本文選題：絲切蛋白 + 電針��； 參考：《福建中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：缺血性卒中已成為世界各地長期神經(jīng)功能障礙和死亡的主要原因,約占所有卒中的80%。盡管已經(jīng)有幾十年的深入研究,但能夠針對性地治療缺血性卒中的措施仍然有限。腦缺血的主要致病機(jī)制包含谷氨酸介導(dǎo)的興奮性毒性,鈣超載,炎癥反應(yīng),血腦屏障破壞和氧化應(yīng)激等。這些病理因素或者環(huán)節(jié)相互影響,互為因果,進(jìn)而引起神經(jīng)細(xì)胞凋亡與壞死,最終導(dǎo)致卒中后認(rèn)知功能障礙。廣泛認(rèn)為半暗帶中的細(xì)胞凋亡是可逆的過程。細(xì)胞凋亡的調(diào)控,在保護(hù)神經(jīng)元和改善認(rèn)知的過程中發(fā)揮著重要的作用。因此,探討神經(jīng)細(xì)胞凋亡的分子機(jī)制,尋找有效的治療方法減少腦損傷,已成為腦血管研究領(lǐng)域的重點(diǎn)和熱點(diǎn)。絲切蛋白(Cofilin)是肌動蛋白結(jié)合蛋白,受LIMK介導(dǎo)的磷酸化而失活。過量谷氨酸,ATP耗竭或氧化應(yīng)激處理原代培養(yǎng)神經(jīng)元,均發(fā)現(xiàn)Cofilinrod結(jié)構(gòu)的形成。產(chǎn)生這種棒狀結(jié)構(gòu)的神經(jīng)元在突觸結(jié)構(gòu)、突觸轉(zhuǎn)運(yùn)和長時程突觸可塑性上都受到了一定程度損傷。Rod的形成主要是由于去磷酸化的Cofilin含量增加引起的,并且僅去磷酸化的Cofilin牽涉線粒體轉(zhuǎn)位。Cofilin線粒體轉(zhuǎn)位在細(xì)胞凋亡程序的啟動中起到關(guān)鍵作用。此外,引起Rod形成的病理性刺激,均是缺血性卒中的重要病理機(jī)制。由此提示,Cofilin及其介導(dǎo)的Rod結(jié)構(gòu)與學(xué)習(xí)記憶等認(rèn)知功能密切相關(guān),同時參與缺血性卒中細(xì)胞凋亡過程中。目前,電針療法已成為腦卒中后康復(fù)治療的一種常見方法。督脈經(jīng)穴"百會"和"神庭"具有益氣升陽、填髓充腦、醒腦開竅之功效,常被用于治療神經(jīng)和精神疾病,如中風(fēng),頭痛和頭暈等。臨床和基礎(chǔ)研究均顯示,電針"百會"和"神庭"穴有利于保護(hù)卒中患者和動物模型的缺血性損傷。本課題組的臨床研究顯示,針刺"百會"、"神庭"穴在認(rèn)知康復(fù)訓(xùn)練基礎(chǔ)上可顯著提高腦卒中患者M(jìn)MSE、MoCA和FIM等認(rèn)知相關(guān)量表的評分。同時,我們以往實(shí)驗(yàn)也發(fā)現(xiàn)電針"百會"、"神庭"穴能夠通過減少腦缺血損傷大鼠的神經(jīng)細(xì)胞凋亡,顯著改善其認(rèn)知功能障礙。然而,電針治療缺血性卒中的分子機(jī)制尚不明確。目的:(1)闡明Cofilin在缺血性卒中后細(xì)胞凋亡中的潛在調(diào)控作用,為將Cofilin作為腦卒中新的治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù);(2)探討電針"百會"、"神庭"穴促進(jìn)腦缺血大鼠認(rèn)知功能恢復(fù)的可能分子機(jī)制,為中醫(yī)康復(fù)臨床實(shí)踐提供一定的理論依據(jù)。方法:第一部分Cofilin在腦缺血大鼠皮質(zhì)梗死區(qū)細(xì)胞凋亡中的作用(1)本研究利用ATP耗竭誘導(dǎo)原代海馬神經(jīng)元,建立體外缺血/缺氧損傷模型,明確Cofilin rod形成和細(xì)胞凋亡。(2)同時,以大腦中動脈閉塞法制備缺血性卒中的實(shí)驗(yàn)動物模型,通過TTC和NeuN染色來評估MCAO大鼠腦梗死的發(fā)展階段。(3)應(yīng)用免疫組化和TUNEL染色,觀察不同時間點(diǎn)(2h,8h,24h和7d)MCAO大鼠腦皮質(zhì)梗死區(qū)Cofilin rod生成和神經(jīng)細(xì)胞凋亡的情況。(4)為進(jìn)一步證實(shí)去磷酸化Cofilin在腦缺血中的作用,我們利用Limk1過表達(dá)抑制Cofilin的活化,觀察MCAO大鼠缺血半暗帶病毒感染區(qū)域Rod形成和Cofilin介導(dǎo)細(xì)胞凋亡的情況。第二部分電針"百會"、"神庭"穴調(diào)控Cofilin線粒體轉(zhuǎn)位減少皮質(zhì)缺血區(qū)細(xì)胞凋亡的機(jī)制研究(1)分別于腦缺血術(shù)后5min和6h,對MCAO大鼠進(jìn)行電針干預(yù)"百會"、"神庭"穴。(2)通過條件恐懼記憶以及神經(jīng)功能缺損得分和腦梗死體積,評價電針"百會"、"神庭"對腦缺血大鼠的神經(jīng)保護(hù)作用。(3)采用Nissl染色和熒光染色以觀察腦缺血組織損傷、Cofilin rod形成、細(xì)胞凋亡和神經(jīng)元丟失情況。(4)并通過Western blot檢測缺血半暗帶Caspase-3的活性,及其胞漿和線粒體中Cofilin和p-cofilin的表達(dá)水平。結(jié)果:第一部分(1)ATP耗竭可誘導(dǎo)原代海馬神經(jīng)元Cofilin rod形成以及細(xì)胞凋亡。(2)TTC和NeuN染色顯示梗死體積從2h增加到24h,然后在MCAO后24h到7d穩(wěn)定。(3)Rod結(jié)構(gòu)在梗死核心區(qū)2h和半暗帶8h開始生成,24h達(dá)到峰值,然后在MCAO后7d減少。而逐步增多的Rod結(jié)構(gòu)可能會逐漸誘導(dǎo)腦卒中后梗死皮層MAP2降解和細(xì)胞凋亡。(4)Limk1過表達(dá)抑制Cofilin的活化,可顯著降低MCAO大鼠缺血區(qū)半暗帶Rod的形成和MAP2降解,并減少Cofilin介導(dǎo)的細(xì)胞凋亡,進(jìn)一步證實(shí)了Cofilin在腦缺血中的作用。第二部分(1)急性期電針"百會"、"神庭"穴治療MCAO大鼠可以顯著減小腦梗死體積,并改善腦損傷引起的神經(jīng)功能缺損癥狀和認(rèn)知功能障礙。(2)Western blot分析表明,電針這兩個穴位還可以明顯抑制Cofilin的線粒體轉(zhuǎn)位和Caspase-3切割。