本文選題：H_2S + 兒茶酚胺��； 參考：《山東大學》2012年碩士論文

【摘要】：研究背景 硫化氫(H2S)是一種具有臭雞蛋氣味的有毒氣體。近年的研究表明,在人體內(nèi),硫化氫(H2S)是一種重要的氣體信號分子,其通過作用于不同細胞的離子通道和受體在哺乳動物組織中發(fā)揮著重要的生理的調(diào)節(jié)功能,并且具有治療心血管、神經(jīng)、生殖和消化等方面疾病的潛在價值。人體內(nèi)的L-半胱氨酸,可在胱硫醚β-合成酶(ystathionine β-synthase, CBS)和胱硫醚γ-裂解酶(cystathionine γ-lyase, CSE)作用下合成硫化氫。據(jù)相關(guān)文獻報道,大鼠腎上腺髓質(zhì)嗜鉻細胞(adrenal chromaffin cells, AMCs)含有CSE。腎上腺髓質(zhì)AMCs通過釋放兒茶酚胺(catecholamine, CA)在應激反應的調(diào)節(jié)中發(fā)揮了關(guān)鍵作用。據(jù)文獻報道缺氧可以導致CSE-/-型乳鼠AMCs的兒茶酚胺分泌減少。目前,對于外源性H2S能否誘導AMCs釋放CA及其離子機制尚無報道。在本研究中,我們使用安培法和全細胞膜片鉗記錄法探討外源性硫化氫能否誘發(fā)AMCs釋放CA及其離子機制。 材料和方法 實驗選用健康雌性Wistar大鼠(200-250g),自由飲食。實驗前麻醉Wistar大鼠,取出腎上腺置于D-Hanks液中,剪除皮質(zhì),將每個髓質(zhì)均分成兩個,置于0.5ml混合酶液中(膠原酶Ⅰ3mg/ml、透明質(zhì)酸酶2.4mg/ml、DNase0.5mg/ml、BSA2.6mg/ml)37℃恒溫孵育70分鐘,吸出酶液加入lml D-Hanks液沖洗組織塊,再用巴斯德管吹打組織塊,顯微鏡下觀察到細胞后停止吹打,將細胞固定于蓋玻片(用PLL處理過)上然后加入DMEM培養(yǎng)基(含10%的FBS),37℃恒溫CO2培養(yǎng)箱中培養(yǎng)24小時。將固定有細胞的蓋玻片放入記錄腔室中,并用細胞外液持續(xù)灌流,速度為lml/min。使用5微米的碳纖維電極檢測兒茶酚胺的釋放。全細胞記錄法檢測嗜鉻細胞的電流變化。 所有數(shù)據(jù)均采用均數(shù)±標準誤表示,采用單因素方差分析比較多組間的差異,t檢驗比較兩組間的差異,以P0.05為顯著性差異界值。 結(jié)果 標準細胞外液中加入外源性的硫氫化鈉(H2S供體)可以誘發(fā)AMCs釋放CA。實驗中使用70mMKCl作為陽性對照,細胞外液作為陰性對照。780mV電壓時可以觀察到碳纖維電極氧化CA產(chǎn)生的尖峰,切換到OmV時尖峰消失。將標準細胞外液換成無鈣的細胞外液,硫氫化鈉不能誘發(fā)AMCs釋放CA。綜上所述,H2S可以誘導AMCs釋放CA。 同時使用安培記錄法和全細胞記錄法檢測AMCs狀態(tài),發(fā)現(xiàn)加入外源性的硫氫化鈉可以誘發(fā)AMCs去極化,并且伴有CA的分泌。 硫氫化鈉可以抑制鈣激活的鉀通道。將細胞外液切換成無鈣溶液時,硫氫化鈉對的抑制作用消失。 結(jié)論 H2S能夠直接誘發(fā)AMCs釋放CA并且抑制K (Ca)電流。這一結(jié)論表明,硫化氫可能是通過調(diào)節(jié)腎上腺髓質(zhì)K (Ca)的電流導致CA的釋放進而參與哺乳類動物機體的應激反應。
[Abstract]:Research background Hydrogen sulfide (H 2 S) is a toxic gas with the smell of rotten eggs. Recent studies have shown that hydrogen sulfide (H _ 2S) is an important gas signaling molecule in human body, which plays an important physiological regulatory function in mammalian tissues through ion channels and receptors acting on different cells. It also has potential value in the treatment of cardiovascular, neurological, reproductive and digestive diseases. Human L-cysteine can synthesize hydrogen sulfide under the action of cystathionine 尾 -synthase (CBS) and cystathionine 緯 lyase (CSE). According to related literature, adrenal chromaffin cells, AMCs) in rat adrenal medullary cells contains CSE. Adrenomedullary AMCs plays a key role in the regulation of stress by releasing catecholamine. Hypoxia has been reported to reduce catecholamine secretion in AMCs of CSE-r-type neonatal mice. At present, whether exogenous H 2S can induce the release of CA from AMCs and its ion mechanism have not been reported. In this study, amperometric method and whole-cell patch clamp recording were used to investigate whether exogenous hydrogen sulfide could induce the release of CA from AMCs and its ion mechanism. Materials and methods Healthy female Wistar rats were fed a free diet of 200-250 g / g. Before the experiment, Wistar rats were anesthetized, the adrenal gland was taken out and placed in D-Hanks solution, the cortex was cut off, and each medulla was divided into two parts. Each medulla was incubated in 0.5ml mixed enzyme solution (collagenase 鈪，

本文編號：1978113