本文選題：幼兔 + 海馬�。� 參考：《山東大學(xué)》2012年博士論文

【摘要】：近年來,雖然神經(jīng)影像技術(shù)的發(fā)展為腦血管病的診斷提供了可靠的依據(jù),同時也提高了人們對兒童腦血管病的認(rèn)識。但目前對兒童腦血管病的基礎(chǔ)與臨床研究仍然較少。蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage, SAH)是常見的出血性腦血管疾病,兒童期主要見于動靜脈畸形和顱內(nèi)動脈瘤破裂。 SAH發(fā)生后,其分解代謝產(chǎn)物極易誘發(fā)腦血管痙攣(cerebral vasospasm,CVS),表現(xiàn)為腦血管不同程度的狹窄,導(dǎo)致腦血流的變化,引發(fā)腦的缺血性改變,狹窄嚴(yán)重者會造成腦的代謝、功能改變,并出現(xiàn)不可逆改變,從而繼發(fā)缺血性腦損傷,為臨床治療帶來極大困難,對患兒生命構(gòu)成極大威脅。由于無法徹底清除SAH后殘存于腦池內(nèi)的積血,這就存在誘發(fā)CVS的可能。SAH后CVS的發(fā)生率在70%左右。研究SAH后CVS的發(fā)病機理和發(fā)生、發(fā)展過程及導(dǎo)致缺血性腦損傷的分子機制,有助于從根本上防治SAH后繼發(fā)缺血性腦損傷,以減少SAH的致死或致殘率。 目前CVS發(fā)生的機制仍然不十分清楚。近年來備受關(guān)注的免疫炎癥反應(yīng)被認(rèn)為在CVS的發(fā)病過程起了重要作用,痙攣血管壁受血管周圍血性液體刺激繼發(fā)的炎癥反應(yīng)及一系列的細胞間信號級聯(lián)反應(yīng)引發(fā)CVS的理論正日益受到人們的重視。 炎癥反應(yīng)的發(fā)生必然有炎性因子的聚集。細胞間粘附分子-1(Intercellular adhesion molecule-1, ICAM-1)的作用就是介導(dǎo)白細胞在痙攣血管壁的粘附和遷移,從而引發(fā)血管壁的炎癥反應(yīng)。炎癥反應(yīng)可以激活作為基因調(diào)控蛋白的核轉(zhuǎn)錄因子-κB (Nuclear transcription factor-κB, NF-κB)而完成對ICAM-1轉(zhuǎn)錄活性的調(diào)控。絲裂原活化蛋白激酶(Mitogen-activated protein kinase, MAPK)是一種細胞信使,它將細胞信號由細胞表面?zhèn)鬟f到細胞核,通過調(diào)控核轉(zhuǎn)錄因子活性發(fā)揮作用。絲裂原活化蛋白激酶p38(p38Mitogen-activated protein kinase, p38MAPK)通路在MAPK傳導(dǎo)通路中起重要作用。對于p38MAPK信號轉(zhuǎn)導(dǎo)通路與NF-κB通路之間的相互關(guān)系、作用,既往研究較少,結(jié)果不一。 目前國內(nèi)外有分別對上述三種因子單獨進行研究的報道,但尚無對三因子在腦血管病相互關(guān)系方面進行共同研究的報道,且以往多是在成年兔進行實驗研究,尚無在幼兔進行上述研究的報道。本實驗試圖通過枕大池二次注血制成幼兔SAH后CVS模型,在此基礎(chǔ)上研究p38MAPK、NF-κB、ICAM-1三者共同在痙攣血管壁和海馬神經(jīng)細胞的表達以及它們與CVS發(fā)生、發(fā)展的關(guān)系,同時探討它們與海馬神經(jīng)細胞代謝變化的關(guān)系。 第一部分幼兔SAH繼發(fā)CVS后p38MAPK、NF-κB、ICAM-1在基底動脈和海馬表達-的研究 目的 研究絲裂原活化蛋白激酶p38(p38Mitogen-activated kinase protein, P38MAPK)、核轉(zhuǎn)錄因子-κB (Nuclear transcription factor-KB, NF-κB)、細胞間粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)在幼兔蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage, SAH)繼發(fā)腦血管痙攣(cerebral vasospasm, CVS)后在基底動脈和海馬的表達變化,以及CVS后海馬神經(jīng)細胞代謝的變化。 方法 通過幼兔寰枕筋膜向枕大池內(nèi)二次注入自體動脈血,制成幼兔SAH后CVS模型。分別在SAH后1、3、5、7、9、11d檢測基底動脈、海馬p38MAPK、 NF-κB、 ICAM-1的表達變化,同時在相同時間段檢測海馬神經(jīng)細胞代謝的變化。 