(3)此外,免疫熒光雙標(biāo)結(jié)果提示,電針"百會"、"神庭"穴治療不僅可以部分抑制Cofilin rod形成和MAP2降解,而且還顯著減少缺血性卒中誘導(dǎo)的腦組織損傷、細(xì)胞凋亡和神經(jīng)元丟失。結(jié)論:(1)體內(nèi)和體外缺血可誘導(dǎo)Cofilin rod形成和神經(jīng)元細(xì)胞凋亡,而Limk1過表達(dá)抑制Cofilin過度激活,可顯著減少腦缺血后皮質(zhì)半暗帶Rod形成、MAP2降解和Cofilin線粒體轉(zhuǎn)位介導(dǎo)的細(xì)胞凋亡,由此可知,Cofilin對缺血性卒中后Rod形成和細(xì)胞凋亡具有重要作用,可作為缺血性卒中潛在的治療靶點(diǎn)。(2)急性期電針"百會"、"神庭"穴可減少Cofilin rod形成和MAP2降解,抑制Cofilin的線粒體轉(zhuǎn)位,減輕缺血性卒中的腦損傷和細(xì)胞凋亡,從而促進(jìn)認(rèn)知功能恢復(fù),這可能是電針療法的新機(jī)制。
[Abstract]:Ischemic stroke has become a major cause of long-term neurological dysfunction and death in all parts of the world. The 80%., which accounts for all strokes, has been studied for decades, but the measures to treat ischemic stroke are still limited. The main pathogenesis of cerebral ischemia includes glutamate mediated excitotoxicity, calcium overload, and inflammation. These pathological factors or links interact with each other, cause cause and effect, cause apoptosis and necrosis of nerve cells, and eventually lead to cognitive impairment after stroke. It is widely considered that apoptosis in the semi dark zone is a reversible process. Apoptosis regulation, in protecting neurons and improving cognition It has played an important role in the process. Therefore, to explore the molecular mechanism of neuronal apoptosis and to find an effective treatment method to reduce brain damage has become the focus and hot spot in the field of cerebral vascular research. Cofilin is actin binding protein, LIMK mediated phosphorylation and inactivation. Excessive glutamate, ATP depletion or oxidative stress The formation of the Cofilinrod structure is found in the primary cultured neurons. The neurons that produce this rod like structure have been damaged to a certain extent in the synaptic structure, the synaptic transport and the long term synaptic plasticity. The formation of the.Rod is mainly due to the increase of the Cofilin content of dephosphorylation, and the Cofilin involvement line is only dephosphorylated. Mitochondrial translocation of granular.Cofilin plays a key role in the initiation of apoptosis procedures. In addition, the pathological stimulation of Rod formation is an important pathological mechanism of ischemic stroke. Thus, it is suggested that Cofilin and its mediated Rod structure are closely related to cognitive functions such as learning and memory, and are involved in the apoptosis of ischemic stroke cells. At present, electroacupuncture has become a common method of rehabilitation after stroke. The meridian meridian "Baihui" and "Shen Ting" have the effect of replenishing qi and Yang, filling the brain into the brain, and activating the brain, often used in the treatment of nerve and mental illness, such as