本文關(guān)鍵詞：碘化-N-正丁基氟哌啶醇對(duì)缺氧心肌細(xì)胞鈣穩(wěn)態(tài)的保護(hù)作用　出處：《汕頭大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 碘化-N-正丁基氟哌啶醇 缺氧/缺血 鈣瞬變

【摘要】：碘化N-正丁基氟哌啶醇(N-n-butyl haloperidol iodide, F_2)是我們課題組在氟哌啶醇的基礎(chǔ)上進(jìn)行結(jié)構(gòu)改造,得到一系列結(jié)構(gòu)全新的氟哌啶醇季銨鹽衍生物,通過(guò)篩選得到的一個(gè)化合物。前期研究發(fā)現(xiàn)F_2作為L(zhǎng)-型鈣通道阻滯劑,劑量依賴地拮抗缺血再灌注所致對(duì)大鼠心臟損傷。心肌細(xì)胞胞內(nèi)鈣穩(wěn)態(tài)失衡與缺血再灌注損傷的發(fā)生密切相關(guān),而鈣瞬變是心肌細(xì)胞中最明顯、最強(qiáng)烈和最典型的胞內(nèi)鈣信號(hào)變化,因此研究F_2在缺氧/缺血時(shí)對(duì)心肌細(xì)胞胞內(nèi)鈣穩(wěn)態(tài)及鈣信號(hào)主要是鈣瞬變的影響的意義顯得尤其重要,這對(duì)于鈣拮抗劑新機(jī)制的探討,豐富作用原理具有重要的指導(dǎo)意義,也為藥物的研發(fā)提供新的理論依據(jù)。本研究擬在缺氧過(guò)程中觀察F_2對(duì)心肌細(xì)胞鈣瞬變的影響,并探討其可能的保護(hù)機(jī)制。 方法 1.采用標(biāo)準(zhǔn)酶解法消化分離成年SD大鼠心室肌細(xì)胞。5μM Fluo-4 AM室溫染色15min標(biāo)記心肌胞內(nèi)Ca~(2+),然后用含有2.5mM Ca~(2+)的臺(tái)氏液沖洗除去多余染料。共聚焦圖像采用Olympus FluoViewFV1000共聚焦顯微鏡記錄,鈣瞬變通過(guò)給予頻率1Hz的方波閾上刺激誘發(fā)。 2.心肌細(xì)胞首先給予正常臺(tái)氏液灌流20min,然后給予充90% N2-10% CO_2的缺氧液灌流30min,灌流通過(guò)重力作用控制,流速為6ml/min,建立缺氧模型,共聚焦顯微鏡依次記錄缺氧10min、20min和30min時(shí)心肌細(xì)胞鈣瞬變圖像,觀察缺氧對(duì)心肌細(xì)胞鈣瞬變的影響。 3.心肌細(xì)胞給予正常臺(tái)氏液灌流20min,然后隨機(jī)給予0.1,1,10μM F_2的缺氧液灌流30min。共聚焦顯微鏡依次記錄含有不同濃度F_2的缺氧液灌流10min,20min和30min時(shí)心肌細(xì)胞鈣瞬變圖像,觀察F_2在缺氧時(shí)對(duì)心肌細(xì)胞鈣瞬變的影響。 4.心肌細(xì)胞首先給予正常臺(tái)氏液灌流20min,然后隨機(jī)給予0,0.1,1,10μMF_2的缺氧液灌流30min后,用微操儀控制自制重力給藥系統(tǒng),快速噴終濃度為15mMcaffeine,檢測(cè)肌質(zhì)網(wǎng)鈣儲(chǔ)量。 5.鈣瞬變過(guò)程中,胞內(nèi)增加的Ca~(2+)主要由肌質(zhì)網(wǎng)鈣泵(SERCA2a)、細(xì)胞膜鈉鈣交換體(NCX)和鈣泵(PMCA)清除。為檢測(cè)肌質(zhì)網(wǎng)鈣泵在鈣瞬變過(guò)程中移除鈣的比例,缺氧液灌流30min后我們給予5μM Thapsigargin(TG)孵育6min阻斷SERCA2a,這時(shí),下降的速率常數(shù)反映了細(xì)胞膜NCX和PMCA的移除速率(VNCX+PMCA)。肌質(zhì)網(wǎng)鈣泵移除Ca~(2+)的速率VSERCA=VTOTAL-VNCX+PMCA。 6.給予5μM Thapsigargin +5μM carboxyeosin (PMCA阻斷劑)室溫下孵育6min,此時(shí)下降的速率常數(shù)反映了細(xì)胞膜NCX的移除速率VNCX。 結(jié)果 1.缺氧30min可以導(dǎo)致心肌細(xì)胞靜息鈣水平升高,鈣瞬變幅度降低,RT25-75、T50和DT75-25時(shí)間延長(zhǎng)。 2.給予含有不同濃度的F_2(0.1,1,10μM)缺氧液灌流30min,可以劑量依賴地抑制心肌細(xì)胞靜息鈣水平升高,鈣瞬變幅度降低,RT25-75、T50和DT75-25時(shí)間延長(zhǎng)。 3.缺氧30min可以導(dǎo)致肌質(zhì)網(wǎng)鈣儲(chǔ)量減少,含有不同濃度F_2(0.1,1,10μM)的缺氧液灌流30min,可以劑量依賴地抑制肌質(zhì)網(wǎng)鈣儲(chǔ)量減少。 4.缺氧30min可以導(dǎo)致肌質(zhì)網(wǎng)鈣泵移除Ca~(2+)的比例降低。給予含有不同濃度F_2(0.1,1,10μM)的缺氧液灌流30min,可以劑量依賴地抑制肌質(zhì)網(wǎng)鈣泵移除Ca~(2+)的比例降低。 5.