發(fā)布時間：2018-06-10 09:19

  本文選題：Kir6.1/K-ATP + 星形膠質(zhì)細胞��； 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：腦卒中是一種發(fā)病迅速的腦血液循環(huán)障礙性疾病,致殘率和致死率高,嚴重威脅人類健康。腦卒中主要包括缺血性腦卒中和出血性腦卒中,其中缺血性腦卒中即腦缺血,是以腦循環(huán)的血流量降低為特征的疾病,臨床約80%的腦卒中患者為缺血性腦卒中。目前臨床仍以重組組織型纖溶酶原激活劑治療為主,但由于療時間窗短和出血的危險限制了其應(yīng)用。近年來旨在尋找神經(jīng)保護藥物治療腦卒中的臨床試驗幾乎均以失敗告終。究其原因,現(xiàn)有研究往往局限于單一神經(jīng)元的保護,而忽視了由神經(jīng)元、星形膠質(zhì)細胞、內(nèi)皮細胞共同組成的神經(jīng)血管單元的研究。因此,發(fā)展新的腦卒中治療策略對于腦卒中患者尤為迫切。星形膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)數(shù)量最多的細胞,是神經(jīng)血管單元的重要組分之一,為神經(jīng)元正常發(fā)揮功能提供保障,具有多種重要的生物學(xué)功能,包括支持、隔離、參與突觸形成、轉(zhuǎn)運神經(jīng)遞質(zhì)、調(diào)節(jié)神經(jīng)元物質(zhì)代謝、維持離子穩(wěn)態(tài)、介導(dǎo)信號轉(zhuǎn)導(dǎo)等。近年來星形膠質(zhì)細胞在腦缺血中的重要性逐漸被認識。星形膠質(zhì)細胞在腦缺血中發(fā)生增殖、活化,特異性標記物膠原纖維酸性蛋白(Glial fibrillary acidic protein,GFAP)表達上調(diào)。星形膠質(zhì)細胞在缺血狀態(tài)下耐受力較強,在腦缺血中具有雙重作用:既可以產(chǎn)生釋放谷胱甘肽和神經(jīng)營養(yǎng)因子、生成促紅細胞生成素等保護神經(jīng)元,也會以釋放炎癥因子、減少縫隙連接和興奮性氨基酸的再攝取等方式加重神經(jīng)元損傷。因此,明確星形膠質(zhì)細胞在腦缺血中的作用及其機制,有效利用星形膠質(zhì)細胞對神經(jīng)元的保護作用,降低損害效應(yīng),將成為腦缺血治療的新思路,對于腦缺血的治療具有重要的科學(xué)意義。ATP 敏感性鉀通道(ATP-sensitive potassium channel,K-ATP)是一類特殊非電壓依賴性的鉀離子通道,耦聯(lián)細胞代謝和電活動,通道的開放或關(guān)閉主要受胞內(nèi)ATP/ADP比值的影響。1K-ATP通道是由內(nèi)向整流鉀通道(inwardly rectifying potassium channel,Kir)和磺酰脲受體(sulphonylurea receptor,SUR)亞單位以4:4組合形成的異構(gòu)八聚體。Kir亞基主要包括Kir6.1和Kir6.2,其中Kir6.1亞基在腦內(nèi)主要表達于星形膠質(zhì)細胞、小膠質(zhì)細胞和神經(jīng)干細胞。本實驗室前期研究發(fā)現(xiàn)Kir6.1/K-ATP通道與缺血性腦卒中發(fā)生發(fā)展相關(guān),Kir6.1基因敲減加重小鼠腦缺血再灌注損傷,而且K-ATP通道開放劑減輕缺糖缺氧/復(fù)糖復(fù)氧(Oxygen-glucose deprivation/reperfusion,OGD/R)引起的星形膠質(zhì)細胞損傷,提示星形膠質(zhì)細胞Kir6.1/K-ATP通道可能參與了缺血性腦卒中的發(fā)生發(fā)展。本文應(yīng)用星形膠質(zhì)細胞Kir6.1條件敲除小鼠制備tMCAO(transient middle cerebral artery occlusion,tMCAO)模型,研究星形膠質(zhì)細胞Kir6.1敲除對小鼠腦缺血再灌注急性期損傷的影響,并培養(yǎng)原代星形膠質(zhì)細胞,觀察Kir6.1表達下調(diào)對OGD/R誘導(dǎo)的星形膠質(zhì)細胞損傷和凋亡的影響,進一步闡明星形膠質(zhì)細胞Kir6.1/K-ATP通道對腦缺血再灌注損傷后內(nèi)質(zhì)網(wǎng)應(yīng)激、自噬和凋亡的調(diào)節(jié)及機制。研究結(jié)果表明,星形膠質(zhì)細胞Kir6.1敲除加重tMCAO引起的小鼠局灶性腦缺血再灌注損傷,增強缺血半影區(qū)內(nèi)質(zhì)網(wǎng)應(yīng)激,上調(diào)自噬和凋亡水平;Kir6.1表達下調(diào)加重OGD/R引起的原代星形膠質(zhì)細胞損傷,增強細胞的內(nèi)質(zhì)網(wǎng)應(yīng)激,增加自噬和細胞凋亡;內(nèi)質(zhì)網(wǎng)應(yīng)激阻斷劑4-苯基丁酸(4-Phenylbutyric acid,4-PBA)可以逆轉(zhuǎn)Kir6.1表達下調(diào)引起的星形膠質(zhì)細胞損傷加重。上述結(jié)果表明星形膠質(zhì)細胞Kir6.1/K-ATP通道通過調(diào)節(jié)內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的細胞自噬和凋亡,在缺血性腦損傷中發(fā)揮重要的作用。目的:研究星形膠質(zhì)細胞Kir6.1/K-ATP通道在小鼠腦缺血再灌注損傷中的作用及機制方法:1)應(yīng)用Cre-Loxp系統(tǒng)構(gòu)建星形膠質(zhì)細胞Kir6.1特異性敲除小鼠。采用 2 月齡對照型(Control,CTL,KCNJ8f/f)及敲除型(Conditonalknockout,CKO,KCNJ8f/fGFAPCre)雄性小鼠,使用線栓法制備小鼠tMCAO模型,缺血1 h后再灌,于再灌后24 h對小鼠進行神經(jīng)功能評分,處死小鼠并對腦梗死體積、腦含水量進行測量,探討星形膠質(zhì)細胞Kir6.1對小鼠大腦局灶短暫性缺血再灌注(tMCAO)損傷的影響;2)制備小鼠tMCAO模型,于再灌注24 h和72 h后將小鼠灌注后腦組織取材,進行冰凍組織切片,應(yīng)用免疫組化技術(shù)研究星形膠質(zhì)細胞Kir6.1敲除對腦缺血后神經(jīng)血管單元各組分(神經(jīng)元、星形膠質(zhì)細胞、小膠質(zhì)細胞、血管基底膜等)的影響;3)制備小鼠tMCAO模型,于再灌注24 h后免疫印跡(Western Blot,WB)法檢測缺血半影區(qū)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白、自噬相關(guān)蛋白和凋亡相關(guān)蛋白的表達;4)培養(yǎng)原代星形膠質(zhì)細胞,轉(zhuǎn)染Kir6.1 siRNA,OGD5h,復(fù)糖復(fù)氧24h,采用WB的方法檢測內(nèi)質(zhì)網(wǎng)應(yīng)激、凋亡相關(guān)蛋白表達。