本文選題：腦缺血再灌注 + VNS　； 參考：《重慶醫(yī)科大學(xué)》2015年博士論文

【摘要】：研究背景與目的目前急性腦卒中已成為中國(guó)人致死性疾病的首位原因,及時(shí)恢復(fù)半暗帶腦血流是最直接有效的治療方式。但早期腦缺血再灌注(ischemia/reperfusion, I/R)后容易誘導(dǎo)產(chǎn)生大量氧自由基、瀑布式炎癥反應(yīng)、壞死和凋亡等級(jí)聯(lián)反應(yīng),多種因素間互相作用,造成二次腦損傷。因此,成功發(fā)展作用于腦卒中多種分子機(jī)制的治療方式是迫切需要的。1997年美國(guó)食品和藥品監(jiān)督局(Food and Drug Administration,FDA)推薦VNS (vagus nerve stimulation, VNS)應(yīng)用于部分發(fā)作性難治性癲癇患者,隨后多個(gè)國(guó)家相繼批準(zhǔn)。近年來(lái),國(guó)外研究發(fā)現(xiàn)VNS對(duì)急性腦缺血再灌注大鼠具有神經(jīng)保護(hù)作用,但其機(jī)制不明確。本研究擬通過(guò)VNS對(duì)急性腦缺血再灌注后氧化應(yīng)激、凋亡、炎癥反應(yīng)的影響及可能分子機(jī)制進(jìn)行深入探討,為其臨床應(yīng)用提供一定理論依據(jù)。研究方法第一部分：采用顱內(nèi)注射antagmiR210抑制內(nèi)源性miR210表達(dá),線栓法制備右側(cè)大腦中動(dòng)脈栓塞／再灌注(Middle cerebral artery occlusion/reperfusion, MCAO/R)模型,梗塞30min后給予VNS干預(yù)。MCAO手術(shù)及電刺激過(guò)程監(jiān)測(cè)心率、尾動(dòng)脈血壓、血?dú)庖约坝覀?cè)大腦中動(dòng)脈供血區(qū)腦血流變化。再灌注24h時(shí),熒光定量PCR檢測(cè)各組miR210表達(dá)。采用Bederson 5分制評(píng)估再灌注24h時(shí)神經(jīng)體征損傷、測(cè)定腦梗死體積；(Tdt-mediated dutp nick end labeling, TUNEL)原位凋亡檢測(cè)技術(shù)檢測(cè)缺血側(cè)皮層神經(jīng)元凋亡數(shù)量；ELISA檢測(cè)缺血側(cè)腦組織氧化應(yīng)激反應(yīng)水平(Superoxide Dismutase, SOD)、(Maleic Dialdehyde assay, MDA)、(Glutathione, GSH)。Western blot技術(shù)檢測(cè)各組缺血側(cè)皮層p-Akt和caspase-3活性蛋白表達(dá)情況,免疫熒光染色測(cè)各組缺血皮層caspase-3陽(yáng)性細(xì)胞表達(dá)。第二部分：采用顱內(nèi)注射PPARyRNAi技術(shù)抑制PPARy (Peroxisome proliferator -activated receptor y, PPARy)表達(dá),構(gòu)建右側(cè)大腦中動(dòng)脈栓塞/再灌注模型,梗塞30min后VNS干預(yù),熒光定量PCR和Western blot技術(shù)檢測(cè)各組缺血側(cè)皮層PPARy基因和蛋白表達(dá)；采用Ludmila Belayev 12分制評(píng)估神經(jīng)功能缺失和腦梗死體積;HE染色顯示各組缺血側(cè)腦組織的病理變化；免疫熒光雙標(biāo)檢測(cè)缺血側(cè)皮層小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞表面a7乙酰膽堿能受體(a7nicotinic acetylcholine receptor, a7nAchR)表達(dá)；ELISA檢測(cè)缺血側(cè)腦組織腫瘤壞死因子a(Tumor necrosis factor α, TNF-α)、白介素-1β(Interleukin 1β, IL-1β)含量水平。研究結(jié)果第一部分：(1)實(shí)驗(yàn)過(guò)程中,VNS對(duì)大鼠心率(Heart rate, HR)、血壓(Blood pressure, BP)、血?dú)饧坝覀?cè)大腦中動(dòng)脈供血區(qū)腦血流無(wú)明顯影響(p0.05)；(2)與缺血組(I/R)相比,VNS進(jìn)一步增加缺血誘導(dǎo)的miR210表達(dá)(p0.05),改善腦缺血再灌注急性期神經(jīng)功能癥狀缺失和減少腦梗死體積,減少缺血側(cè)皮層神經(jīng)元凋亡數(shù)量(p0.05),沉默miR210后,進(jìn)一步加重缺血導(dǎo)致的腦組織損傷同時(shí)也減少VNS神經(jīng)保護(hù)作用(p0.05),提示miR210參與VNS的神經(jīng)保護(hù)效應(yīng)。(3)與假手術(shù)組(sham I/R)相比,缺血后導(dǎo)致大鼠缺血側(cè)皮層SOD活性下降、GSH含量減少和MAD含量增加；與缺血組(I/R)相比,VNS明顯抑制缺血側(cè)腦組織氧化應(yīng)激水平(p0.05)；沉默miR210后,進(jìn)一步加重再灌注誘導(dǎo)的氧化應(yīng)激反應(yīng)(p0.05),同時(shí)削弱VNS抗氧化應(yīng)激效應(yīng)(p0.05)。(4)與假手術(shù)組(sham I/R)相比,缺血再灌注后缺血側(cè)腦組織p-Akt含量減少(p0.05),VNS干預(yù)后逆轉(zhuǎn)缺血抑制p-Akt表達(dá)的效應(yīng)(p0.05),并且p-Akt蛋白變化與antagomiR210干擾無(wú)明顯關(guān)聯(lián)性(p0.05)。(5)與假手術(shù)組(sham I/R)相比,缺血組(I/R)缺血側(cè)皮層caspase 3活性蛋白表達(dá)明顯增加(p0.05)；沉默miR210后,進(jìn)一步增加缺血側(cè)腦組織caspase3活性(p0.05),而VNS后可抑制缺血側(cè)皮層caspase 3活性蛋白表達(dá)(p0.05)；沉默miR210后,明顯減弱VNS抑制凋亡反應(yīng)效應(yīng)(p0.05),各組免疫熒光caspase 3活性細(xì)胞變化趨勢(shì)同蛋白表達(dá)基本一致,提示miR210參與VNS對(duì)氧化應(yīng)激和凋亡的調(diào)控過(guò)程。第二部分：(1)熒光定量PCR和Western blot法檢測(cè)顱內(nèi)注射PPARyRNAi干預(yù)后各組PPARy表達(dá)示：與缺血+陰性病毒對(duì)照組(I/R+ LV-control)相比,缺血+電刺激+陰性病毒對(duì)照組(I/R+VNS+LV-control)中PPARγ基因和蛋白表達(dá)明顯上調(diào)(p0.05)；PPARyRNAi干擾后,缺血+干擾組(I/R+LV-shPPARr)和缺血+電刺激+干擾組(I/R+VNS+LV-shPPARr)兩組基因蛋白表達(dá)明顯下降(p0.05)。