本文選題：阿米巴狀小膠質(zhì)細胞 + Kv1.2　； 參考：《昆明醫(yī)學院》2008年博士論文

【摘要】： 小膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)的“感受器”,當腦內(nèi)微環(huán)境發(fā)生變化時它首先被激活,活化后的小膠質(zhì)細胞通過釋放細胞因子和活性氧(Reactive OxygenSpecies,ROS)等對周圍環(huán)境進行調(diào)整;此時其膜電生理學特性的改變對其生物學行為起到調(diào)控作用;小膠質(zhì)細胞表達多種離子通道,它們協(xié)調(diào)運轉(zhuǎn)控制著小膠質(zhì)細胞膜的生物學特性。業(yè)已證實小膠質(zhì)細胞表達電壓依從性鉀離子通道(Voltage gated potassium channel,Kv),它們所控制的外向延遲整合鉀離子電流是小膠質(zhì)細胞活化的特征,其開放所致的細胞內(nèi)鉀離子濃度降低是上調(diào)細胞因子表達的關(guān)鍵步驟。研究表明小膠質(zhì)細胞表達Kv1.3、Kv1.5,它們與小膠質(zhì)細胞的增殖、遷移、釋放細胞因子和ROS等多種生理功能有關(guān);使用這些通道的阻斷劑能夠降低小膠質(zhì)細胞活化的程度,減輕神經(jīng)元損傷。因此深入研究小膠質(zhì)細胞膜上Kv的表達及其可能的功能,對闡明小膠質(zhì)細胞活化的分子機制,尋找調(diào)控其活化的靶點具有重要意義。目的:本研究旨在探索鉀離子通道Kv1.2在小膠質(zhì)細胞的表達及其可能的功能。方法:采用免疫熒光雙重標記法觀察大鼠不同發(fā)育階段腦內(nèi)阿米巴狀小膠質(zhì)細胞表達Kv1.2的情況;并通過免疫熒光雙重標記和real time PCR技術(shù),觀察體外培養(yǎng)的小膠質(zhì)細胞Kv1.2表達的情況。體內(nèi)建立大鼠缺氧動物模型,觀察不同年齡大鼠缺氧后小膠質(zhì)細胞表達Kv1.2的變化;體外采用脂多糖(Lipopolysaccharide,LPS)、缺氧(Hypoxia exposure,HPE)、三磷酸腺苷(Adenosine 5'-triphosphate,ATP)誘導激活小膠質(zhì)細胞,通過real time PCR觀察小膠質(zhì)細胞Kv1.2、白細胞介素1β(Interleukin-1β,IL-1β)和腫瘤壞死因子(Tumornecrosis factor,TNFα)mRNA表達的變化;并采用特異性結(jié)合鉀離子的熒光探針(Potassium-binding benzofuran isophthalate acetoxymethyl ester,PBFI AM)作為標記物,檢測小膠質(zhì)激活后細胞內(nèi)鉀離子濃度的變化。最后,當小膠質(zhì)細胞被LPS、HPE、ATP誘導激活之后,使用東亞鉗蝎毒提取物(rTityustoxin-Kα,TsTx)阻斷Kv1.2通道再次觀察上述指標的變化;并用LPS激活細胞以及TsTx阻斷通道后通過流式細胞儀觀察小膠質(zhì)細胞內(nèi)ROS產(chǎn)生的改變。結(jié)果:體內(nèi)外小膠質(zhì)細胞均表達Kv1.2;體內(nèi)Kv1.2的表達伴隨發(fā)育過程出現(xiàn)變化,P1-10大鼠腦內(nèi)阿米巴狀小膠質(zhì)細胞表達Kv1.2,P14開始Kv1.2的表達出現(xiàn)下調(diào),P21的大鼠腦內(nèi)突起型小膠質(zhì)細胞幾乎不能觀察到Kv1.2的表達。P1大鼠缺氧后小膠質(zhì)細胞表達Kv1.2顯著增加;體外real time PCR分析的結(jié)果亦證實,當細胞被LPS、缺氧、ATP誘導后Kv1.2 mRNA的表達出現(xiàn)上調(diào);同時小膠質(zhì)細胞表達IL-1β、TNFα增加;并伴隨細胞內(nèi)鉀離子濃度降低。通過TsTx特異性阻斷Kv1.2通道電流后,細胞內(nèi)鉀離子濃度出現(xiàn)恢復,同時使得LPS、缺氧、ATP誘導細胞后的IL-1β和TNFα的表達降低,LPS誘導的小膠質(zhì)細胞ROS產(chǎn)生出現(xiàn)減少。結(jié)論:①本研究首次證實小膠質(zhì)細胞表達Kv1.2,并隨動物發(fā)育而逐漸消失,提示Kv1.2的表達與發(fā)育早期動物腦內(nèi)的微環(huán)境需求有關(guān)。②Kv1.2的表達參與了小膠質(zhì)細胞活化的過程。③Kv1.2的表達在LPS誘導的ROS生成中具有重要作用。④缺氧后小膠質(zhì)細胞釋放IL-1β和TNFα增多可能因缺氧后小膠質(zhì)細胞表達Kv1.2上調(diào),鉀離子外流增多,細胞內(nèi)鉀離子濃度降低所致。⑤Kv1.2的表達在ATP激活小膠質(zhì)細胞的過程中發(fā)揮作用。⑥Kv1.2通道的特異阻斷劑TsTx使得小膠質(zhì)細胞的活化程度減輕。因此,進一步研究該通道的功能,優(yōu)化該通道阻斷劑TsTx的劑量,阻斷該通道電流,能夠為控制因小膠質(zhì)細胞過度活化而引起的炎癥反應(yīng)提供有力的實驗室證據(jù)。
[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.

【學位授予單位】：昆明醫(yī)學院
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R33

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