5)給予內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-PBA,OGD5h,復(fù)糖復(fù)氧24h,應(yīng)用MTT法檢測細胞活力;采用Annexin-V和PI雙染流式細胞儀檢測凋亡率;結(jié)果:1)星形膠質(zhì)細胞Kir6.1敲除加重tMCAO模型小鼠神經(jīng)運動功能障礙,增大梗死體積,加重腦水腫;2)星形膠質(zhì)細胞Kir6.1敲除加重缺血皮層半影區(qū)神經(jīng)元的丟失,增強星形膠質(zhì)細胞和小膠質(zhì)細胞的增殖活化,破壞基底膜完整性;3)星形膠質(zhì)細胞Kir6.1敲除加劇缺血半影區(qū)內(nèi)質(zhì)網(wǎng)應(yīng)激,增加自噬和凋亡相關(guān)蛋白的表達;4)轉(zhuǎn)染Kir6.1 siRNA加劇OGD/R引起的星形膠質(zhì)細胞內(nèi)質(zhì)網(wǎng)應(yīng)激和凋亡;5)Kir6.1表達下調(diào)加劇OGD/R引起的星形膠質(zhì)細胞的損傷,增加細胞凋亡,4-PBA逆轉(zhuǎn)星形膠質(zhì)細胞Kir6.1表達下調(diào)引起的細胞損傷和凋亡。結(jié)論:星形膠質(zhì)細胞Kir6.1敲除促進內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡加重腦缺血再灌注損傷。綜上所述,本文工作的創(chuàng)新之處在于:揭示星形膠質(zhì)細胞Kir6.1/K-ATP通道通過調(diào)節(jié)內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡發(fā)揮腦缺血損傷保護作用。
[Abstract]:Cerebral apoplexy is a rapid onset of cerebral blood circulation disorder with high morbidity and mortality, which seriously threatens human health. Cerebral apoplexy mainly includes ischemic stroke and hemorrhagic stroke, among which ischemic stroke is cerebral ischemia, which is characterized by the decrease of blood flow in the brain circulation, and about 80% of patients in the clinic are clinically Ischemic stroke. Currently, recombinant tissue type plasminogen activator is still the main treatment, but its application is limited due to the short time window and the risk of bleeding. In recent years, the clinical trials aimed at finding neuroprotective drugs for stroke have almost failed. Therefore, the development of a new stroke treatment strategy is particularly urgent for stroke patients. Astrocytes are the most important cells in the central nervous system, one of the important components of the neurovascular unit, and the nerve yuan Zheng Zheng. It has many important biological functions, including supporting, isolating, participating in synapse formation, transporting neurotransmitters, regulating the metabolism of neurons, maintaining ionic homeostasis, and mediating signal transduction. In recent years, the importance of astrocytes in cerebral ischemia is gradually recognized. Astrocytes are in the middle of cerebral ischemia. The expression of Glial fibrillary acidic protein (GFAP) is up regulated by the proliferation, activation and specific markers of collagen fibrillary acidic protein (GFAP). Astrocytes have a strong tolerance in ischemic state, and have double effects in cerebral ischemia: it can produce both glutathione and God management factor, and produce erythropoietin and other protective neurons. It will also aggravate neuronal damage by releasing inflammatory factors, reducing gap junctions and reuptake of excitatory amino acids. Therefore, it is a new idea for the treatment of cerebral ischemia to clarify the role and mechanism of astrocytes in cerebral ischemia and to effectively utilize the protective effects of astrocytes on neurons and reduce damage