(2)再灌注24h時(shí),VNS改善神經(jīng)功能癥狀缺失,減少腦梗死體積,減輕腦組織病理?yè)p傷,抑制缺血側(cè)皮層炎癥因子(TNF-α、IL-1β)水平(p0.05)；PPARyRNAi干擾后,VNS保護(hù)效應(yīng)明顯下降,炎癥因子水平增加(p0.05),提示PPAR γ參與VNS對(duì)炎癥反應(yīng)的抑制過(guò)程。(3)免疫熒光顯示VNS同時(shí)可以激活缺血側(cè)皮層小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞表面a7nAchR,調(diào)控神經(jīng)免疫細(xì)胞形態(tài)和功能,發(fā)揮抗炎作用。結(jié)論(1)VNS改善急性腦缺血再灌注大鼠神經(jīng)功能癥狀缺失和減少腦梗死體積。(2)VNS減少急性腦缺血再灌注大鼠缺血半暗帶神經(jīng)元凋亡數(shù)量。(3)VNS減少急性腦缺血再灌注大鼠缺血半暗帶氧化應(yīng)激和凋亡反應(yīng)。(4)電刺激迷走神經(jīng)減少急性腦缺血再灌注大鼠缺血半暗帶炎癥反應(yīng)。(5)miR210表達(dá)活動(dòng)影響氧化應(yīng)激和凋亡反應(yīng),參與了電刺激迷走神經(jīng)對(duì)急性腦缺血再灌注大鼠腦保護(hù)作用。PPARy表達(dá)活動(dòng)調(diào)節(jié)腦缺血再灌注后炎癥反應(yīng)參與了電刺激迷走神經(jīng)對(duì)急性腦缺血再灌注大鼠的腦保護(hù)作用。
[Abstract]:Background and objective acute cerebral apoplexy has become the first cause of fatal disease in China. It is the most direct and effective way to restore the cerebral blood flow in the semi dark zone in time. However, early cerebral ischemia reperfusion (ischemia/reperfusion, I/R) easily induces a large number of oxygen free radicals, waterfall inflammation, necrosis and apoptosis. A variety of factors interact with each other to cause two brain damage. Therefore, the successful development of a multiple molecular mechanism for stroke is an urgent need for the.1997 and Drug Administration (FDA) and the recommendation of the VNS (vagus nerve stimulation, VNS) for partial seizures of intractable epilepsy. In recent years, foreign studies have found that VNS has a neuroprotective effect on acute cerebral ischemia reperfusion rats, but its mechanism is not clear. This study intends to explore the effects and possible molecular mechanisms of oxidative stress, apoptosis, inflammatory response and the possible molecular mechanism of acute cerebral ischemia reperfusion in order to provide the clinical application of VNS. Part 1: the first part of the study: using intracranial injection of antagmiR210 to inhibit endogenous miR210 expression, and to prepare the right middle cerebral artery embolism / reperfusion (Middle cerebral artery occlusion/reperfusion, MCAO/R) model by the thread thrombus method. After the infarct 30min, the heart rate and the tail artery were monitored by the VNS dry pre.MCAO operation and the electrical stimulation process. Blood pressure, blood gas and the cerebral blood flow in the right middle cerebral artery supply area. MiR210 expression in each group was detected by fluorescence quantitative PCR at 24h. Bederson 5 fraction was used to evaluate the neurological damage, and the volume of cerebral infarction was measured; (Tdt-mediated dUTP nick end labeling, TUNEL) in situ apoptosis detection technique for the detection of ischemic cortex God The number of apoptotic cells, the level of oxidative stress response (Superoxide Dismutase, SOD), (Maleic Dialdehyde assay, MDA), (Glutathione, GSH).Western blot techniques were detected