結(jié)果 腦血管造影顯示對照組及生理鹽水(normal saline,NS)組基底動脈光滑、平直,沒有明顯的縮窄性改變。SAH組第1-7d狹窄程度逐漸加重,管壁增厚,內(nèi)皮細胞腫脹,內(nèi)壁皺縮,中層平滑肌細胞增大、扭曲,到第7d狹窄最為嚴(yán)重,第9d逐漸緩解,第11d基本恢復(fù)正常。隨痙攣程度的不同, p38MAPK、NF-κB、IC AM-1在基底動脈及海馬的表達也出現(xiàn)變化。SAH后1-7d, ICAM-1的表達呈逐漸增高的趨勢,第7d時出現(xiàn)強烈表達,與CVS的發(fā)展過程相一致.p38MAPK、NF-κB的表達反應(yīng)出現(xiàn)較早,在第5d出現(xiàn)強烈表達,其中p38MAPK的表達更為強烈。P38MAPK、 NF-κB、 ICAM-1的表達強弱有時相上的先后性。SAH后1-7d,隨痙攣程度的加重,海馬神經(jīng)細胞過氧化物歧化酶(Superoxide Dismutase, SOD)含量總體呈逐漸減少趨勢,而丙二醛(Malondialdehyde, MDA)呈逐漸增多趨勢。之后隨痙攣程度的減輕,上述變化逐漸緩解。 結(jié)論 SAH繼發(fā)CVS后,在基底動脈和海馬神經(jīng)細胞可能存在由P38MAPK、 NF-κB調(diào)控,ICAM-1介導(dǎo)的免疫炎癥反應(yīng)。這一級聯(lián)反應(yīng)同時影響了海馬神經(jīng)細胞的代謝功能。 第二部分p38MAPK、NF-κB、ICAM-1拮抗劑治療CVS的研究 目的確定SAH繼發(fā)CVS后的基底動脈和海馬神經(jīng)細胞是否存在由p38MAPK、 NF-κB調(diào)控,ICAM-1介導(dǎo)的免疫炎癥反應(yīng)。阻斷這一級聯(lián)反應(yīng)后CVS的發(fā)展會出現(xiàn)怎樣的變化?海馬神經(jīng)細胞的代謝功能發(fā)生怎樣的變化? 方法 通過幼兔寰枕筋膜向枕大池內(nèi)二次注入自體動脈血,制成幼兔SAH后CVS模型,之后連續(xù)3d分別向枕大池內(nèi)注入ICAM-1單克隆抗體、NF-κB拮抗劑、p38MAPK抑制劑,并在SAH后第5d行頸內(nèi)動脈置管椎動脈造影,觀察CVS程度后處死動物并留取標(biāo)本,觀察p38MAPK、NF-κB、ICAM-1在基底動脈、海馬神經(jīng)細胞的表達變化,同時在相同時間段檢測海馬神經(jīng)細胞代謝的變化。 結(jié)果 在基底動脈,經(jīng)p38MAPK抑制劑治療后,p38MAPK、NF-κB、ICAM-1的表達主要局限在內(nèi)膜、內(nèi)膜下,表達微弱。經(jīng)NF-κB拮抗劑治療后,NF-κB、 ICAM-1在內(nèi)膜和內(nèi)膜下有微弱表達,而p38MAPK仍然在內(nèi)膜、中膜出現(xiàn)表達。經(jīng)ICAM-1單克隆抗體治療后,p38MAPK、NF-κB在內(nèi)膜、中膜均有表達,ICAM-1僅在內(nèi)膜和內(nèi)膜下有微弱表達。經(jīng)生理鹽水治療后,p38MAPK、NF-κB、 ICAM-1在內(nèi)膜、中膜均有強烈的表達。在海馬神經(jīng)細胞,經(jīng)p38MAPK抑制劑治療后,p38MAPK、NF-κB、ICAM-1表達均明顯抑制；經(jīng)NF-κB拈抗劑治療后,NF-κB、ICAM-1表達均明顯抑制,呈弱陽性,但p38MAPK仍有較強表達；在ICAM-1單克隆抗體治療組,ICAM-1的表達受到抑制,ICAM-1呈微弱表達,而p38MAPK、NF-κB表達并未受到影響,仍然呈陽性表達。經(jīng)生理鹽水治療后,p38MAPK、NF-κB、ICAM-1在海馬神經(jīng)細胞呈強陽性表達。p38MAPK、 NF-κB、ICAM-1治療組海馬腦組織SOD含量較對照組明顯升高,MDA含量明顯降低,p38MAPK、NF-κB治療組效果更為明顯。 結(jié)論 SAH繼發(fā)CVS后,在基底動脈和海馬神經(jīng)細胞確實存在由p38MAPK、 NF-κB調(diào)控,ICAM-1介導(dǎo)的免疫炎癥反應(yīng)。阻斷這一級聯(lián)反應(yīng)的任一環(huán)節(jié)都可以有效地緩解CVS,同時改善海馬神經(jīng)細胞的代謝功能。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R748

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本文編號：2075839