實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎模型的建立及MMP-9的表達(dá)
發(fā)布時(shí)間：2019-04-12 09:20
【摘要】：目的 1、建立實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎(EAE)模型。 2、觀察EAE大鼠行為學(xué)及中樞神經(jīng)系統(tǒng)(CNS)病理學(xué)改變,檢測(cè)CNS中基質(zhì)金屬蛋白酶9(MMP-9)的分布與表達(dá)。 3、為EAE、多發(fā)性硬化(MS)的治療提供新的思路。 方法 將雌性Wistar大鼠隨機(jī)分為模型組(EAE組)和對(duì)照組(CFA組)。給予EAE組大鼠四肢足墊皮內(nèi)注射誘導(dǎo)乳劑豚鼠脊髓勻漿-完全福氏佐劑(GPSCH-CFA)0.4ml,同時(shí)左后肢足背皮下注射百日咳疫苗(BPV)0.2ml,制備大鼠EAE模型。給予CFA組注射等量的生理鹽水和完全福氏佐劑(NS+CFA)。對(duì)兩組大鼠分別進(jìn)行神經(jīng)功能評(píng)分,測(cè)量體重變化,并動(dòng)態(tài)觀察EAE組大鼠腦及脊髓組織的病理學(xué)變化及MMP-9的表達(dá)情況。 結(jié)果 1、行為學(xué)變化：EAE組大鼠在注射GPSCH-CFA后8-12天相繼發(fā)病,主要表現(xiàn)為雙后肢無(wú)力、癱瘓,尾部肌張力減低、消失,大鼠以前肢朝前爬行時(shí),劍突以下的身體部分著地,雙后肢癱瘓,拖拉在后,足跖面朝上,部分大鼠伴有大小便失禁的情況。發(fā)病后3-5天臨床表現(xiàn)達(dá)到高峰,嚴(yán)重者出現(xiàn)前肢肌張力降低、四肢癱瘓,處于瀕死狀態(tài),發(fā)病后7天左右EAE癥狀開始恢復(fù),病情恢復(fù)較快。EAE組大鼠體重較CFA組明顯下降,神經(jīng)功能評(píng)分較CFA組明顯增高,最高評(píng)分為5分。CFA組大鼠均未出現(xiàn)EAE癥狀,一般情況同正常動(dòng)物,活動(dòng)、飲食正常,體重逐漸增加。 2、病理學(xué)變化：光鏡下所示,腦、脊髓實(shí)質(zhì)內(nèi)有大量的炎性細(xì)胞浸潤(rùn),主要為淋巴細(xì)胞,以分布在小血管周圍為主,呈典型的“袖套”狀改變,以白質(zhì)和灰白交界處較為明顯。KB染色顯示發(fā)病高峰期腦及脊髓實(shí)質(zhì)內(nèi)小靜脈周圍白質(zhì)出現(xiàn)大片髓鞘脫失,恢復(fù)期可見髓鞘再生現(xiàn)象。Bodian染色顯示整個(gè)病程中軸索保留相對(duì)良好。免疫組化染色顯示EAE組大鼠發(fā)病早期腦、脊髓內(nèi)有大量MMP-9陽(yáng)性細(xì)胞浸潤(rùn),典型的MMP-9陽(yáng)性細(xì)胞胞漿呈棕褐色,主要為淋巴細(xì)胞、單核細(xì)胞,發(fā)病14天后陽(yáng)性細(xì)胞表達(dá)開始減少,1月后未見明顯的MMP-9表達(dá)。電鏡下,EAE組大鼠腦、脊髓實(shí)質(zhì)毛細(xì)血管內(nèi)皮細(xì)胞線粒體腫脹,多見管腔狹窄閉鎖,出現(xiàn)髓樣小體,神經(jīng)細(xì)胞及神經(jīng)膠質(zhì)細(xì)胞內(nèi)內(nèi)質(zhì)網(wǎng)及線粒體腫脹,有髓神經(jīng)纖維軸索內(nèi)微絲微管稀疏,鞘膜扭曲或?qū)用娣蛛x。對(duì)照組未見明顯的組織病理學(xué)改變。 結(jié)論 1、采用豚鼠脊髓勻漿—完全福氏佐劑(GPSCH-CFA),輔以百日咳疫苗(BPV)誘導(dǎo)Wistar大鼠,可以成功制備EAE模型。 2、EAE組大鼠的病理學(xué)改變主要表現(xiàn)為腦脊髓實(shí)質(zhì)內(nèi)小血管周圍大量的炎性細(xì)胞浸潤(rùn),呈典型的“袖套”狀改變,可觀察到白質(zhì)脫髓鞘現(xiàn)象,而軸索保留相對(duì)完好,與人類多發(fā)性硬化(MS)病理學(xué)改變相一致。 3、EAE組大鼠腦脊髓實(shí)質(zhì)內(nèi)MMP-9表達(dá)明顯增加,且與病情嚴(yán)重程度相一致,表明MMP-9在EAE發(fā)病機(jī)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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R744.51;R-332
本文編號(hào)：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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R744.51;R-332
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