实验性变态反应性脑脊髓炎模型的建立及MMP-9的表达
发布时间：2019-04-12 09:20
【摘要】：目的 1、建立实验性变态反应性脑脊髓炎(EAE)模型。 2、观察EAE大鼠行为学及中枢神经系统(CNS)病理学改变,检测CNS中基质金属蛋白酶9(MMP-9)的分布与表达。 3、为EAE、多发性硬化(MS)的治疗提供新的思路。 方法 将雌性Wistar大鼠随机分为模型组(EAE组)和对照组(CFA组)。给予EAE组大鼠四肢足垫皮内注射诱导乳剂豚鼠脊髓匀浆-完全福氏佐剂(GPSCH-CFA)0.4ml,同时左后肢足背皮下注射百日咳疫苗(BPV)0.2ml,制备大鼠EAE模型。给予CFA组注射等量的生理盐水和完全福氏佐剂(NS+CFA)。对两组大鼠分别进行神经功能评分,测量体重变化,并动态观察EAE组大鼠脑及脊髓组织的病理学变化及MMP-9的表达情况。 结果 1、行为学变化：EAE组大鼠在注射GPSCH-CFA后8-12天相继发病,主要表现为双后肢无力、瘫痪,尾部肌张力减低、消失,大鼠以前肢朝前爬行时,剑突以下的身体部分着地,双后肢瘫痪,拖拉在后,足跖面朝上,部分大鼠伴有大小便失禁的情况。发病后3-5天临床表现达到高峰,严重者出现前肢肌张力降低、四肢瘫痪,处于濒死状态,发病后7天左右EAE症状开始恢复,病情恢复较快。EAE组大鼠体重较CFA组明显下降,神经功能评分较CFA组明显增高,最高评分为5分。CFA组大鼠均未出现EAE症状,一般情况同正常动物,活动、饮食正常,体重逐渐增加。 2、病理学变化：光镜下所示,脑、脊髓实质内有大量的炎性细胞浸润,主要为淋巴细胞,以分布在小血管周围为主,呈典型的“袖套”状改变,以白质和灰白交界处较为明显。KB染色显示发病高峰期脑及脊髓实质内小静脉周围白质出现大片髓鞘脱失,恢复期可见髓鞘再生现象。Bodian染色显示整个病程中轴索保留相对良好。免疫组化染色显示EAE组大鼠发病早期脑、脊髓内有大量MMP-9阳性细胞浸润,典型的MMP-9阳性细胞胞浆呈棕褐色,主要为淋巴细胞、单核细胞,发病14天后阳性细胞表达开始减少,1月后未见明显的MMP-9表达。电镜下,EAE组大鼠脑、脊髓实质毛细血管内皮细胞线粒体肿胀,多见管腔狭窄闭锁,出现髓样小体,神经细胞及神经胶质细胞内内质网及线粒体肿胀,有髓神经纤维轴索内微丝微管稀疏,鞘膜扭曲或层面分离。对照组未见明显的组织病理学改变。 结论 1、采用豚鼠脊髓匀浆—完全福氏佐剂(GPSCH-CFA),辅以百日咳疫苗(BPV)诱导Wistar大鼠,可以成功制备EAE模型。 2、EAE组大鼠的病理学改变主要表现为脑脊髓实质内小血管周围大量的炎性细胞浸润,呈典型的“袖套”状改变,可观察到白质脱髓鞘现象,而轴索保留相对完好,与人类多发性硬化(MS)病理学改变相一致。 3、EAE组大鼠脑脊髓实质内MMP-9表达明显增加,且与病情严重程度相一致,表明MMP-9在EAE发病机制中起到一定作用。
[Abstract]:Objective 1. To establish (EAE) model of experimental allergic encephalomyelitis. 2. To observe the behavioral changes and the pathological changes of central nervous system (CNS) in EAE rats, and to detect the distribution and expression of matrix metalloproteinase-9 (MMP-9) in CNS. 3, to provide a new idea for the treatment of EAE, multiple sclerosis (MS). Methods female Wistar rats were randomly divided into model group (EAE group) and control group (CFA group). Rats in the EAE group were given intradermal injection of inducible emulsion of guinea pig spinal cord homogenate-complete Freund's adjuvant (GPSCH-CFA) 0.4 ml, and pertussis vaccine (BPV) 0.2 ml was injected subcutaneously into the dorsum of the left hind leg. The model of EAE was made in rats. The CFA group was injected with the same amount of saline and complete Freund's adjuvant (NS CFA). The changes of body weight and pathological changes of brain and spinal cord in EAE group were observed. The expression of MMP-9 in brain and spinal cord of rats in the two groups were measured. Results 1Behavioral changes: 8 days after injection of GPSCH-CFA, the rats in the EAE group developed one after another, the main manifestations were weakness of the hind limbs, paralysis, hypotension of the tail muscle and disappearance, while the forelimbs of the rats crawled forward. The part of the body below the swords touched the ground, paralyzed both hind limbs, dragged behind, with the metatarsal face facing upward, and some rats with urinary incontinence. 3 days after onset, the clinical manifestations reached a peak at 5 days after onset. In severe cases, the tension of forelimb muscle decreased, quadriplegia and near-death. The symptoms of EAE began to recover around 7 days after onset, and the condition recovered quickly. The weight of rats in the EAE group was significantly lower than that in the CFA group. The neurological function score was significantly higher in CFA group than that in CFA group, and the highest score was 5. There were no symptoms of EAE in CFA group, but the normal condition was the same as that of normal animals, activity, normal diet and body weight gain gradually. 