白介素-1信号通路在异氟醚预处理保护脊髓损伤中的机制研究

发布时间：2018-06-17 14:08

本文选题：脊髓损伤 + 异氟醚预处理　；参考：《第四军医大学》2014年硕士论文

【摘要】：脊髓损伤（SCI）是一个具有严重破坏性而且病理生理比较复杂的临床疾病，引起SCI的原因很多，其中主要原因为外伤，由于中枢神经系统的不可再生，SCI带来的损伤往往也是不可逆的。有研究表明SCI发生后，缺血是引起损伤加重的一个不可忽视的角色。脊髓损伤后存在两个缺血区域，一个区域紧靠损伤位点，该区域大部分神经元正在或者已经坏死或者凋亡，属于已经不可挽救区域；另一个区域离损伤位点比较远，紧靠第一个损伤区，该区域的神经元尚未发生凋亡或坏死，属于可挽救区域，因此如果能及时挽救该区域处于早期变性的神经元，就能很大程度的缓解脊髓损伤，从而促进神经功能的恢复[1]。异氟醚作为吸入麻醉药的一种已经广泛用运于临床，我们实验室的前期实验已经证实了异氟醚预处理可以明显改善大脑局部缺血损伤时导致的神经功能的破坏[2]，也有部分实验证实异氟醚预处理可以改善脊髓缺血损伤导致的神经功能损伤[3-4]。在临床实际操作中，异氟醚预处理已经用于保护心脏手术引起的心肌缺血性损伤[5]。然而异氟醚预处理是否可以缓解脊髓损伤导致的缺血损伤尚未报道，因此异氟醚预处理是否可以用于减轻SCI，有必要进一步进行研究和探讨。异氟醚预处理产生神经保护作用所涉及的机制很多，离子通道的抑制，抑制凋亡，抑制炎症反应等。我们实验室的前期实验证实异氟醚预处理减轻脑缺血再灌注损伤是通过抑制TLR4一髓样分化因子88(MyD88)信号通路，从而抑制炎症和免疫应答的级联反应。白介素-1β（IL-1β）作为白介素-1的主要组成部分在引起炎症反应中发挥重要作用，而IL-1β又是通过激活核转录因子（NF-κB）引起多种炎症因子的激活引起炎症反应。因此，本研究拟探讨异氟醚预处理对大鼠SCI能否产生保护作用及其可能的机制是否与IL-1信号通路有关，为以后SCI的治疗及麻醉提供实验基础。目的：观察异氟醚预处理对大鼠SCI神经行为学的变化、脊髓组织病理学的改变和脊髓组织IL-1β、NF-κB表达变化，探讨异氟醚预处理对大鼠SCI产生保护作用及其可能的机制是否与IL-1信号通路有关，为SCI的治疗提供理论基础。方法：选取健康成年雄性SD大鼠30只，随机分为3组（n=10）。假手术组（Sham组）：只暴露脊髓组织不做打击损伤；损伤组（SCI组）：分离脊髓并进行打击损伤；异氟醚预处理组（ISO组）：吸入2%异氟醚2h，预处理结束后24h时做脊髓损伤模型。术后连续七天进行行为学评分，观察结束后取脊髓组织进行HE染色计数存活神经元数量。随后再选取SD大鼠40只进行第二部分实验，随机将大鼠分为4组（n=10）：假手术组（Sham组）：只暴露脊髓组织不做打击损伤；损伤组（SCI组）：分离脊髓并进行打击损伤；异氟醚预处理组（ISO组）：吸入2%异氟醚2h，预处理结束后24h时做脊髓损伤模型:；IL-1抑制剂组（IL-Ra组）：先做脊髓损伤模型，用含2g/L白介素-1抑制剂明胶海绵放于损伤处2h。免疫组化染色观察IL-1β的表达变化。通过Western-blot法观察脊髓组织IL-1β、NF-κB的变化。结果：（1）BBB评分结果显示：随着时间的推移SCI组和ISO组大鼠后肢功能逐渐恢复，，但是异氟醚预处理组BBB评分较单纯损伤组增加更为明显。7天后ISO组神经行为学评分比SCI组明显增高，差异有统计学意义（P0.05）。（2）HE染色结果显示：SCI组脊髓组织水肿，细胞核固缩。ISO组脊髓组织水肿减轻，存活细胞数量较多。存活神经元计数，ISO组存活神经元数量较SCI组明显增高，差异具有统计学意义（P0.05）。（3）免疫组织化学实验结果显示：本实验主要观察脊髓组织中IL-1β的表达量的变化，其中认为胞浆、间质被染成棕褐色为阳性。在Sham组中几乎检测不到IL-1β的表达。SCI组中可见IL-1β在细胞质及胞外基质中大量表达。而ISO组以及IL-1Ra组中细胞质及间质中IL-1β表达量减少。（4）Western-blot结果显示：分别在2h、6h、24h、48h取SCI组脊髓组织进行IL-1β的蛋白测定，发现在6h时IL-1β的表达量最高。在Sham组中，IL-1β及NF-κB蛋白表达与其他组相比较少。SCI组内IL-1β及NF-κB蛋白表达量最高，IL-1β及NF-κB蛋白表达在ISO组和IL-1Ra组表达明显下降，其差异具有统计学意义（P0.05）。结论：异氟醚预处理能明显降低脊髓损伤后神经行为学障碍，增加损伤脊髓组织存活神经元数量，说明异氟醚预处理对脊髓损伤产生保护作用。ISO组IL-1β及NF-κB蛋白表达量明显降低，说明异氟醚预处理对脊髓产生保护作用是通过抑制IL-1信号通路产生作用的。
[Abstract]:Spinal cord injury ( SCI ) is a kind of clinical disease with severe damage and complicated pathological physiology , which causes many reasons . The main reason is trauma . Due to the non - regeneration of the central nervous system , the injury caused by SCI is often irreversible . There are two ischemic regions after SCI . One region is close to the injury site . Most of the neurons in the region are either already necrosis or apoptosis , which belong to the region which has not been saved ;
The other region is far from the injury site , close to the first lesion region , and the neurons in the region have not yet undergone apoptosis or necrosis , and belong to the salvage region , so that if the region in early degeneration can be saved in time , the spinal cord injury can be relieved to a large extent , thereby promoting the restoration of the neurological function . It has been proved that isoflurane preconditioning can improve the neurological deficit caused by ischemia injury in the brain , and some experiments prove that isoflurane preconditioning can improve the neurological deficit caused by ischemia injury in the brain . However , whether isoflurane preconditioning can alleviate ischemia injury caused by spinal cord injury has not been reported , and it is necessary to study and discuss whether isoflurane preconditioning can be used to alleviate SCI .

