脈泵注后翻正反射消失(lost of righting reflex,LORR)的時(shí)間縮短并維持該狀態(tài),即a TRN毀損組大鼠對丙泊酚的敏感性有明顯且持續(xù)的提高;a TRN毀損組大鼠對性質(zhì)不同的靜脈麻醉藥物(右美托咪定)的敏感性無明顯變化。(2)p TRN毀損組大鼠相較于對照組大鼠接受丙泊酚及右美托咪定靜脈泵注后LORR時(shí)間均無明顯變化。(2)a TRN與p TRN的GABA能神經(jīng)元在清醒、麻醉和復(fù)蘇三個(gè)階段呈現(xiàn)相反的功能狀態(tài)(1)a TRN的GABA能神經(jīng)元在清醒時(shí)活躍,而在丙泊酚麻醉后處于相對抑制狀態(tài)。(2)p TRN的GABA能神經(jīng)元在丙泊酚麻醉后被明顯激活,而撤藥蘇醒后則處于相對抑制狀態(tài)。(3)a TRN和p TRN均表達(dá)丙泊酚作用靶點(diǎn)GABAa R-β3且a TRN的表達(dá)量較p TRN更為豐富且密集(1)采用免疫印跡法驗(yàn)證抗GABAa R-β3抗體的特異性并使用該抗體檢測GABAa R-β3在a TRN、p TRN、丘腦、海馬、皮層等腦組織的表達(dá)。結(jié)果顯示a TRN和p TRN區(qū)域均存在GABAa R-β3的蛋白表達(dá)。(2)免疫熒光檢測結(jié)果顯示,GABAa R-β3在a TRN和p TRN均有一定分布;在相同視野下,GABAa R-β3在a TRN的表達(dá)量較p TRN更為豐富且密集。(4)拮抗a TRN的GABAa R及下調(diào)a TRN處GABAa R-β3的表達(dá)使大鼠對丙泊酚敏感性下降(1)大鼠雙側(cè)a TRN處注射GABAa受體拮抗劑BIC阻斷該受體,動(dòng)物即刻行為活躍,活動(dòng)度明顯增加。(2)BIC注射組大鼠相較于對照組大鼠在接受丙泊酚靜脈泵注后LORR時(shí)間明顯延長,即BIC注射組大鼠對丙泊酚敏感性下降。(3)針對GABAa R-β3的si RNA病毒下調(diào)a TRN處該蛋白的表達(dá),病毒干擾組大鼠較對照組大鼠在接受丙泊酚靜脈泵注后LORR時(shí)間明顯延長,即病毒干擾組大鼠對丙泊酚敏感性下降。結(jié)論:a TRN的神經(jīng)元被化學(xué)性毀損后,動(dòng)物對丙泊酚的敏感性有顯著且持續(xù)的提高,而毀損其它部位則無類似變化。這提示a TRN極有可能參與丙泊酚對中樞神經(jīng)系統(tǒng)發(fā)揮麻醉作用的過程。C-Fos和GAD-67免疫熒光共標(biāo)發(fā)現(xiàn)a TRN與p TRN的GABA能神經(jīng)元在清醒、麻醉、復(fù)蘇三個(gè)階段顯示出相反的功能狀態(tài),表明TRN各亞核團(tuán)在丙泊酚麻醉過程中發(fā)生了不同的功能變化。BIC阻斷a TRN的GABAa R后動(dòng)物即刻表現(xiàn)出亢奮狀態(tài),即a TRN的GABA能神經(jīng)元興奮性升高使動(dòng)物呈現(xiàn)與麻醉相反的行為學(xué)表現(xiàn)。BIC注射組相較于對照組,大鼠對丙泊酚敏感性下降。推測一方面a TRN處的GABAa R可能是丙泊酚的作用靶點(diǎn),其被拮抗劑阻斷后使丙泊酚麻醉效能降低;另一方面a TRN的GABA能神經(jīng)元興奮性升高可能對抗丙泊酚的麻醉作用。免疫熒光結(jié)果顯示GABAa R-β3在a TRN處相較于p TRN有著更為豐富且密集的表達(dá),在形態(tài)學(xué)角度支持a TRN作為丙泊酚麻醉神經(jīng)網(wǎng)絡(luò)相關(guān)核團(tuán)的特殊性。利用si RNA病毒下調(diào)GABAa R-β3的表達(dá),病毒干擾組大鼠相較于對照組大鼠對丙泊酚敏感性下降。進(jìn)一步證明a TRN處的GABAa R-β3對丙泊酚發(fā)揮麻醉作用至關(guān)重要。綜上所述,我們推測丙泊酚極有可能通過與位于a TRN的GABAa R-β3結(jié)合后,抑制了該處GABA能神經(jīng)元(a TRN的GABA能神經(jīng)元興奮性下降),從而使其下游、處于p TRN的GABA能神經(jīng)元去抑制。p TRN的GABA能神經(jīng)元興奮性升高,其軸突末梢發(fā)放更多的抑制性信號(hào)到丘腦和皮層,最終產(chǎn)生麻醉鎮(zhèn)靜作用(參見假說示意圖)。
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圖片說明：假說示意圖
[Abstract]:The background and purpose of the study were that, with the approval of the U.S. FDA, the general anesthetic propofol was officially launched in 1989 and was a landmark event. Since then, the propofol has the advantages of quick action, no accumulation, rapid recovery and the like, and is widely applied in clinic. In that present world, there are ten million surgical procedures each year under the anesthesia of propofol. Propofol has become the most widely and indispensable general intravenous anesthesia drug in clinical application. The anesthesia mechanism of propofol, however, is still not clear to date, as is the case with other panacea. The mechanism of general anesthesia is the dream of neuroscience and anesthesiology. The reversible consciousness caused by general anesthesia disappears as a special non-physiological state of the brain, and is closely related to the high-level functions of the brain such as learning memory and sleep wakefulness[2]. In recent years, more and more research results support the mechanism of the general anesthesia drug, which is not the general non-specific inhibition of the whole brain, but by the various anatomical positions, the complex network of the neural circuits with different functional characteristics can participate in the reversible change of the function of the brain caused by the anesthesia drug (3-4], that is, the narcotic drug acts on a specific brain region, blocks the functional contact of different brain regions, and breaks the positive of the information in the high-level center, And a negative feedback balance is used to play the role of anesthesia. The study of Ying suggested that thalamic reticulate nucleus (TRN) played an important role in the general anesthesia mechanism of propofol[5]. However, whet or not that propofol and how to act on the TRN is still not clear. The