幼兔SAH后继发脑损伤p38MAPK、NF-κB、ICAM-1作用的研究

发布时间：2018-06-28 00:13

本文选题：幼兔 + 海马　；参考：《山东大学》2012年博士论文

【摘要】：近年来,虽然神经影像技术的发展为脑血管病的诊断提供了可靠的依据,同时也提高了人们对儿童脑血管病的认识。但目前对儿童脑血管病的基础与临床研究仍然较少。蛛网膜下腔出血(subarachnoid hemorrhage, SAH)是常见的出血性脑血管疾病,儿童期主要见于动静脉畸形和颅内动脉瘤破裂。 SAH发生后,其分解代谢产物极易诱发脑血管痉挛(cerebral vasospasm,CVS),表现为脑血管不同程度的狭窄,导致脑血流的变化,引发脑的缺血性改变,狭窄严重者会造成脑的代谢、功能改变,并出现不可逆改变,从而继发缺血性脑损伤,为临床治疗带来极大困难,对患儿生命构成极大威胁。由于无法彻底清除SAH后残存于脑池内的积血,这就存在诱发CVS的可能。SAH后CVS的发生率在70%左右。研究SAH后CVS的发病机理和发生、发展过程及导致缺血性脑损伤的分子机制,有助于从根本上防治SAH后继发缺血性脑损伤,以减少SAH的致死或致残率。目前CVS发生的机制仍然不十分清楚。近年来备受关注的免疫炎症反应被认为在CVS的发病过程起了重要作用,痉挛血管壁受血管周围血性液体刺激继发的炎症反应及一系列的细胞间信号级联反应引发CVS的理论正日益受到人们的重视。炎症反应的发生必然有炎性因子的聚集。细胞间粘附分子-1(Intercellular adhesion molecule-1, ICAM-1)的作用就是介导白细胞在痉挛血管壁的粘附和迁移,从而引发血管壁的炎症反应。炎症反应可以激活作为基因调控蛋白的核转录因子-κB (Nuclear transcription factor-κB, NF-κB)而完成对ICAM-1转录活性的调控。丝裂原活化蛋白激酶(Mitogen-activated protein kinase, MAPK)是一种细胞信使,它将细胞信号由细胞表面传递到细胞核,通过调控核转录因子活性发挥作用。丝裂原活化蛋白激酶p38(p38Mitogen-activated protein kinase, p38MAPK)通路在MAPK传导通路中起重要作用。对于p38MAPK信号转导通路与NF-κB通路之间的相互关系、作用,既往研究较少,结果不一。目前国内外有分别对上述三种因子单独进行研究的报道,但尚无对三因子在脑血管病相互关系方面进行共同研究的报道,且以往多是在成年兔进行实验研究,尚无在幼兔进行上述研究的报道。本实验试图通过枕大池二次注血制成幼兔SAH后CVS模型,在此基础上研究p38MAPK、NF-κB、ICAM-1三者共同在痉挛血管壁和海马神经细胞的表达以及它们与CVS发生、发展的关系,同时探讨它们与海马神经细胞代谢变化的关系。第一部分幼兔SAH继发CVS后p38MAPK、NF-κB、ICAM-1在基底动脉和海马表达-的研究目的研究丝裂原活化蛋白激酶p38(p38Mitogen-activated kinase protein, P38MAPK)、核转录因子-κB (Nuclear transcription factor-KB, NF-κB)、细胞间粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)在幼兔蛛网膜下腔出血(subarachnoid hemorrhage, SAH)继发脑血管痉挛(cerebral vasospasm, CVS)后在基底动脉和海马的表达变化,以及CVS后海马神经细胞代谢的变化。方法通过幼兔寰枕筋膜向枕大池内二次注入自体动脉血,制成幼兔SAH后CVS模型。分别在SAH后1、3、5、7、9、11d检测基底动脉、海马p38MAPK、 NF-κB、 ICAM-1的表达变化,同时在相同时间段检测海马神经细胞代谢的变化。结果脑血管造影显示对照组及生理盐水(normal saline,NS)组基底动脉光滑、平直,没有明显的缩窄性改变。SAH组第1-7d狭窄程度逐渐加重,管壁增厚,内皮细胞肿胀,内壁皱缩,中层平滑肌细胞增大、扭曲,到第7d狭窄最为严重,第9d逐渐缓解,第11d基本恢复正常。随痉挛程度的不同, p38MAPK、NF-κB、IC AM-1在基底动脉及海马的表达也出现变化。