小胶质细胞表达钾离子通道Kv1.2及其对细胞因子和活性氧生成的调控作用

发布时间：2018-05-06 05:27

本文选题：阿米巴状小胶质细胞 + Kv1.2　；参考：《昆明医学院》2008年博士论文

【摘要】： 小胶质细胞是中枢神经系统的“感受器”,当脑内微环境发生变化时它首先被激活,活化后的小胶质细胞通过释放细胞因子和活性氧(Reactive OxygenSpecies,ROS)等对周围环境进行调整;此时其膜电生理学特性的改变对其生物学行为起到调控作用;小胶质细胞表达多种离子通道,它们协调运转控制着小胶质细胞膜的生物学特性。业已证实小胶质细胞表达电压依从性钾离子通道(Voltage gated potassium channel,Kv),它们所控制的外向延迟整合钾离子电流是小胶质细胞活化的特征,其开放所致的细胞内钾离子浓度降低是上调细胞因子表达的关键步骤。研究表明小胶质细胞表达Kv1.3、Kv1.5,它们与小胶质细胞的增殖、迁移、释放细胞因子和ROS等多种生理功能有关;使用这些通道的阻断剂能够降低小胶质细胞活化的程度,减轻神经元损伤。因此深入研究小胶质细胞膜上Kv的表达及其可能的功能,对阐明小胶质细胞活化的分子机制,寻找调控其活化的靶点具有重要意义。目的:本研究旨在探索钾离子通道Kv1.2在小胶质细胞的表达及其可能的功能。方法:采用免疫荧光双重标记法观察大鼠不同发育阶段脑内阿米巴状小胶质细胞表达Kv1.2的情况;并通过免疫荧光双重标记和real time PCR技术,观察体外培养的小胶质细胞Kv1.2表达的情况。体内建立大鼠缺氧动物模型,观察不同年龄大鼠缺氧后小胶质细胞表达Kv1.2的变化;体外采用脂多糖(Lipopolysaccharide,LPS)、缺氧(Hypoxia exposure,HPE)、三磷酸腺苷(Adenosine 5'-triphosphate,ATP)诱导激活小胶质细胞,通过real time PCR观察小胶质细胞Kv1.2、白细胞介素1β(Interleukin-1β,IL-1β)和肿瘤坏死因子(Tumornecrosis factor,TNFα)mRNA表达的变化;并采用特异性结合钾离子的荧光探针(Potassium-binding benzofuran isophthalate acetoxymethyl ester,PBFI AM)作为标记物,检测小胶质激活后细胞内钾离子浓度的变化。最后,当小胶质细胞被LPS、HPE、ATP诱导激活之后,使用东亚钳蝎毒提取物(rTityustoxin-Kα,TsTx)阻断Kv1.2通道再次观察上述指标的变化;并用LPS激活细胞以及TsTx阻断通道后通过流式细胞仪观察小胶质细胞内ROS产生的改变。结果:体内外小胶质细胞均表达Kv1.2;体内Kv1.2的表达伴随发育过程出现变化,P1-10大鼠脑内阿米巴状小胶质细胞表达Kv1.2,P14开始Kv1.2的表达出现下调,P21的大鼠脑内突起型小胶质细胞几乎不能观察到Kv1.2的表达。P1大鼠缺氧后小胶质细胞表达Kv1.2显著增加;体外real time PCR分析的结果亦证实,当细胞被LPS、缺氧、ATP诱导后Kv1.2 mRNA的表达出现上调;同时小胶质细胞表达IL-1β、TNFα增加;并伴随细胞内钾离子浓度降低。通过TsTx特异性阻断Kv1.2通道电流后,细胞内钾离子浓度出现恢复,同时使得LPS、缺氧、ATP诱导细胞后的IL-1β和TNFα的表达降低,LPS诱导的小胶质细胞ROS产生出现减少。结论:①本研究首次证实小胶质细胞表达Kv1.2,并随动物发育而逐渐消失,提示Kv1.2的表达与发育早期动物脑内的微环境需求有关。②Kv1.2的表达参与了小胶质细胞活化的过程。③Kv1.2的表达在LPS诱导的ROS生成中具有重要作用。④缺氧后小胶质细胞释放IL-1β和TNFα增多可能因缺氧后小胶质细胞表达Kv1.2上调,钾离子外流增多,细胞内钾离子浓度降低所致。⑤Kv1.2的表达在ATP激活小胶质细胞的过程中发挥作用。⑥Kv1.2通道的特异阻断剂TsTx使得小胶质细胞的活化程度减轻。因此,进一步研究该通道的功能,优化该通道阻断剂TsTx的剂量,阻断该通道电流,能够为控制因小胶质细胞过度活化而引起的炎症反应提供有力的实验室证据。
[Abstract]:Microglia is a "receptor" of the central nervous system. When the microenvironment changes in the brain, it is activated first. The activated microglia is adjusted by releasing cytokines and active oxygen (Reactive OxygenSpecies, ROS), and the changes in the electrophysiological characteristics of the membrane play an important role in the biological behavior. The microglia expressed a variety of ion channels that coordinated the biological characteristics of the microglia membrane. It has been proved that microglia expressed voltage dependent potassium channel (Voltage gated potassium channel, Kv), and the delayed delayed integrated potassium current controlled by them was a characteristic of microglia activation. The decrease in intracellular potassium concentration caused by its opening is a key step in increasing the expression of cytokines. The study shows that microglia express Kv1.3, Kv1.5, which are related to the proliferation, migration, release of cytokine and ROS of microglia, and the use of these passages can reduce the process of microglia activation. The study of the expression of Kv on microglia membrane and its possible functions is of great significance to elucidate the molecular mechanism of microglia activation and to find the targets to regulate its activation. Objective: This study aims to explore the expression of potassium channel Kv1.2 in microglia and its possible functions. The expression of Kv1.2 in the brain amoeba like microglia in different developmental stages of rats was observed by double immunofluorescent labeling, and the expression of Kv1.2 in microglia cultured in vitro was observed by double immunofluorescence double labeling and real