本文選題：TDAG8　切入點：BTB09089　出處：《江蘇大學》2017年碩士論文

【摘要】：腦卒中是最嚴重的和最致命的神經(jīng)系統(tǒng)疾病,是全世界導致死亡的第二大原因,并且是導致大多數(shù)地區(qū)成年人嚴重殘疾的最主要的因素,嚴重影響著患者的生活質(zhì)量。缺血性腦卒中是最主要的腦卒中類型,主要是由于大腦中動脈阻塞(middle cerebral artery occlusion,MCAO)引起。T細胞死亡相關基因8(T cell death associated gene 8,TDAG8)是一種質(zhì)子敏感的G蛋白偶聯(lián)受體(G protein coupled receptor,GPCR),主要在免疫細胞通過組氨酸殘基與細胞外質(zhì)子相互作用從而調(diào)控細胞內(nèi)生物學活動。最近研究表明TDAG8在大鼠腦組織廣泛表達,尤其是前腦邊緣區(qū)域,比如下丘腦、杏仁核、海馬、額葉皮層和紋狀體等,并被特異性激動劑BTB09089激活。盡管活化的TDAG8廣泛參與炎癥、免疫、疼痛和凋亡等的調(diào)節(jié),但是其是否參與腦缺血再灌注損傷的調(diào)節(jié)及其潛在的作用機制并不十分清楚。本研究擬在建立的大鼠短暫性腦缺血再灌注損傷模型中,觀察TDAG8及其可能的下游蛋白在大鼠腦皮層缺血半暗帶區(qū)域的表達變化;其次通過側腦室注射BTB09089激動TDAG8或應用基因干擾技術抑制TDAG8的表達,觀察其對大鼠腦梗體積、神經(jīng)行為學及其下游蛋白表達的影響;最后探討TDAG8調(diào)節(jié)腦缺血再灌注損傷的可能機制。本研究將通過探討TDAG8對大鼠腦缺血再灌注損傷的影響及其作用機制,為治療腦缺血再灌注損傷提供新的治療靶點及理論依據(jù)。目的(1)探索TDAG8在大鼠腦缺血再灌注損傷中的表達變化。(2)探索BTB09089經(jīng)大鼠側腦室注射后對TDAG8表達的影響。(3)探索SiRNA經(jīng)大鼠側腦室注射后對TDAG8表達的影響。(4)探索TDGA8及其下游Akt信號通路對大鼠腦缺血再灌注誘導的神經(jīng)元凋亡和缺血后炎癥反應的影響。方法(1)清潔級健康成年雄性SD大鼠24只,體重270-280 g。按隨機數(shù)字表法隨機分為5組(n=4):假手術組(sham組),大腦中動脈阻塞再灌注3 h組(I/R3 h組),I/R 6 h組,I/R 12 h組和I/R 24 h組。Sham組不做任何處理;各灌注組行大腦中動脈栓塞2 h后再灌注相應的時間后斷頭取腦,提取缺血半暗帶腦組織。此外,原代皮層神經(jīng)元在氧糖剝奪處理后(ogd)的3h,6h,12h和24h提取神經(jīng)元蛋白。用westernblot和qrt-pcr檢測缺血半暗帶tdag8的表達變化。(2)清潔級健康成年雄性sd大鼠16只,體重270-280g。按隨機數(shù)字表法隨機分為4組(n=4):正常組(normal組),btb090895μm組(btb5μm組),btb10μm組和btb20μm組。normal組不做任何處理,btb5μm組側腦室注射5μm的btb09089,btb10μm組側腦室注射10μm的btb09089和btb20μm組側腦室注射20μm的btb09089,每組給予8μl。藥物處理6h后斷頭取腦,提取前腦邊緣區(qū)域皮層腦組織。用westernblot和qrt-pcr檢測tdag8的表達變化。此外,為了檢測btb09089是否具有細胞毒性,分別用不同濃度的btb09089作用于神經(jīng)元,在指定的時間點檢測神經(jīng)元細胞活性。(3)清潔級健康成年雄性sd大鼠20只,體重270-280g。按隨機數(shù)字表法隨機分為5組(n=4):正常組(normal組),轉染試劑組(vehicle組),sirna陰性對照組(sirna-c組),sirna組和sirna陽性對照組(sirna-β-actin組)。normal組不做任何處理,vehicle組側腦室只注射等量的轉染試劑,sirna-c組只注射4μg序列錯配的sirna,sirna組注射4μg針對tdag8特異性設計的sirna,sirna-β-actin組注射4μg針對β-actin特異性設計的sirna。處理24h后斷頭取腦,提取前腦邊緣區(qū)域皮層腦組織。用westernblot和qrt-pcr檢測tdag8的表達變化。(4)清潔級健康成年雄性sd大鼠48只,體重270-280g。按隨機數(shù)字表法分為8組(n=6):sham組,mcao組,vehicle組,btb5μm組,btb10μm組,btb20μm組,sirna-c組,sirna組和sirna+btb20μm。btb5μm組,btb10μm組,btb20μm組在mcao前6h經(jīng)側腦室分別注射8μl5μm、10μm和20μm的btb09089;sirna-c組、sirna組和sirna+btb20μm組在mcao前24h經(jīng)側腦室分別注射4μg(8μl)的sirna-c、sirna和sirna,其中sirna+btb20μm組再在mcao前6h經(jīng)側腦室注射8μl20μm的btb09089;sham組僅做手術不栓塞大腦中動脈,mcao組短暫堵塞大腦中動脈2h后再灌注,vehicle組在缺血再灌注前24h給予適量轉染試劑。在再灌注后6h斷頭取腦,用westernblot和qrt-pcr檢測缺血半暗帶促凋亡因子caspase-3、和cleavedcaspase-3、抗凋亡因子bcl-2和tdag8下游相關蛋白磷酸化akt(p-akt)和磷酸化creb(p-creb)的表達,以及用qrt-pcr檢測炎癥因子tnf-α,mcp-1和il-1β的表達。分別在再灌注后24h和72h評估神經(jīng)功能缺陷評分和檢測腦梗體積。結果(1)在mcao模型:與sham組比較,tdag8在i/r3h左右表達增加,持續(xù)到24h,并在i/r6h左右達到峰值。在ogd模型:與sham組比較,tdag8在復氧3h左右表達增加,持續(xù)到24h,并在復氧24h左右達到峰值。(2)與normal組比較,btb5μm組tdag8的表達增加,與btb5μm組比較,btb10μm組tdag8的表達增加,與btb10μm組比較,btb20μm組tdag8的表達上調(diào)。與normal組比較,btb090895μm組、10μm組、20μm組和40μm組細胞活性均大于95%。