【摘要】：目的：建立少突膠質(zhì)細(xì)胞糖蛋白多肽35-55（myelin oligodendrocyteglycoprotein,MOG35-55)誘導(dǎo)的實(shí)驗(yàn)性自身免疫性腦脊髓炎（experimentalautoimmune encephalomyelitis,EAE）小鼠模型，研究cathepsin C在急性和慢性期EAE模型中的表達(dá)。 方法： 1.EAE小鼠模型的建立:成年C57BL/6雌性小鼠，采用MOG35-55多肽與完全弗氏佐劑（Freund’s adjuvant complete，CFA）混合的乳化抗原皮下注射免疫，并腹腔注射百日咳毒素建立EAE模型，正常C57BL/6小鼠作為空白對照組，用不含有MOG多肽的混合乳劑作為實(shí)驗(yàn)對照組。注射后觀察各組小鼠體重變化，并根據(jù)小鼠行為學(xué)改變進(jìn)行臨床評分。 2.組織病理學(xué)檢測：HE染色觀察中樞神經(jīng)系統(tǒng)(central nervous system，CNS）炎癥細(xì)胞浸潤；髓鞘堿性蛋白（myelin basic protein，MBP）免疫組化染色和Black Gold髓鞘染色觀察髓鞘脫失情況； Iba-1和髓過氧化物酶（myeloperoxidase，MPO）免疫組化染色分別觀察小膠質(zhì)細(xì)胞活化和中性粒細(xì)胞浸潤情況。 3.Cathepsin C表達(dá)及細(xì)胞定位：于MOG誘導(dǎo)模型建立后的第21天(急性期)和第41天（慢性期），利用免疫組化染色檢測cathepsin C在腦和脊髓中的表達(dá)；cathepsin C原位雜交染色后進(jìn)行MPO和Iba-1免疫組化染色，確定cathepsin C的細(xì)胞定位。 結(jié)果： 1.MOG誘導(dǎo)模型小鼠一般狀態(tài)及行為學(xué)評分 MOG誘導(dǎo)組小鼠于免疫后平均10.3±1.58天開始出現(xiàn)精神萎靡，體重下降，尾部張力消失，繼而出現(xiàn)后肢無力、后肢癱瘓等癥狀。臨床癥狀平均評分為1.7±0.9分，21天癥狀最為嚴(yán)重，此后癥狀持續(xù)，無明顯緩解。實(shí)驗(yàn)對照組與空白對照組均未發(fā)現(xiàn)體重明顯減輕和行為異常。 2.組織病理學(xué)檢測 （1）HE染色顯示：急性期和慢性期小鼠腦和脊髓組織中均出現(xiàn)以白質(zhì)受累為主的神經(jīng)炎癥性病理改變：炎性細(xì)胞浸潤，擴(kuò)張的小血管周圍形成“袖套”樣改變，急性期較慢性期明顯。 （2）MBP免疫組化與Black Gold的髓鞘染色顯示：急性期EAE小鼠腦區(qū)未見明顯髓鞘脫失，脊髓區(qū)域出現(xiàn)片狀髓鞘脫失。慢性期EAE小鼠的腦和脊髓均有脫髓鞘改變。 （3）MPO和Iba-1免疫組化染色顯示：在腦和脊髓組織的炎性浸潤及髓鞘脫失區(qū)域周圍，可見中性粒細(xì)胞浸潤及小膠質(zhì)細(xì)胞的集聚與活化。實(shí)驗(yàn)對照組和空白對照組沒有發(fā)現(xiàn)中性粒細(xì)胞浸潤和小膠質(zhì)細(xì)胞的改變。 3.Cathepsin C表達(dá)及細(xì)胞定位： 實(shí)驗(yàn)對照組和空白對照組小鼠cathepsin C的表達(dá)僅限于海馬CAII區(qū)神經(jīng)元，其他腦區(qū)未發(fā)現(xiàn)陽性表達(dá)。在EAE模型中，cathepsin C在腦和脊髓組織中髓鞘脫失區(qū)域有明顯陽性表達(dá)。cathepsin C原位雜交后MPO和Iba-1的免疫組化雙染顯示：cathepsin C在急性EAE模型腦和脊髓的小膠質(zhì)細(xì)胞和中性粒細(xì)胞均有表達(dá)，，在慢性期主要由中性粒細(xì)胞表達(dá)。 結(jié)論： 1.本研究MOG35-55誘導(dǎo)小鼠EAE模型成功，中樞神經(jīng)系統(tǒng)神經(jīng)炎癥和髓鞘脫失呈持續(xù)性，無明顯緩解期。 2.Cathepsin C在急、慢性期EAE模型中表達(dá)增強(qiáng)，可能參與CNS炎癥反應(yīng)及髓鞘脫失。
[Abstract]:Objective: to establish a mouse model of experimental autoimmune encephalomyelitis (experimentalautoimmune encephalomyelitis, EAE) induced by oligodendrocyte glycoprotein polypeptide 35-55 (myelin oligodendrocyteglycoprotein, MOG35-55) and to study the expression of cathepsin C in the acute and chronic EAE model.