SAH后1-7d, ICAM-1的表达呈逐渐增高的趋势,第7d时出现强烈表达,与CVS的发展过程相一致.p38MAPK、NF-κB的表达反应出现较早,在第5d出现强烈表达,其中p38MAPK的表达更为强烈。P38MAPK、 NF-κB、 ICAM-1的表达强弱有时相上的先后性。SAH后1-7d,随痉挛程度的加重,海马神经细胞过氧化物歧化酶(Superoxide Dismutase, SOD)含量总体呈逐渐减少趋势,而丙二醛(Malondialdehyde, MDA)呈逐渐增多趋势。之后随痉挛程度的减轻,上述变化逐渐缓解。结论 SAH继发CVS后,在基底动脉和海马神经细胞可能存在由P38MAPK、 NF-κB调控,ICAM-1介导的免疫炎症反应。这一级联反应同时影响了海马神经细胞的代谢功能。第二部分p38MAPK、NF-κB、ICAM-1拮抗剂治疗CVS的研究目的确定SAH继发CVS后的基底动脉和海马神经细胞是否存在由p38MAPK、 NF-κB调控,ICAM-1介导的免疫炎症反应。阻断这一级联反应后CVS的发展会出现怎样的变化?海马神经细胞的代谢功能发生怎样的变化? 方法通过幼兔寰枕筋膜向枕大池内二次注入自体动脉血,制成幼兔SAH后CVS模型,之后连续3d分别向枕大池内注入ICAM-1单克隆抗体、NF-κB拮抗剂、p38MAPK抑制剂,并在SAH后第5d行颈内动脉置管椎动脉造影,观察CVS程度后处死动物并留取标本,观察p38MAPK、NF-κB、ICAM-1在基底动脉、海马神经细胞的表达变化,同时在相同时间段检测海马神经细胞代谢的变化。结果在基底动脉,经p38MAPK抑制剂治疗后,p38MAPK、NF-κB、ICAM-1的表达主要局限在内膜、内膜下,表达微弱。经NF-κB拮抗剂治疗后,NF-κB、 ICAM-1在内膜和内膜下有微弱表达,而p38MAPK仍然在内膜、中膜出现表达。经ICAM-1单克隆抗体治疗后,p38MAPK、NF-κB在内膜、中膜均有表达,ICAM-1仅在内膜和内膜下有微弱表达。经生理盐水治疗后,p38MAPK、NF-κB、 ICAM-1在内膜、中膜均有强烈的表达。在海马神经细胞,经p38MAPK抑制剂治疗后,p38MAPK、NF-κB、ICAM-1表达均明显抑制；经NF-κB拈抗剂治疗后,NF-κB、ICAM-1表达均明显抑制,呈弱阳性,但p38MAPK仍有较强表达；在ICAM-1单克隆抗体治疗组,ICAM-1的表达受到抑制,ICAM-1呈微弱表达,而p38MAPK、NF-κB表达并未受到影响,仍然呈阳性表达。经生理盐水治疗后,p38MAPK、NF-κB、ICAM-1在海马神经细胞呈强阳性表达。p38MAPK、 NF-κB、ICAM-1治疗组海马脑组织SOD含量较对照组明显升高,MDA含量明显降低,p38MAPK、NF-κB治疗组效果更为明显。结论 SAH继发CVS后,在基底动脉和海马神经细胞确实存在由p38MAPK、 NF-κB调控,ICAM-1介导的免疫炎症反应。阻断这一级联反应的任一环节都可以有效地缓解CVS,同时改善海马神经细胞的代谢功能。
[Abstract]:In recent years, although the development of neuroimaging technology has provided a reliable basis for the diagnosis of cerebrovascular disease, it has also improved the understanding of cerebral vascular disease in children. However, the basic and clinical study of cerebral vascular disease in children is still less. Subarachnoid hemorrhage (SAH) is a common hemorrhagic cerebrovascular disease. The disease is mainly seen in arteriovenous malformations and ruptured intracranial aneurysms in childhood.