缺氧30min,F_2對(duì)鈉鈣交換體(NCX)移除Ca~(2+)的比例沒(méi)有影響。 結(jié)論 1.缺氧可以導(dǎo)致心肌細(xì)胞胞內(nèi)鈣穩(wěn)態(tài)失衡,包括靜息鈣水平升高,鈣瞬變幅度降低,RT25-75、DT75-25和T50延長(zhǎng)。 2.F_2可以抑制缺氧導(dǎo)致的心肌細(xì)胞胞內(nèi)鈣信號(hào)的改變,包括缺氧導(dǎo)致的靜息鈣水平升高,鈣瞬變幅度降低,RT25-75、DT75-25和T50延長(zhǎng)。 3.F_2對(duì)肌質(zhì)網(wǎng)鈣泵SERCA2a的保護(hù)作用是其加速缺氧時(shí)鈣瞬變移除速率及影響肌質(zhì)網(wǎng)鈣儲(chǔ)量的主要機(jī)制。 4.F_2對(duì)肌質(zhì)網(wǎng)鈣儲(chǔ)量的影響間接調(diào)節(jié)雷諾定受體的活性,即鈣瞬變RT25-75。 5.F_2在心肌缺氧時(shí)抑制跨膜Ca~(2+)內(nèi)流的主要作用靶點(diǎn)不是鈉鈣交換體(NCX),而是L-型鈣通道。 6.F_2防止鈣超載和肌質(zhì)網(wǎng)鈣儲(chǔ)量減少及對(duì)肌質(zhì)網(wǎng)鈣泵和雷諾定受體活性的保護(hù)作用是其影響鈣瞬變幅度和T50的主要原因。
[Abstract]:N-n-butyl haloperidol iodide N- (N-n-butyl haloperidol iodide, F_2) is our group structural transformation in haloperidol on the basis of a series of novel quaternary ammonium salt derivatives, through a compound were obtained. The preliminary study found that F_2 as the L- type calcium channel blockers, antagonist dose dependently induced by ischemia reperfusion on the heart injury in rats. Myocardial intracellular calcium homeostasis and ischemia reperfusion injury is closely related to the occurrence of calcium transients are most obvious in myocardial cells, calcium signal changes of the strongest and most typical in the cell, so the research of F_2 in hypoxia / ischemia on calcium homeostasis and calcium signaling in cardiac cells mainly is the effect of calcium transients, the significance is particularly important, to explore the new mechanism of the calcium antagonist, has an important guiding significance to enrich the effect principle, also for drug development. This study intends to observe the effect of F_2 on calcium transient in cardiac myocytes during the hypoxia process and to explore the possible protective mechanism.
Method
1. by standard enzymatic digestion of isolated adult rat ventricular myocytes SD.5 M Fluo-4 AM 15min labeled myocardial intracellular staining at room temperature Ca~ (2+), and then with 2.5mM containing Ca~ (2+) Tyrode's solution rinse to remove the excess dye. Confocal images using Olympus FluoViewFV1000 confocal microscope recorded by calcium transients given the frequency of 1Hz square wave induced by suprathreshold stimulation.