stroke, headache, dizziness and so on. Clinical and basic studies all show the electric acupuncture "hundred meeting" and "Shen Ting" points. It is beneficial to protect the ischemic injury of stroke patients and animal models. The clinical study of this group has shown that acupuncture of "Baihui" and "Shen Ting" point can significantly improve the score of cognitive related scales of stroke patients, such as MMSE, MoCA and FIM on the basis of cognitive rehabilitation training. Reduce the apoptosis of neurons in rats with cerebral ischemia and improve their cognitive impairment. However, the molecular mechanism of electroacupuncture in the treatment of ischemic stroke is not clear. Objective: (1) to clarify the potential regulation of Cofilin in cell apoptosis after ischemic stroke, and to provide experimental basis for Cofilin as a new target for stroke treatment; (2) To discuss the possible molecular mechanism of "Baihui" and "Shen Ting" point to promote the recovery of cognitive function in rats with cerebral ischemia, and provide some theoretical basis for the clinical practice of TCM rehabilitation. Method: the role of Cofilin in the apoptosis of cortical infarct area of cerebral ischemia rats (1) this study used the depletion of ATP to induce the primary hippocampal neurons to establish in vitro deficiency. Blood / hypoxia damage model, Cofilin rod formation and apoptosis. (2) at the same time, the experimental animal model of ischemic stroke was prepared by middle cerebral artery occlusion, and the development stage of cerebral infarction in MCAO rats was evaluated by TTC and NeuN staining. (3) immunohistochemistry and TUNEL staining were used to observe the cerebral cortex of MCAO rats at different time points (2h, 8h, 24h and 7D). The formation of Cofilin rod and the apoptosis of nerve cells in the dead tissue of the stem. (4) to further confirm the role of dephosphorylation of Cofilin in cerebral ischemia, we use Limk1 overexpression to inhibit the activation of Cofilin, and observe the situation of Rod formation and Cofilin mediated apoptosis in the ischemic penumbra virus infected region of MCAO rats. The second part of the electroacupuncture "hundred meetings", " The mechanism of "Shen Ting" regulating Cofilin mitochondrial transposition to reduce cell apoptosis in cortical ischemic areas (1) respectively 5min and 6h after cerebral ischemia, electroacupuncture intervention in MCAO rats, "Baihui", "Shen Ting" point. (2) through the memory of the condition of fear and the score of nerve function defect and cerebral infarction volume, the evaluation of the "hundred meetings" of electroacupuncture, "God court" to cerebral ischemia rats Neuroprotective effect. (3) Nissl staining and fluorescence staining were used to observe