实验性自身免疫性脑脊髓炎模型的建立及Cathepsin C在急、慢性期模型中的表达

发布时间：2018-07-27 16:42

【摘要】：目的：建立少突胶质细胞糖蛋白多肽35-55（myelin oligodendrocyteglycoprotein,MOG35-55)诱导的实验性自身免疫性脑脊髓炎（experimentalautoimmune encephalomyelitis,EAE）小鼠模型，研究cathepsin C在急性和慢性期EAE模型中的表达。方法： 1.EAE小鼠模型的建立:成年C57BL/6雌性小鼠，采用MOG35-55多肽与完全弗氏佐剂（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小鼠作为空白对照组，用不含有MOG多肽的混合乳剂作为实验对照组。注射后观察各组小鼠体重变化，并根据小鼠行为学改变进行临床评分。 2.组织病理学检测：HE染色观察中枢神经系统(central nervous system，CNS）炎症细胞浸润；髓鞘碱性蛋白（myelin basic protein，MBP）免疫组化染色和Black Gold髓鞘染色观察髓鞘脱失情况； Iba-1和髓过氧化物酶（myeloperoxidase，MPO）免疫组化染色分别观察小胶质细胞活化和中性粒细胞浸润情况。 3.Cathepsin C表达及细胞定位：于MOG诱导模型建立后的第21天(急性期)和第41天（慢性期），利用免疫组化染色检测cathepsin C在脑和脊髓中的表达；cathepsin C原位杂交染色后进行MPO和Iba-1免疫组化染色，确定cathepsin C的细胞定位。结果： 1.MOG诱导模型小鼠一般状态及行为学评分 MOG诱导组小鼠于免疫后平均10.3±1.58天开始出现精神萎靡，体重下降，尾部张力消失，继而出现后肢无力、后肢瘫痪等症状。临床症状平均评分为1.7±0.9分，21天症状最为严重，此后症状持续，无明显缓解。实验对照组与空白对照组均未发现体重明显减轻和行为异常。 2.组织病理学检测（1）HE染色显示：急性期和慢性期小鼠脑和脊髓组织中均出现以白质受累为主的神经炎症性病理改变：炎性细胞浸润，扩张的小血管周围形成“袖套”样改变，急性期较慢性期明显。（2）MBP免疫组化与Black Gold的髓鞘染色显示：急性期EAE小鼠脑区未见明显髓鞘脱失，脊髓区域出现片状髓鞘脱失。慢性期EAE小鼠的脑和脊髓均有脱髓鞘改变。（3）MPO和Iba-1免疫组化染色显示：在脑和脊髓组织的炎性浸润及髓鞘脱失区域周围，可见中性粒细胞浸润及小胶质细胞的集聚与活化。实验对照组和空白对照组没有发现中性粒细胞浸润和小胶质细胞的改变。 3.Cathepsin C表达及细胞定位：实验对照组和空白对照组小鼠cathepsin C的表达仅限于海马CAII区神经元，其他脑区未发现阳性表达。在EAE模型中，cathepsin C在脑和脊髓组织中髓鞘脱失区域有明显阳性表达。cathepsin C原位杂交后MPO和Iba-1的免疫组化双染显示：cathepsin C在急性EAE模型脑和脊髓的小胶质细胞和中性粒细胞均有表达，，在慢性期主要由中性粒细胞表达。结论： 1.本研究MOG35-55诱导小鼠EAE模型成功，中枢神经系统神经炎症和髓鞘脱失呈持续性，无明显缓解期。 2.Cathepsin C在急、慢性期EAE模型中表达增强，可能参与CNS炎症反应及髓鞘脱失。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R-332;R744

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