Method:
The establishment of 1.EAE mouse model: adult C57BL/6 female mice were immunized subcutaneously with MOG35-55 polypeptide and complete Freund's adjuvant (Freund 's adjuvant complete, CFA), and a EAE model was established by intraperitoneal injection of pertussis toxin. Normal C57BL/6 mice were used as blank control group, and mixed emulsion that did not contain MOG polypeptide was used as a mixed emulsion. In the experimental control group, the weight changes of mice in each group were observed after injection, and the clinical scores were scored according to behavioral changes in mice.
2. histopathological examination: HE staining was used to observe the infiltration of central nervous system (CNS); myelin basic protein (myelin basic protein, MBP) and Black Gold myelin staining were used to observe the loss of myelin sheath; Iba-1 and medullary peroxidase immunohistochemical staining was observed respectively. Activation of microglia and infiltration of neutrophils.
3.Cathepsin C expression and cell location: twenty-first days (acute) and forty-first days (chronic phase) after the establishment of MOG induced model, the expression of cathepsin C in the brain and spinal cord was detected by immunohistochemical staining; MPO and Iba-1 immunohistochemical staining was performed after cathepsin C in situ hybridization, and the localization of cathepsin C was determined.
Result:
1. General state and behavioral score of MOG-induced mice
The mice induced by MOG began to be depressed, the weight decline, the tail tension disappearing, and then the weakness of the hind limbs and the paralysis of the hind limbs. The average score of the clinical symptoms was 1.7 + 0.9, and the symptoms were the most serious in 21 days. The symptoms continued and no obvious remission was found. Both the experimental control group and the blank control group had not been found. Significant reduction in weight and abnormal behavior.
2. histopathological detection
(1) HE staining showed that there were inflammatory pathological changes in the brain and spinal cord tissues in both the acute and chronic stage of the brain and spinal cord of mice. Inflammatory cells infiltrated, and the small vessels of the dilated small vessels formed "cuff" like changes. The acute phase was more obvious than the chronic stage.
(2) the immunohistochemical staining of MBP and the myelin staining of Black Gold showed that there was no obvious demyelination in the brain area of the acute phase of EAE mice and the lamellar myelin sheath in the spinal region. The demyelinating changes in the brain and spinal cord of the EAE mice of the chronic stage were all demyelinated.
(3) MPO and Iba-1 immunohistochemical staining showed that neutrophil infiltration and aggregation and activation of microglia were seen around the inflammatory infiltration of brain and spinal cord tissue and the area of myelin loss, and no neutrophilic infiltration and microglia were found in the experimental and blank control groups.
3.Cathepsin C expression and cell location:
The expression of cathepsin C in the experimental control group and the blank control group was limited to the neurons in the hippocampal CAII region, and the other brain regions were not found positive. In the EAE model, cathepsin C had a significant positive expression in the myelinating region in the brain and spinal cord tissues. The immunohistochemical double staining of MPO and Iba-1 after.Cathepsin C in situ hybridization showed that cathepsin C was in the EAE model. In acute EAE model, microglia and neutrophils were expressed in brain and spinal cord, mainly expressed by neutrophils in chronic phase.
Conclusion:
1. in this study, MOG35-55 induced mouse EAE model was successful, and the central nervous system neuroinflammation and myelin demeligmentation were persistent without obvious remission.
2.Cathepsin C expression is enhanced in acute and chronic EAE models, which may be involved in CNS inflammation and myelin depletion.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R-332;R744

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