the injury of cerebral ischemia tissue, Cofilin rod formation, apoptosis and neuron loss. (4) the activity of Caspase-3 in the ischemic penumbra and the expression level of Cofilin and p-cofilin in the cytoplasm and mitochondria were detected by Western blot. Results: the first part (1) ATP consumption The depletion can induce the formation of Cofilin rod and apoptosis in the primary hippocampal neurons. (2) TTC and NeuN staining showed that the infarct volume increased from 2H to 24h, and then 24h to 7d after MCAO. (3) Rod structure was generated in the 2H and semi dark band of the infarct core area, then reached its peak, and then decreased. Induced MAP2 degradation and apoptosis in infarct cortex after cerebral apoplexy. (4) overexpression of Limk1 inhibits the activation of Cofilin, which can significantly reduce the formation of Rod in the dark zone and MAP2 degradation in the ischemic zone of MCAO rats, and reduce the apoptosis of Cofilin mediated cells, and further confirm the role of Cofilin in cerebral ischemia. The second part (1) the acute phase of electroacupuncture "hundred meetings", "the divine court" "Acupoint treatment of MCAO rats can significantly reduce the volume of cerebral infarction and improve the neurological deficits and cognitive impairment caused by brain damage. (2) Western blot analysis shows that the two points of electroacupuncture can also significantly inhibit the mitochondrial translocation and Caspase-3 cutting of Cofilin. (3) the results of double immunofluorescent labeling suggest that the Electroacupuncture of" 100 meetings "," "Acupoint therapy" not only partially inhibits Cofilin rod formation and MAP2 degradation, but also significantly reduces cerebral tissue damage induced by ischemic stroke, cell apoptosis and neuron loss. Conclusion: (1) in vivo and in vitro ischemia can induce the formation of Cofilin rod and neuronal cell death, while Limk1 overexpression inhibits Cofilin overactivation, which is significant Reduce the formation of Rod formation, MAP2 degradation and Cofilin mitochondrial Translocation Induced Apoptosis after cerebral ischemia. Thus, Cofilin plays an important role in Rod formation and cell apoptosis after ischemic stroke. It can be used as a potential therapeutic target for ischemic stroke. (2) the acute phase electroacupuncture "hundred meetings", "Shen Ting" point can reduce the formation of Cofilin rod and MA P2 degradation, inhibiting the mitochondrial translocation of Cofilin, alleviates brain damage and apoptosis of ischemic stroke, thus promoting the recovery of cognitive function, which may be a new mechanism of electroacupuncture therapy.

【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R743.3

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