2, pathological changes: under light microscope, there are a large number of inflammatory cell infiltration in brain and spinal cord parenchyma, mainly lymphocytes, mainly distributed around the small blood vessels, showing a typical "sleeve"-like changes. KB staining showed a large amount of demyelination of the white matter around the venules of the brain and spinal cord during the peak period of the onset of the disease, especially at the junction of the white matter and the gray-white matter. Myelin sheath regeneration was observed in convalescence. Bodian staining showed that axonal retention was relatively good throughout the course of the disease. Immunohistochemical staining showed that there were a large number of MMP-9 positive cells infiltrating in the spinal cord of rats in the early stage of onset of EAE, and the cytoplasm of typical MMP-9 positive cells was brown, mainly lymphocytes and monocytes. After 14 days of onset, the expression of positive cells began to decrease, but no obvious expression of MMP-9 was observed one month later. Under electron microscope, the mitochondria of capillary endothelial cells in brain and spinal cord parenchyma of rats in EAE group were swollen, the lumen stenosis and atresia, myeloid bodies, endoplasmic reticulum and mitochondria swelling in nerve cells and glial cells were observed. The microtubules in the axons of myelinated nerve fibers are sparse, the sheath is twisted or the plane is separated. No significant histopathological changes were observed in the control group. Conclusion 1. Using guinea pig spinal cord homogenate-complete Freund's adjuvant (GPSCH-CFA) and pertussis vaccine (BPV) to induce Wistar rats, EAE model can be successfully established. 2. 2. In the EAE group, the pathological changes were mainly characterized by the infiltration of inflammatory cells around the small vessels in the parenchyma of the brain and spinal cord, which showed a typical "sleeve" change, and the demyelination of the white matter could be observed, while the axons remained relatively intact. It is consistent with the pathological changes of (MS) in human multiple sclerosis. 3. The expression of MMP-9 in the brain and spinal cord parenchyma of rats in EAE group was significantly increased, which was consistent with the severity of the disease, suggesting that MMP-9 may play a role in the pathogenesis of EAE.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R744.51;R-332
本文编号：2456897
[Abstract]:Objective 1. To establish (EAE) model of experimental allergic encephalomyelitis. 2. To observe the behavioral changes and the pathological changes of central nervous system (CNS) in EAE rats, and to detect the distribution and expression of matrix metalloproteinase-9 (MMP-9) in CNS. 3, to provide a new idea for the treatment of EAE, multiple sclerosis (MS). Methods female Wistar rats were randomly divided into model group (EAE group) and control group (CFA group). Rats in the EAE group were given intradermal injection of inducible emulsion of guinea pig spinal cord homogenate-complete Freund's adjuvant (GPSCH-CFA) 0.4 ml, and pertussis vaccine (BPV) 0.2 ml was injected subcutaneously into the dorsum of the left hind leg. The model of EAE was made in rats. The CFA group was injected with the same amount of saline and complete Freund's adjuvant (NS CFA). The changes of body weight and pathological changes of brain and spinal cord in EAE group were observed. The expression of MMP-9 in brain and spinal cord of rats in the two groups were measured. Results 1Behavioral changes: 8 days after injection of GPSCH-CFA, the rats in the EAE group developed one after another, the main manifestations were weakness of the hind limbs, paralysis, hypotension of the tail muscle and disappearance, while the forelimbs of the rats crawled forward. The part of the body below the swords touched the ground, paralyzed both hind limbs, dragged behind, with the metatarsal face facing upward, and some rats with urinary incontinence. 