by ELISA, and the expression of active protein in the ischemic cortex of each group was detected by Glutathione, GSH.Western blot, and the immunofluorescence staining was used to detect the positive cells of ischemic cortex The second part: the second part: using the intracranial injection of PPARyRNAi technique to inhibit the expression of PPARy (Peroxisome proliferator -activated receptor y, PPARy), construct the right middle cerebral artery embolization / reperfusion model, the VNS intervention after the infarct 30min, and the fluorescence quantitative PCR and Western techniques to detect the gene and protein expression of the ischemic side cortex. The Ludmila Belayev 12 score was used to evaluate the loss of nerve function and the volume of cerebral infarction; HE staining showed the pathological changes of the ischemic brain tissue in each group; the expression of A7 acetylcholinergic receptor (a7nicotinic acetylcholine receptor, a7nAchR) on the surface of ischemic lateral cortex and astrocytes was detected by double labeling immunofluorescence; ELISA detection was absent. The level of tumor necrosis factor A (Tumor necrosis factor A, TNF- alpha) and interleukin -1 beta (Interleukin 1 beta, IL-1 beta) in the cerebral tissue of the blood side. Results the first part: (1) in the experimental process, VNS has no obvious effect on the heart rate (Heart rate, HR), blood pressure, blood gas and the blood flow of the right middle cerebral artery in the rat. (2) compared with the ischemic group (I/R), VNS further increased the miR210 expression (P0.05) induced by ischemia, improved the loss of nerve function symptoms and reduced the volume of cerebral infarction in the acute phase of cerebral ischemia reperfusion, reduced the number of neuronal apoptosis in the ischemic lateral cortex (P0.05). After silent miR210, the brain tissue damage caused by ischemia was further aggravated and the VNS God was also reduced. The protective effect (P0.05) showed that miR210 was involved in the neuroprotective effect of VNS. (3) compared with the sham operation group (sham I/R), the ischemic side cortex SOD activity decreased, the GSH content decreased and the MAD content increased. Compared with the ischemic group (I/R), VNS significantly inhibited the level of oxidative stress in the ischemic side brain tissue (P0.05); after silencing miR210, further further Aggravated reperfusion induced oxidative stress response (P0.05) and weakened VNS antioxidant stress effect (P0.05). (4) compared with the sham group (sham I/R), the ischemic side cerebral tissue p-Akt content decreased (P0.05) after ischemia-reperfusion, and VNS intervention reversed the inhibitory effect of ischemia on the inhibitory effect of p-Akt (P0.05), and p-Akt protein changes and antagomiR210 interference were not clear. Significant correlation (P0.05). (5) compared with the sham group (sham I/R), the expression of caspase 3 active protein in the ischemic side cortex of ischemic group (I/R) increased significantly (P0.05). After silence miR210, Caspase3 activity (P0.05) was further increased in ischemic side brain tissue, while VNS could inhibit the expression of caspase 3 active protein (P0.05) in the ischemic side cortex after VNS. Weak VNS inhibits apoptosis response effect (P0.05), and the change trend of immunofluorescent caspase 3 active cells is basically the same as protein expression, suggesting that miR210 participates in the regulatory process of VNS on oxidative stress and apoptosis. The second part: (1) fluorescence quantitative PCR and Western blot method for the detection of PPARy expression in each group after the intervention of intracranial injection PPARyRNAi: and ischemia + Compared with the negative virus control group (I/R+ LV-control), the PPAR gamma gene and protein expression in the ischemic + electrical stimulation + negative virus control group (I/R+VNS+LV-control) increased significantly (P0.05). After PPARyRNAi interference, the expression of gene protein in the two groups of ischemic + interference group (I/R+LV-shPPARr) and ischemia + Electrical stimulation + interference group (I/R+VNS+LV-shPPARr) decreased significantly (P0.05). (2) when reperfusion of 24h, VNS improved the lack of neurological symptoms, reduced the volume of cerebral infarction, reduced the pathological damage of brain tissue, and inhibited the level of inflammatory factors (TNF-, IL-1 beta) in the ischemic lateral cortex (P0.05). After PPARyRNAi interference, the protective effect of VNS was significantly decreased, and the level of inflammatory factors increased (P0.05), suggesting that PPAR gamma participates in the inhibition of the inflammatory reaction of VNS. (3) immunofluorescence showed that VNS could activate the a7nAchR of the microglia and astrocytes on the ischemic side of the ischemic cortex, regulate the morphology and function of the neurocyte and play an anti-inflammatory effect. Conclusion (1) VNS can improve the lack of neurological symptoms and reduce the volume of cerebral infarction in the rats with acute cerebral ischemia reperfusion. (2) VNS reduces the acute cerebral ischemia reperfusion The number of apoptosis in the rat ischemic penumbra neurons. (3) VNS reduces the oxidative stress and apoptosis response in the ischemic penumbra of rats with acute cerebral ischemia reperfusion. (4) electric stimulation of the vagus nerve to reduce the inflammatory response in the ischemic penumbra of rats with acute cerebral ischemia reperfusion. (5) miR210 expression activity affects oxidative stress and apoptosis response, and participates in the electrical stimulation of the vagus. The protective effect of.PPARy on acute cerebral ischemia reperfusion rats regulates the inflammatory response after cerebral ischemia reperfusion and the protective effect of electric stimulation of the vagus nerve on the rats with acute cerebral ischemia reperfusion.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R743.3

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