(3)normal組、vehicle組和sirna-c組tdag8的表達差異無統(tǒng)計學意義。與normal組比較,sirna組抑制tdag8的表達;而與sirna組比較,sirna-β-actin組抑制β-actin的表達。(4)與sham組比較,mcao組腦梗體積增大,神經(jīng)功能缺陷評分降低,tnf-α、mcp-1、il-1β、cleavedcaspase-3、p-akt和p-creb表達增高,bcl-2表達降低;btb5μm組和mcao組差異無統(tǒng)計學意義;而與mcao組比較,btb10μm組腦梗體積降低,神經(jīng)功能缺陷評分升高,tnf-α、mcp-1、il-1β和cleavedcaspase-3表達降低,bcl-2、p-akt和p-creb表達升高;與btb10μm組比較,btb20μm組腦梗體積降低,神經(jīng)功能缺陷評分增高,tnf-α、mcp-1、il-1β和cleavedcaspase-3表達降低,bcl-2、p-akt和p-creb表達升高;mcao組和vehicle組、sirna-c組差異無統(tǒng)計學意義;與mcao組比較,sirna組腦梗體積升高,神經(jīng)功能缺陷評分降低,tnf-α、mcp-1、il-1β和cleavedcaspase-3表達升高,bcl-2、p-akt和p-creb表達降低;sirna+btb20μm和sirna組差異無統(tǒng)計學意義。結論(1)tdag8在大鼠腦缺血再灌注損傷中有意義的表達上調(diào)。(2)大鼠側腦室注射btb09089能夠促進tdag8的表達,沒有明顯的細胞毒性。(3)大鼠側腦室注射TDAG8-siRNA能夠抑制TDAG8的表達。(4)活化TDAG8可能通過Akt信號通路抑制大鼠腦缺血再灌注誘導的神經(jīng)元凋亡和缺血后炎癥反應。
[Abstract]:Stroke is the most serious and deadly diseases of the nervous system, is the world's second leading cause of death, and is the most important factor leading to the majority of adults severely disabled, seriously affect the quality of life of patients with ischemic stroke. Stroke is the most common type, mainly due to middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO.T) induced cell death related gene 8 (T cell death associated gene 8, TDAG8) is a proton sensitive G protein coupled receptors (G protein coupled receptor, GPCR), mainly in immune cells by histidine residues and extracellular proton interactions to regulate the biological activity in cells. Recent studies show that TDAG8 is widely expressed in rat brain tissue, especially limbic forebrain regions such as hypothalamus, amygdala, hippocampus, frontal cortex and striatum, and be specific Agonist activation of BTB09089. Although the activation of TDAG8 are involved in inflammation, immune regulation, pain and apoptosis, but whether it is involved in cerebral ischemia reperfusion injury and its potential regulation mechanism is not very clear. This study in rats with transient cerebral ischemia reperfusion injury model was established, to observe the expression change TDAG8 and its downstream protein may in the cerebral cortex ischemia penumbra region; followed by intracerebroventricular injection of BTB09089 agonist TDAG8 or application of gene interference to inhibit the expression of TDAG8, to observe the cerebral infarction volume in rats, the influence of God behavior and expression of downstream proteins; finally, the regulation of TDAG8 on cerebral ischemia reperfusion injury the possible mechanism of this study. Through the study of TDAG8 on cerebral ischemia reperfusion injury in rats and its effect mechanism, for the treatment of cerebral ischemia reperfusion injury provides a new therapeutic target 鐐瑰強鐞嗚渚濇嵁.鐩殑(1)鎺㈢儲TDAG8鍦ㄥぇ榧犺剳緙鴻鍐嶇亴娉ㄦ崯浼や腑鐨勮〃杈懼彉鍖，

本文編號：1654862