effects. The treatment of cerebral ischemia has important scientific significance, the.ATP sensitive potassium channel (ATP-sensitive potassium channel, K-ATP) is a special type of non voltage dependent potassium channel, coupled with cell metabolism and electrical activity, the opening or closing of the channel is mainly influenced by the intracellular ATP/ADP ratio, and the.1K-ATP channel is an endogenous rectifier potassium channel (inwardl). The Y rectifying potassium channel, Kir) and the sulfonylurea receptor (sulphonylurea receptor, SUR) subunits formed by the 4:4 combination of the isomeric eight polymer.Kir subunits mainly include Kir6.1 and Kir6.2, which are mainly expressed in astrocytes, microglia and neural stem cells in the brain. The TP channel is associated with the development of ischemic stroke, and Kir6.1 gene knockout aggravates the cerebral ischemia reperfusion injury in mice, and the K-ATP channel opener reduces the astrocyte damage caused by the lack of glucose hypoxia / reoxygenation (Oxygen-glucose deprivation/reperfusion, OGD/R), and the Kir6.1/K-ATP channel of astrocytes may be involved. The development of ischemic stroke. In this paper, tMCAO (transient middle cerebral artery occlusion, tMCAO) model of astrocyte Kir6.1 knockout mice was used to study the effect of Kir6.1 knockout on the acute stage of cerebral ischemia reperfusion injury in mice, and the primary astrocytes were cultured, and the expression of Kir6.1 was observed under Kir6.1. The effect of OGD/R induced astrocyte damage and apoptosis, and further elucidate the regulation and mechanism of endoplasmic reticulum stress, autophagy and apoptosis after cerebral ischemia reperfusion injury by Kir6.1/K-ATP channel in astrocytes. The results show that Kir6.1 knockout in astrocytes aggravates the focal cerebral ischemia reperfusion injury in mice caused by tMCAO Injury, enhanced endoplasmic reticulum stress in the ischemic penumbra, up-regulated the level of autophagy and apoptosis; Kir6.1 expression down-regulation aggravated OGD/R induced astrocyte damage, enhanced endoplasmic reticulum stress, increased autophagy and apoptosis; endoplasmic reticulum stress blocker 4- phenyl butyric acid (4-Phenylbutyric acid, 4-PBA) could reverse the downregulation of Kir6.1 expression The results show that the Kir6.1/K-ATP channel of star glial cells can play an important role in ischemic brain injury by regulating endoplasmic reticulum stress mediated autophagy and apoptosis. Objective: To study the role and mechanism of astrocyte Kir6.1/K-ATP channel in cerebral ischemia reperfusion injury in mice. Method: 1) the Cre-Loxp system was used to construct Kir6.1 specific knockout mice of astrocytes. 2 month old control (Control, CTL, KCNJ8f/f) and knockout (Conditonalknockout, CKO, KCNJ8f/fGFAPCre) male mice were used to prepare the mice tMCAO model using the thread thrombus method. The blood deficiency was 1 h after reperfusion. The neurological function score of the mice was scored in 24 h after reperfusion. The mice were killed and the volume of cerebral infarction and the water content of brain were measured. The effects of astrocyte Kir6.1 on cerebral focal transient ischemia and reperfusion (tMCAO) injury in mice were investigated. 