Necroptosis在脊髓损伤后的表达及作用的实验研究

发布时间：2018-03-31 11:41

  本文选题：脊髓损伤　切入点：necroptosi　出处：《中国人民解放军医学院》2015年博士论文

【摘要】：目的：建立胸10节段挫伤的大鼠模型,检测necroptosis关键蛋白在脊髓损伤(spinal cord injury, SCI)后随时间变化而发生的动态表达,及发生necroptosis的细胞类型。采用其特异性抑制剂necrostatin-1(Nec-1)进行干预,观察其对SCI大鼠模型的影响,同时对内在的分子机制进行探索,来初步确定necroptosis在SCI急性期病理改变中的作用。方法：SD大鼠共计一百四十四只,分成四组：(1)Sham组：剪除椎板,无脊髓的损伤；(2)SCI组：只损伤脊髓,不给任何药物处理；(3)SCI+Nec-1组：打击脊髓前15min,鞘内注射Nec-1 (20uL,40mg/mL);(4)SCI+溶剂组(SCI+Veh组)：损伤脊髓前给予同等剂量Nec-1的溶剂(DMSO与0.9%的NaCI等体积混合)。其中SCI组根据不同时间点分为多个亚组,用于检测necroptosis相关蛋白RIP1和RIP3的动态表达。采用的实验方法主要有(1)PI标记+免疫荧光：观察发生necroptosi s的细胞种类；(2)TTC染色：显示SCI后24h脊髓的血供状态；(3)伊文氏蓝渗透实验：观察损伤24h后血-脊髓屏障通透性的改变；(4)Western-blot:观察各种蛋白水平的变化；(5)EL ISA:脊髓局部炎症相关因子的浓度测定；(6)感觉及运动神经功能评价。结果：(1)SCI后necroptosis存在时空变化：RIP1和RIP3的表达均上调均于损伤后48h达到高峰(P0.01),且与凋亡相关的cleaved caspase-3和自噬标志蛋白LC3BⅡ的表达峰值时间点一致。免疫荧光结果表明,MAP-2、GFAP及Olig2与PI均有共定位的情况出现。(2)Nec-1预处理可以减轻损伤造成的组织结构破坏,主要表现为SCI+Nec-1组脊髓组织病理评分显著低于SCI组(P0.01),损伤1w后前角运动神经元数量显著多于SCI组(P0.01),脱髓鞘改变得到缓解(P0.01)。(3)Nec-1预处理可以促进大鼠神经功能的恢复,主要表现为SCI+Nec-1组的BBB评分从损伤后1w开始明显高于SCI组(P0.01)。(4)Nec-1预处理可以通过多种机制发挥神经保护作用：(a)减轻损伤造成的脊髓水肿,表现为SCI+Nec-1组的湿/干比重明显低于SCI组(P0.01)；(b)改善脊髓的微循环：SCI+Nec-1组脊髓组织内伊文氏蓝含量显著低于SCI组(P0.01),前一组的脊髓缺血面积明显小于后一组(P0.01)：(c) Nec-1干预可以同时抑制细胞凋亡和自噬相关蛋白的表达(P0.01)。结论：Necroptosis在大鼠SCI后病理演变过程中发挥了重要的作用。对动物模型给予necroptosis抑制剂Nec-1干预,可有效保护残存的神经组织,促进功能恢复。这将为创伤性SCI的临床试验提供有效的实验数据支持,给SCI患者的治疗带来新的希望。
[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，

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