status of TRN in the regulation of brain information is extremely special. It is composed of GABA-capable inhibitory neurons, and the control of the thalamus-cortical loop is a part of the mesh up-up system. Since 2011, it has been reported that there are different functional sub-networks in TRN, that is, the GABA-energy neurons of the TRN subnuclei are projected to different regions of the thalamus and the cortex and play different physiological functions[6]. Because of the special anatomy of TRN, the physiological function, and the function of the brain, such as sleep, wakefulness, consciousness, and so on, we have inspired and led us to explore the role of it in the special non-physiological state of anesthesia, unconsciousness, unconsciousness, and the like. The purpose of this study is to explore the specific molecular, cellular, network and behavioral mechanism of the participation of the internal subnuclei of TRN in the anesthesia of propofol, and provide a new basis and direction for the network theory of the central anesthesia of propofol and the location of the related nuclear clusters. Methods: (1) The rats of TRN were chemically damaged by using the goose extract, and the response and the sensitivity of the rats to the anesthesia of propofol were detected. (2) The functional states of the GABA-ergic neurons of the TRN head-end (a TRN) and the tail-end of the TRN were detected by the immediate expression of the C-Fos and the GAD-67 immunofluorescence. (3) The existence and distribution of GABAa receptor-3 subunit (GABAa receptor-Sup3 subunit, GABAa R-Sup3) in the TRN subnuclei were verified by molecular immunology. (4) A competitive antagonist of the GABAa receptor (bicuculline, BIC) was injected at the two-side a TRN of the rat to block the receptor and the behavior of the receptor was observed. After the state of the rat was stable, the response and sensitivity of propofol anesthesia were detected. (5) Preparation of small interfering RNA (si RNA) virus for GABAa R-Sup3 and down-regulation of the expression of GABAa R-Sup3 at a TRN. And the effect of the virus interference is verified by the immunoblotting method. And the response and the sensitivity of the rat to the propofol anesthesia were detected after the effect of the virus. The results of the study: (1) After the chemical damage of the TRN subnuclei, the sensitivity of only a TRN damaged group to the propofol was significantly and continuously increased (1) a (1) a TRN damaged group was reflected and disappeared after the injection of the propofol intravenous pump compared with the control group (lost of right reflex, The time of LORR was shortened and maintained, i.e., the sensitivity of a TRN damaged group to propofol was significantly and continuously increased; a TRN damaged group of rats had no significant change in the sensitivity of the different intravenous anesthesia drugs (dexmedetomidine). (2) There was no significant change in the time of LORR after injection of propofol and dexmedetomidine in the p-TRN damaged group compared with the control group. (2) The GABA-ergic neurons of a TRN and p-TRN exhibited the opposite functional states (1) a TRN in the three stages of conscious, anesthesia and resuscitation, while the GABA-ergic neurons of a TRN were active in the awake period, and were in a state of relative inhibition after the anesthesia of propofol. (2) The GABA-energy neurons of p-TRN were significantly activated after the anesthesia of propofol, and were in a state of relative inhibition after the withdrawal of the drug. (3) The expression of a TRN and p TRN was more abundant and the expression of a TRN was more abundant and the expression of a TRN was more abundant and dense (1) The specificity of the anti-GABAa R-Sup3 antibody was verified by the immunoblotting method and the antibody was used to detect the specificity