Interleukin - 1尾 ( IL - 1尾 ) plays an important role in the inflammatory response , and IL - 1尾 ( IL - 1尾 ) plays an important role in inflammatory response by inhibiting the activation of NF - 魏B . IL - 1尾 ( IL - 1尾 ) is an important component of IL - 1 . IL - 1尾 ( IL - 1尾 ) is an important component of IL - 1 .

Therefore , it is proposed to investigate whether the protective effects of isoflurane preconditioning on SCI in rats and possible mechanisms are related to IL - 1 signaling pathway , which provides an experimental basis for the treatment and anesthesia of SCI .

Purpose :

The changes of SCI neural behavior in rats , the changes of spinal cord tissue pathology and the expression of IL - 1尾 and NF - 魏B in the spinal cord were observed . The protective effects of isoflurane preconditioning on SCI in rats and possible mechanisms were discussed .

Method :

30 male SD rats were randomly divided into 3 groups ( n = 10 ) , sham operation group ( Sham group ) : only exposed spinal cord tissue did not fight injury ;
Injury group ( SCI group ) : spinal cord was isolated and injured ;
The rats were randomly divided into 4 groups ( n = 10 ) : sham operation group ( Sham group ) .
Injury group ( SCI group ) : spinal cord was isolated and injured ;
isoflurane preconditioning group ( iso group ) : inhalation of 2 % isoflurane for 2 h , spinal cord injury model at 24 h after pretreatment :
IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 , IL - 1尾 ,

Results :

( 1 ) BBB scores showed that the function of hindlimb of SCI group and ISO group gradually recovered over time , but the BBB score of isoflurane preconditioning group was significantly higher than that in SCI group ( P0.05 ) .

( 2 ) The results of HE staining showed that the spinal cord tissue edema and cell nuclear fixation in SCI group were significantly decreased and the number of viable cells was more . The number of viable neurons in ISO group was significantly higher than that in SCI group ( P0.05 ) .

( 3 ) The results of immunohistochemistry showed that the expression of IL - 1尾 in the spinal cord tissue was mainly observed in this experiment . The expression of IL - 1尾 in the cytoplasm and extracellular matrix was almost undetectable in the Sham group , but the expression of IL - 1尾 in the cytoplasm and stroma of the ISO group and the IL - 1Ra group was decreased .

( 4 ) Western - blot showed that the expression of IL - 1尾 and NF - 魏B in SCI group was the highest at 2h , 6h , 24h and 48h . In Sham group , the expression of IL - 1尾 and NF - 魏B in SCI group was the highest , and the expression of IL - 1尾 and NF - 魏B in SCI group was the highest , and the expression of IL - 1尾 and NF - 魏B was significantly decreased in the ISO group and IL - 1Ra group , and the difference was statistically significant ( P0.05 ) .

Conclusion :

The effect of isoflurane preconditioning on spinal cord injury was significantly decreased , and the protective effect of isoflurane preconditioning on spinal cord was decreased by inhibiting IL - 1 signaling pathway .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

【参考文献】