本文選題：脊髓損傷　切入點：necroptosi　出處：《中國人民解放軍醫(yī)學(xué)院》2015年博士論文

【摘要】：目的：建立胸10節(jié)段挫傷的大鼠模型,檢測necroptosis關(guān)鍵蛋白在脊髓損傷(spinal cord injury, SCI)后隨時間變化而發(fā)生的動態(tài)表達(dá),及發(fā)生necroptosis的細(xì)胞類型。采用其特異性抑制劑necrostatin-1(Nec-1)進行干預(yù),觀察其對SCI大鼠模型的影響,同時對內(nèi)在的分子機制進行探索,來初步確定necroptosis在SCI急性期病理改變中的作用。方法：SD大鼠共計一百四十四只,分成四組：(1)Sham組：剪除椎板,無脊髓的損傷；(2)SCI組：只損傷脊髓,不給任何藥物處理；(3)SCI+Nec-1組：打擊脊髓前15min,鞘內(nèi)注射Nec-1 (20uL,40mg/mL);(4)SCI+溶劑組(SCI+Veh組)：損傷脊髓前給予同等劑量Nec-1的溶劑(DMSO與0.9%的NaCI等體積混合)。其中SCI組根據(jù)不同時間點分為多個亞組,用于檢測necroptosis相關(guān)蛋白RIP1和RIP3的動態(tài)表達(dá)。采用的實驗方法主要有(1)PI標(biāo)記+免疫熒光：觀察發(fā)生necroptosi s的細(xì)胞種類；(2)TTC染色：顯示SCI后24h脊髓的血供狀態(tài)；(3)伊文氏藍(lán)滲透實驗：觀察損傷24h后血-脊髓屏障通透性的改變；(4)Western-blot:觀察各種蛋白水平的變化；(5)EL ISA:脊髓局部炎癥相關(guān)因子的濃度測定；(6)感覺及運動神經(jīng)功能評價。結(jié)果：(1)SCI后necroptosis存在時空變化：RIP1和RIP3的表達(dá)均上調(diào)均于損傷后48h達(dá)到高峰(P0.01),且與凋亡相關(guān)的cleaved caspase-3和自噬標(biāo)志蛋白LC3BⅡ的表達(dá)峰值時間點一致。免疫熒光結(jié)果表明,MAP-2、GFAP及Olig2與PI均有共定位的情況出現(xiàn)。(2)Nec-1預(yù)處理可以減輕損傷造成的組織結(jié)構(gòu)破壞,主要表現(xiàn)為SCI+Nec-1組脊髓組織病理評分顯著低于SCI組(P0.01),損傷1w后前角運動神經(jīng)元數(shù)量顯著多于SCI組(P0.01),脫髓鞘改變得到緩解(P0.01)。(3)Nec-1預(yù)處理可以促進大鼠神經(jīng)功能的恢復(fù),主要表現(xiàn)為SCI+Nec-1組的BBB評分從損傷后1w開始明顯高于SCI組(P0.01)。(4)Nec-1預(yù)處理可以通過多種機制發(fā)揮神經(jīng)保護作用：(a)減輕損傷造成的脊髓水腫,表現(xiàn)為SCI+Nec-1組的濕/干比重明顯低于SCI組(P0.01)；(b)改善脊髓的微循環(huán)：SCI+Nec-1組脊髓組織內(nèi)伊文氏藍(lán)含量顯著低于SCI組(P0.01),前一組的脊髓缺血面積明顯小于后一組(P0.01)：(c) Nec-1干預(yù)可以同時抑制細(xì)胞凋亡和自噬相關(guān)蛋白的表達(dá)(P0.01)。結(jié)論：Necroptosis在大鼠SCI后病理演變過程中發(fā)揮了重要的作用。對動物模型給予necroptosis抑制劑Nec-1干預(yù),可有效保護殘存的神經(jīng)組織,促進功能恢復(fù)。這將為創(chuàng)傷性SCI的臨床試驗提供有效的實驗數(shù)據(jù)支持,給SCI患者的治療帶來新的希望。
[Abstract]:Objective: to establish a rat model of thoracic 10 segment contusion, to detect the dynamic expression of necroptosis key protein in spinal cord injuryand the cell types of necroptosis after spinal cord injury. The effects of necroptosis on SCI rat model were observed and the intrinsic molecular mechanism was explored to determine the role of necroptosis in the pathological changes of SCI in acute stage. Methods 144 rats were divided into four groups: the spinal lamina was cut off, the laminae were cut off, Sci group without spinal cord injury: only spinal cord injury, No drug treatment was given to the sci Nec-1 group: 15 minutes before the spinal cord was struck, the sci Veh group was treated with intrathecal injection of Nec-1 20 渭 L of 40 mg / m L + 4% sci. The SCI group was divided into several subgroups according to the different time points according to the different time points, and the same dose of Nec-1 was given before the spinal cord injury, and the same dose of Nec-1 was given to the sci Veh group. The main methods used to detect the dynamic expression of necroptosis related protein RIP1 and RIP3. The main experimental methods were PI-labeled immunofluorescence: observing the cell type of necroptosi s: TTC staining: showing the blood supply status of spinal cord 24 hours after SCI and Yi Wen's blue osmosis. Permeation experiment: observe the changes of blood-spinal barrier permeability after 24 hours of injury; observe the changes of various protein levels; observe the changes of various protein levels; determine the concentration of local inflammatory related factors in spinal cord, and evaluate the sensory and motor nerve function. Results necroptosis was stored after 1% sci. The expression of RIP1 and RIP3 reached the peak at 48h after injury, and was consistent with that of apoptosis-related cleaved caspase-3 and autophagy marker protein LC3B 鈪，

本文編號：1690583