2) the mouse tMCAO model was prepared. After reperfusion of 24 h and 72 h, the brain tissue of the mice was taken from the cerebral tissue, and the frozen tissue sections were sectioning. The effect of Kir6.1 knockout on astrocytes (astrocytes, astrocytes, microglia, basilar membrane and so on) after cerebral ischemia; 3) to prepare tMCAO model in mice, and to detect the endoplasmic reticulum stress related protein, autophagy related protein and withering in the penumbra of the penumbra after 24 h reperfusion. Expression of apoptosis related protein; 4) cultured primary astrocytes, transfection Kir6.1 siRNA, OGD5h, reoxygenation 24h, WB method to detect endoplasmic reticulum stress, apoptosis related protein expression.5), give endoplasmic reticulum stress inhibitor 4-PBA, OGD5h, reoxygenation 24h, use MTT method to detect cell viability; Annexin-V and PI double dye flow cytometry was used. The results were as follows: 1) 1) astrocyte knockout aggravated the neuromotor dysfunction in tMCAO model mice, increased infarct volume and aggravated brain edema; 2) Kir6.1 knockout in astrocytes aggravated the loss of neurons in the shadow area of the ischemic cortex, enhanced the proliferation and activation of astrocytes and microglia, and destroyed the integrity of the basilar membrane; 3) Kir6.1 knockout in astrocytes aggravated the endoplasmic reticulum stress in the ischemic penumbra and increased the expression of autophagy and apoptosis related proteins; 4) transfection of Kir6.1 siRNA aggravated astrocyte endoplasmic reticulum stress and apoptosis induced by OGD/R; 5) down regulation of Kir6.1 increased the damage of astrocytes caused by OGD/R, increased apoptosis, and reversed the astrocyte of 4-PBA. Conclusion: Kir6.1 knockout in astrocytes promotes endoplasmic reticulum stress mediated apoptosis and aggravates cerebral ischemia reperfusion injury. To sum up, the innovation of this work is to reveal the apoptosis mediated by the regulation of endoplasmic reticulum stress in astrocytes Kir6.1/K-ATP channels. The protective effect of cerebral ischemia injury.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743.3

【參考文獻】

相關(guān)期刊論文 前3條

1 Stéphane Mathis;Philippe Couratier;Adrien Julian;Philippe Corcia;Gwendal Le Masson;;Current view and perspectives in amyotrophic lateral sclerosis[J];Neural Regeneration Research;2017年02期

2 Robert C.Rennert;J.Scott Pannell;Alexander A.Khalessi;;Advances in minimally invasive treatment of hemorrhagic and ischemic stroke[J];Neural Regeneration Research;2016年02期

3 Wenqi Chen;Yinyi Sun;Kangyong Liu;Xiaojiang Sun;;Autophagy: a double-edged sword for neuronal survival after cerebral ischemia[J];Neural Regeneration Research;2014年12期

，

本文編號：2002719