甲强龙通过调控锌离子水平促进小胶质细胞自噬性死亡改善脊髓损伤的作用机制

发布时间：2018-02-12 08:18

本文关键词： 甲强龙脊髓损伤锌离子自噬性死亡 ZIP8　出处：《辽宁医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的探讨甲强龙(MP)在治疗脊髓损伤方面的作用,及其确切的分子机制,最终为临床治疗提供理论依据。方法取成年雄性SD大鼠110只,随机分为2组,每组根据时间点又分为不同亚组,每亚组随机分配5只。选用改良Allen’s法建立大鼠脊髓(T10)损伤模型,术前及术后3d,7d,21d,60d做BBB功能评分;术后72h取材做尼氏染色观察神经元状况;术后12h、18h取材做免疫组化检测Iba1表达情况;术后不同时间点采取原子分光光度技术检测组织和血液中锌离子的改变。在体外实验中,我们通过免疫荧光观察细胞Iba1表达情况;通过MTT检测不同处理因素对细胞存活率的影响;通过ELISA检测细胞液中IL-6,IL-10,TNFα的表达水平;通过Western blot检测i NOS,LC-3B,ZIP8,NF-κβ和Beclin-1的表达情况;最后我们用Fluo Zin-3-AM检测了细胞内锌离子的变化。结果研究发现MP作用于脊髓损伤后,Basso,Beattie and Bresnahan(BBB)评分和大鼠脊髓损伤区运动神经元的数量均得到提高。此外,我们在体外实验中发现MP导致大鼠脊髓损伤区的小胶质细胞数量显著减少,并且MP诱导了小胶质细胞发生自噬性死亡。更进一步的是,我们发现MP能显著减少小胶质细胞激活的相关因子。同时,MP可引起胞内锌离子和膜通道蛋白ZIP8表达水平的增高,而TPEN可以逆转MP引起小胶质细胞发生自噬性死亡的结果。最后,我们还发现在LPS诱导的体外模型中,MP可以抑制NF-κβ。结论MP会导致小胶质细胞发生自噬性死亡,其主要通过增加细胞内锌离子的浓度,此外还可能还和NF-κβ的抑制有关。MP对脊髓损伤起保护作用。
[Abstract]:Objective to investigate the role of MPM in the treatment of spinal cord injury and its molecular mechanism, and to provide theoretical basis for clinical treatment. Methods 110 adult male SD rats were randomly divided into two groups. Each group was divided into different subgroups according to time points, and 5 rats were randomly assigned to each subgroup. The model of spinal cord T10 injury was established by modified Allen's method. The BBB functional score was evaluated at the end of 21d before and 3 days after operation, and the neuronal status was observed by Nissl staining at 72 h after operation. The expression of Iba1 was detected by immunohistochemistry at 18h after operation, the changes of zinc ions in tissues and blood were detected by atomic spectrophotometry at different time points after operation. In vitro, the expression of Iba1 was observed by immunofluorescence. The effect of different treatment factors on cell survival was detected by MTT, the expression of IL-10 TNF- 伪 in cell fluid was detected by ELISA, the expression of NF- 魏尾 and Beclin-1 was detected by Western blot. Finally, Fluo Zin-3-AM was used to detect the changes of zinc ions in cells. Results it was found that the scores of bassoBeattie and Bresnahanna neurons and the number of motor neurons in the injured area of spinal cord were increased after spinal cord injury. We found in vitro that MP resulted in a significant decrease in the number of microglia in the injured area of the spinal cord in rats, and that MP induced autophagic death of microglia. We found that MP could significantly reduce the related factors of microglial activation. At the same time, MP could increase the expression of zinc ion and membrane channel protein ZIP8, while TPEN could reverse the autophagic death of microglia induced by MP. We also found that in vitro model induced by LPS, MP could inhibit NF- 魏尾. Conclusion MP can induce autophagic death of microglia, mainly by increasing the concentration of zinc ions in the cells. It may also be related to the inhibition of NF- 魏尾. MP may play a protective role in spinal cord injury.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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