time PCR technique. The rat model of oxygen deficiency animal was established in vivo, and the hypoxia of rats of different ages was observed. The changes in Kv1.2 were expressed in the microglia. In vitro, Lipopolysaccharide, LPS, Hypoxia exposure, HPE, and adenosine triphosphate (Adenosine 5'-triphosphate, ATP) were used to induce the activation of microglia. The microglia was observed by real time PCR, and the interleukin 1 beta (beta) and tumor necrosis factor were observed by time PCR. The changes in the expression of umornecrosis factor, TNF alpha) and the fluorescence probe of the specific binding potassium ion (Potassium-binding benzofuran isophthalate acetoxymethyl ester, PBFI AM) as a marker to detect the change in the concentration of potassium ions in the cells after the microglia activation. Finally, when the microglia is activated by LPS, induction, activation, RTityustoxin-K alpha (TsTx) was used to observe the changes of the above index by blocking the Kv1.2 channel, and using LPS to activate cells and the blocking channel of TsTx to observe the changes of ROS in microglia through flow cytometry. Results: the microglia in and outside the body expressed Kv1.2, and the expression of Kv1.2 in the body was accompanied by the development process. Changes in the expression of amoeba microglia in the brain of P1- rats were Kv1.2, and the expression of Kv1.2 was down regulated at the beginning of P14. The expression of Kv1.2 in the rat brain of P21 was almost impossible to observe the expression of Kv1.2 in the rat.P1 rats, and the expression of Kv1.2 in the microglia was significantly increased after the hypoxia, and the real time PCR analysis in vitro also confirmed that when the cells were LPS, the results of PCR analysis of the real time were also confirmed. Hypoxia, the expression of Kv1.2 mRNA was up-regulated after ATP induction, while microglia expressed IL-1 beta, TNF alpha and decreased intracellular potassium concentration. After TsTx specific blocking of Kv1.2 channel current, the concentration of potassium ions in cells recovered, while LPS, oxygen deficiency, and ATP induced cells decreased the expression of IL-1 beta and TNF alpha. The induced microglia ROS production decreased. Conclusion: (1) this study first demonstrated that microglia expressed Kv1.2 and disappeared gradually with the development of animals, suggesting that the expression of Kv1.2 was related to the microenvironment requirements in the brain of early developing animals. (2) the expression of Kv1.2 was involved in the process of microglia activation. (3) the expression of Kv1.2 was induced by LPS. ROS production plays an important role. (4) the release of IL-1 beta and TNF alpha in microglia after hypoxia may be due to the up regulation of Kv1.2 in the microglia after hypoxia, the increase of potassium ion Exodus and the decrease of the concentration of potassium ions in the cells. 5. Kv1.2 expression plays a role in the process of ATP activation of microglia. 6. The specific blocker TsTx of the Kv1.2 channel, TsTx Therefore, the activation of microglia is reduced. Therefore, further study of the function of the channel, optimizing the dose of the channel blocker TsTx, blocking the current of the channel, can provide powerful laboratory evidence for controlling the inflammatory reaction caused by the excessive activation of microglia.

【学位授予单位】：昆明医学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R33

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