After the occurrence of SAH, its metabolites can easily induce cerebral vasospasm (cerebral vasospasm, CVS), which is characterized by varying degrees of stenosis of cerebral blood vessels, causing changes in cerebral blood flow and causing ischemic changes in the brain. The severe stenosis will cause brain metabolism, function change, and irreversibly change, thus secondary ischemic brain injury and clinical treatment. Treatment brings great difficulties and poses a great threat to the life of children. Because of the inability to completely remove the accumulation of blood in the brain pool after SAH, there is a incidence of CVS after the possible.SAH induced by CVS. The pathogenesis and occurrence of CVS after SAH, the development process and the molecular mechanism that lead to ischemic brain damage are helpful to the fundamental prevention. After treatment of SAH, ischemic brain damage can be reduced to reduce the mortality or disability rate of SAH.
At present, the mechanism of the occurrence of CVS is still not very clear. In recent years, the immune and inflammatory reactions that have attracted much attention have been considered to play an important role in the pathogenesis of CVS. The theory of the inflammatory reaction secondary to the blood vessels of spastic vessels and a series of intercellular signal cascade reactions to CVS is being paid more and more attention.
The occurrence of inflammatory reactions is necessarily an aggregation of inflammatory factors. The role of the intercellular adhesion molecule -1 (Intercellular adhesion molecule-1, ICAM-1) is to mediate the adhesion and migration of leukocytes in the spasmodic vascular walls, thus triggering the inflammatory reaction of the vascular walls. The inflammatory reaction can activate the nuclear factor - kappa B (Nucle) as a gene regulatory protein. Ar transcription factor- kappa B, NF- kappa B) regulates the transcriptional activity of ICAM-1. Mitogen activated protein kinase (Mitogen-activated protein kinase, MAPK) is a cell messenger, which transfers cell signals from the cell surface to the nucleus and plays a role by regulating nuclear transcriptional activity. Mitogen activated protein kinase p38. The togen-activated protein kinase, p38MAPK) pathway plays an important role in the MAPK pathway. The interaction between the p38MAPK signal transduction pathway and the NF- kappa B pathway is not one of the previous studies.
At present, there are separate reports on the above three factors, but there is no report on the mutual relationship between the three factors in cerebrovascular disease and the previous experimental study in adult rabbits. There is no report on the above study in the young rabbits. This experiment attempts to make the young rabbit S through the two blood injection of the pillow big pool. After AH CVS model, the expression of p38MAPK, NF- kappa B, ICAM-1 three together in the spasmodic vascular wall and hippocampal neurons, their relationship with the occurrence and development of CVS, and the relationship between them and the metabolic changes of hippocampal neurons were investigated.
Part one: the expression of p38MAPK, NF- B and ICAM-1 in basilar artery and hippocampus after secondary CVS in young rabbits SAH.
objective
The mitogen activated protein kinase p38 (p38Mitogen-activated kinase protein, P38MAPK), nuclear factor kappa B (Nuclear transcription factor-KB, NF- kappa B) were studied. After L vasospasm (CVS), the expression changes in basilar artery and hippocampus and the metabolism of hippocampal neurons after CVS were observed.
Method
After two injections of autologous arterial blood into the occipital large cistern of the young rabbit, the CVS model of the young rabbit was made after SAH. After SAH, the basilar artery, the hippocampus p38MAPK, the NF- kappa B, the ICAM-1 expression were detected, and the changes of the hippocampus nerve cell metabolism were detected at the same time.