2. myocardial cells give first normal Tyrode's solution perfusion 20min, hypoxia and then give 90% N2-10% liquid filling CO_2 perfusion 30min and perfusion control by gravity, the flow rate was 6ml/min, the establishment of hypoxia model, confocal microscope were recorded by hypoxia 10min, myocardial cell calcium transient images of 20min and 30min, to observe the effect of hypoxia myocardial cell calcium transients.
3. myocardial cells with normal Tyrode's solution perfusion 20min, hypoxia solution and then randomly given 0.1,1,10 M F_2 perfusion 30min. confocal microscope were recorded by hypoxia liquid with different concentrations of F_2 perfusion 10min, myocardial cell calcium transient images of 20min and 30min, to observe the effect of F_2 on myocardial intracellular calcium transients in hypoxia.
4. myocardial cells give first normal Tyrode's solution perfusion 20min, hypoxia solution and then randomly given 0,0.1,1,10 MF_2 perfusion after 30min with micro gravity control instrument homemade drug delivery system, rapid injection at final concentrations of 15mMcaffeine, detection of sarcoplasmic reticulum calcium reserves.
5. calcium transients, and increased the intracellular Ca~ (2+) is mainly composed of calcium pump of sarcoplasmic reticulum (SERCA2a), sodium calcium exchange (NCX) and calcium pump (PMCA) removed. For the detection of calcium pump of sarcoplasmic reticulum calcium transients in removing calcium during hypoxia perfusion ratio we give the flow after 30min 5 M Thapsigargin (TG) were incubated with 6min blocking SERCA2a, at this time, the rate constant decreased reflects the cell membrane NCX and PMCA removal rate (VNCX+PMCA). Calcium pump of sarcoplasmic reticulum (2+) of the Ca~ removal rate of VSERCA=VTOTAL-VNCX+PMCA.
6. a 5 mu M Thapsigargin +5 mu M carboxyeosin (PMCA blocker) was incubated at room temperature for 6min, and the rate constant of the decrease reflected the removal rate VNCX. of the cell membrane NCX.
Result
1. anoxic 30min could lead to the increase of resting calcium level in cardiac myocytes, the decrease of calcium transient amplitude, and prolonged time of RT25-75, T50 and DT75-25.
2., 30min was injected with different concentrations of F_2 (0.1,1,10 M) anoxic solution, which could inhibit the increase of resting calcium level and decrease the calcium transient amplitude, and prolong the time of RT25-75, T50 and DT75-25.
3., hypoxia 30min can reduce the calcium storage of sarcoplasmic reticulum, and 30min with different concentrations of F_2 (0.1,1,10 M) can reduce the calcium storage of sarcoplasmic reticulum in a dose dependent manner.
4., hypoxia 30min can lead to a decrease in the proportion of Ca~ (2+) removed by sarcoplasmic reticulum calcium pump. The perfusion of 30min with different concentration of F_2 (0.1,1,10 M M) can reduce the proportion of Ca~ (2+) of calcium pump in sarcoplasmic reticulum in a dose dependent manner.
5. anoxic 30min, F_2 did not affect the ratio of sodium calcium exchanger (NCX) to the removal of Ca~ (2+).
conclusion
1. anoxia can lead to the imbalance of intracellular calcium homeostasis, including elevated resting calcium levels, reduced calcium transient amplitude, and prolonged RT25-75, DT75-25 and T50.
2.F_2 can inhibit the change of intracellular calcium signal induced by hypoxia, including the increase of resting calcium level caused by hypoxia, the decrease of calcium transient amplitude, and the prolongation of RT25-75, DT75-25 and T50.
The protective effect of 3.F_2 on the calcium pump SERCA2a of the sarcoplasmic reticulum is the main mechanism for accelerating the calcium transient removal rate and affecting the calcium reserves of the sarcoplasmic reticulum.
The effect of 4.F_2 on the calcium reserves of the sarcoplasmic reticulum indirectly regulates the activity of Reynolds receptor, that is, calcium transient RT25-75.
The main target of 5.F_2 to inhibit the transmembrane Ca~ (2+) inflow in myocardial anoxia is not the sodium calcium exchanger (NCX), but the L- type calcium channel.
6.F_2 prevents calcium overload and sarcoplasmic reticulum calcium storage, and protects the sarcoplasmic reticulum calcium pump and Renault receptor activity, which is the main reason for its influence on calcium transient amplitude and T50.

【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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本文編號(hào)：1380506