3 days after onset, the clinical manifestations reached a peak at 5 days after onset. In severe cases, the tension of forelimb muscle decreased, quadriplegia and near-death. The symptoms of EAE began to recover around 7 days after onset, and the condition recovered quickly. The weight of rats in the EAE group was significantly lower than that in the CFA group. The neurological function score was significantly higher in CFA group than that in CFA group, and the highest score was 5. There were no symptoms of EAE in CFA group, but the normal condition was the same as that of normal animals, activity, normal diet and body weight gain gradually. 2, pathological changes: under light microscope, there are a large number of inflammatory cell infiltration in brain and spinal cord parenchyma, mainly lymphocytes, mainly distributed around the small blood vessels, showing a typical "sleeve"-like changes. KB staining showed a large amount of demyelination of the white matter around the venules of the brain and spinal cord during the peak period of the onset of the disease, especially at the junction of the white matter and the gray-white matter. Myelin sheath regeneration was observed in convalescence. Bodian staining showed that axonal retention was relatively good throughout the course of the disease. Immunohistochemical staining showed that there were a large number of MMP-9 positive cells infiltrating in the spinal cord of rats in the early stage of onset of EAE, and the cytoplasm of typical MMP-9 positive cells was brown, mainly lymphocytes and monocytes. After 14 days of onset, the expression of positive cells began to decrease, but no obvious expression of MMP-9 was observed one month later. Under electron microscope, the mitochondria of capillary endothelial cells in brain and spinal cord parenchyma of rats in EAE group were swollen, the lumen stenosis and atresia, myeloid bodies, endoplasmic reticulum and mitochondria swelling in nerve cells and glial cells were observed. The microtubules in the axons of myelinated nerve fibers are sparse, the sheath is twisted or the plane is separated. No significant histopathological changes were observed in the control group. Conclusion 1. Using guinea pig spinal cord homogenate-complete Freund's adjuvant (GPSCH-CFA) and pertussis vaccine (BPV) to induce Wistar rats, EAE model can be successfully established. 2. 2. In the EAE group, the pathological changes were mainly characterized by the infiltration of inflammatory cells around the small vessels in the parenchyma of the brain and spinal cord, which showed a typical "sleeve" change, and the demyelination of the white matter could be observed, while the axons remained relatively intact. It is consistent with the pathological changes of (MS) in human multiple sclerosis. 3. The expression of MMP-9 in the brain and spinal cord parenchyma of rats in EAE group was significantly increased, which was consistent with the severity of the disease, suggesting that MMP-9 may play a role in the pathogenesis of EAE.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R744.51;R-332
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