of the anti-GABAa R-Sup3 antibody and to use the antibody to detect the expression of a TRN, p TRN, thalamus, and hippocampus. The expression of the brain tissue, such as the cortex. The results showed that the expression of GABAa R-Sup3 in a TRN and p TRN regions. (2) The results of immunofluorescence test showed that GABAa R-CD3 had a certain distribution in a TRN and p TRN; in the same visual field, the expression of GABAa R-CD3 in a TRN was more abundant and dense. (4) The expression of GABAa R and the down-regulation a TRN of a TRN inhibited the expression of the GABAa receptor antagonist BIC at the two-side a TRN of the rat on the decrease of the sensitivity of propofol (1). (2) The LRR time of the BBIC injection group was significantly prolonged compared with that of the control group in the control group, that is, the sensitivity of the BBIC injection group to the propofol was decreased. (3) The expression of the protein at a TRN was down-regulated for the si-RNA virus of the GABAa R-Sup3, and the time of the LORR in the control group of the virus-interference group was significantly prolonged after the injection of the propofol intravenous pump, that is, the sensitivity of the group of the virus to the propofol was decreased. Conclusion: After the neurons of a TRN were chemically damaged, the sensitivity of the animals to propofol was significantly and continuously increased, and no similar changes were observed in other parts. This suggests that a TRN is most likely to be involved in the anesthesia of the central nervous system by propofol. C-Fos and GAD-67 immunofluorescence co-standard showed that the GABA-ergic neurons of a TRN and p-TRN showed the opposite functional states in the three stages of conscious, anesthesia and resuscitation, indicating that the TRN subnuclei had different functional changes in the course of propofol anesthesia. After the BIC block a TRN, the animals showed a hyperactive state immediately after the GABAa R of a TRN. In the BIC injection group, the sensitivity of propofol to propofol was decreased compared with the control group. It is suggested that, on the one hand, the GABAa R at a TRN may be the target of the action of propofol, which is blocked by the antagonist, so that the anesthesia efficiency of propofol is reduced; on the other hand, the excitability of the GABA of a TRN can antagonize the anesthesia effect of propofol. The results showed that GABAa R-CD3 had a more abundant and intensive expression at a TRN than p TRN, and it was supported a TRN at a morphological angle as the particularity of the related nuclear group of propofol anesthesia neural network. The expression of GABAa R-Sup3 was down-regulated by the si-RNA virus, and the sensitivity of propofol to propofol was decreased in the virus-disturbed group compared with the control group. It is further proved that the GABAa R-Sup3 at a TRN is essential for the anesthesia of propofol. In conclusion, we have speculated that, after the combination of the GABAa R-Sup3 located in a TRN, it is possible to inhibit the GABA-energy neurons (a TRN in the GABA-energy neurons of a TRN) from decreasing the excitability of the GABA-ergic neurons in the region, so that the GABA-energy neurons in the downstream and in the p-TRN can be inhibited. The GABAergic neurons of p-TRN increase the excitability of the neurons, and the axons of their axons release more inhibitory signals to the thalamus and the cortex, resulting in the anesthesia and sedation (see the hypothesis).
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R614.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;GABA_A receptor partially mediated propofol-induced hyperalgesia at superspinal level and analgesia at spinal cord level in rats[J];Acta Pharmacologica Sinica;2004年12期

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本文編號(hào)：2511885