碘化-N-正丁基氟哌啶醇对缺氧心肌细胞钙稳态的保护作用

发布时间：2018-01-04 23:11

本文关键词：碘化-N-正丁基氟哌啶醇对缺氧心肌细胞钙稳态的保护作用　出处：《汕头大学》2011年博士论文　论文类型：学位论文

【摘要】：碘化N-正丁基氟哌啶醇(N-n-butyl haloperidol iodide, F_2)是我们课题组在氟哌啶醇的基础上进行结构改造,得到一系列结构全新的氟哌啶醇季铵盐衍生物,通过筛选得到的一个化合物。前期研究发现F_2作为L-型钙通道阻滞剂,剂量依赖地拮抗缺血再灌注所致对大鼠心脏损伤。心肌细胞胞内钙稳态失衡与缺血再灌注损伤的发生密切相关,而钙瞬变是心肌细胞中最明显、最强烈和最典型的胞内钙信号变化,因此研究F_2在缺氧/缺血时对心肌细胞胞内钙稳态及钙信号主要是钙瞬变的影响的意义显得尤其重要,这对于钙拮抗剂新机制的探讨,丰富作用原理具有重要的指导意义,也为药物的研发提供新的理论依据。本研究拟在缺氧过程中观察F_2对心肌细胞钙瞬变的影响,并探讨其可能的保护机制。方法 1.采用标准酶解法消化分离成年SD大鼠心室肌细胞。5μM Fluo-4 AM室温染色15min标记心肌胞内Ca~(2+),然后用含有2.5mM Ca~(2+)的台氏液冲洗除去多余染料。共聚焦图像采用Olympus FluoViewFV1000共聚焦显微镜记录,钙瞬变通过给予频率1Hz的方波阈上刺激诱发。 2.心肌细胞首先给予正常台氏液灌流20min,然后给予充90% N2-10% CO_2的缺氧液灌流30min,灌流通过重力作用控制,流速为6ml/min,建立缺氧模型,共聚焦显微镜依次记录缺氧10min、20min和30min时心肌细胞钙瞬变图像,观察缺氧对心肌细胞钙瞬变的影响。 3.心肌细胞给予正常台氏液灌流20min,然后随机给予0.1,1,10μM F_2的缺氧液灌流30min。共聚焦显微镜依次记录含有不同浓度F_2的缺氧液灌流10min,20min和30min时心肌细胞钙瞬变图像,观察F_2在缺氧时对心肌细胞钙瞬变的影响。 4.心肌细胞首先给予正常台氏液灌流20min,然后随机给予0,0.1,1,10μMF_2的缺氧液灌流30min后,用微操仪控制自制重力给药系统,快速喷终浓度为15mMcaffeine,检测肌质网钙储量。 5.钙瞬变过程中,胞内增加的Ca~(2+)主要由肌质网钙泵(SERCA2a)、细胞膜钠钙交换体(NCX)和钙泵(PMCA)清除。为检测肌质网钙泵在钙瞬变过程中移除钙的比例,缺氧液灌流30min后我们给予5μM Thapsigargin(TG)孵育6min阻断SERCA2a,这时,下降的速率常数反映了细胞膜NCX和PMCA的移除速率(VNCX+PMCA)。肌质网钙泵移除Ca~(2+)的速率VSERCA=VTOTAL-VNCX+PMCA。 6.给予5μM Thapsigargin +5μM carboxyeosin (PMCA阻断剂)室温下孵育6min,此时下降的速率常数反映了细胞膜NCX的移除速率VNCX。结果 1.缺氧30min可以导致心肌细胞静息钙水平升高,钙瞬变幅度降低,RT25-75、T50和DT75-25时间延长。 2.给予含有不同浓度的F_2(0.1,1,10μM)缺氧液灌流30min,可以剂量依赖地抑制心肌细胞静息钙水平升高,钙瞬变幅度降低,RT25-75、T50和DT75-25时间延长。 3.缺氧30min可以导致肌质网钙储量减少,含有不同浓度F_2(0.1,1,10μM)的缺氧液灌流30min,可以剂量依赖地抑制肌质网钙储量减少。 4.缺氧30min可以导致肌质网钙泵移除Ca~(2+)的比例降低。给予含有不同浓度F_2(0.1,1,10μM)的缺氧液灌流30min,可以剂量依赖地抑制肌质网钙泵移除Ca~(2+)的比例降低。 5.缺氧30min,F_2对钠钙交换体(NCX)移除Ca~(2+)的比例没有影响。结论 1.缺氧可以导致心肌细胞胞内钙稳态失衡,包括静息钙水平升高,钙瞬变幅度降低,RT25-75、DT75-25和T50延长。 2.F_2可以抑制缺氧导致的心肌细胞胞内钙信号的改变,包括缺氧导致的静息钙水平升高,钙瞬变幅度降低,RT25-75、DT75-25和T50延长。 3.F_2对肌质网钙泵SERCA2a的保护作用是其加速缺氧时钙瞬变移除速率及影响肌质网钙储量的主要机制。 4.F_2对肌质网钙储量的影响间接调节雷诺定受体的活性,即钙瞬变RT25-75。 5.F_2在心肌缺氧时抑制跨膜Ca~(2+)内流的主要作用靶点不是钠钙交换体(NCX),而是L-型钙通道。 6.F_2防止钙超载和肌质网钙储量减少及对肌质网钙泵和雷诺定受体活性的保护作用是其影响钙瞬变幅度和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.

【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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