Result
Cerebral angiography showed that the basilar artery in the control group and the normal saline (NS) group was smooth and straight, without obvious narrowing. The degree of 1-7d stenosis was gradually aggravated in the.SAH group, the thickening of the tube wall, the swelling of the endothelial cells, the contraction of the inner wall, the enlargement of the middle smooth muscle cells and the distortion, the most serious 7d stenosis, the gradual relief of 9D and 11d basic. The expression of p38MAPK, NF- kappa B, IC AM-1 in the basilar artery and hippocampus also changed.SAH 1-7d, and the expression of ICAM-1 increased gradually with the difference of the degree of spasticity, and the expression of ICAM-1 increased gradually after.SAH. The expression of APK was more strongly.P38MAPK, NF- kappa B, and the expression of ICAM-1 was strong and weak at times of.SAH, and the content of peroxidase (Superoxide Dismutase, SOD) in hippocampal neurons was gradually decreasing with the aggravation of spasticity, and the trend of malondialdehyde (Malondialdehyde, MDA) increased gradually. The change of the above changes gradually relieved.
conclusion
After SAH secondary CVS, there may be an immune inflammatory response mediated by P38MAPK, NF- kappa B and ICAM-1 mediated in the basilar and hippocampal neurons. This cascade reaction simultaneously affects the metabolic function of the hippocampal neurons.
The second part is the study of p38MAPK, NF- kappa B and ICAM-1 antagonists in the treatment of CVS.
Objective to determine whether there is an immune inflammatory response mediated by p38MAPK, NF- kappa B and ICAM-1 in the basilar and hippocampal neurons after SAH secondary CVS. How can the development of CVS after this cascade reaction be blocked? How does the metabolic function of the hippocampal neurons change?
Method
After two injections of autologous arterial blood into the occipital large cistern of the young rabbit, the CVS model of the young rabbit was made after SAH, and then the ICAM-1 monoclonal antibody, NF- kappa B antagonist and p38MAPK inhibitor were injected into the large cistern of the occipital 3D, and the arteriography of the vertebral artery was placed in the internal carotid artery at 5D after SAH, and the animals were killed and the specimens were left after the CVS, and P3 was observed. P3 observed P3. The expression of 8MAPK, NF- kappa B, ICAM-1 in the basilar artery and hippocampal neurons was detected, and the metabolism of hippocampal neurons was detected at the same time.
Result
In the basilar artery, after the treatment of p38MAPK inhibitor, the expression of p38MAPK, NF- kappa B, ICAM-1 is mainly confined to the intima and under the intima, and the expression is weak. After the treatment of NF- kappa B antagonist, NF- kappa B, ICAM-1 are weak expression in the intima and intima, while p38MAPK still is in the intima and the middle membrane is expressed. The membrane and middle membrane were expressed, and ICAM-1 was only weakly expressed in the intima and intima. After the treatment of physiological saline, p38MAPK, NF- kappa B and ICAM-1 were strongly expressed in the intima and middle membrane. In the hippocampal neurons, the expression of p38MAPK, NF- kappa B and ICAM-1 were obviously suppressed after the treatment of p38MAPK inhibitors; after the treatment of NF- kappa B, the expression was expressed. In the treatment group of ICAM-1 monoclonal antibody, the expression of ICAM-1 was inhibited and the expression of ICAM-1 was weak, while the expression of p38MAPK, NF- kappa B was not affected, and the expression of ICAM-1 was still positive. After the treatment of physiological saline, p38MAPK, NF- kappa B, ICAM-1 were expressed strongly positive for.P38MA in the hippocampal neurons. In PK, NF- kappa B, ICAM-1 treatment group, the content of SOD in hippocampus tissue was significantly higher than that in control group, MDA content decreased significantly, p38MAPK, NF- kappa B treatment group was more effective.
conclusion
After SAH secondary CVS, there is indeed an immune inflammatory response mediated by p38MAPK, NF- kappa B and ICAM-1 mediated in the basilar and hippocampal neurons, which can effectively alleviate CVS and improve the metabolic function of hippocampal neurons.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R748

【参考文献】