粒细胞—血管内皮细胞炎性黏附参与自闭症发病机制及药物调控研究

发布时间：2018-03-05 03:39

本文选题：BTBR　切入点：双光子活体成像　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:自闭症是一种复杂的神经系统失调导致的神经发育障碍性疾病,以严重的沟通技能损害、重复刻板行为、兴趣和活动减少为主要特征。近年来自闭症的发病率越来越高,严重影响病患家庭的生活质量。但是目前自闭症的发病机制仍然不明,临床上缺乏有针对性的治疗方法和手段。脑神经炎症反应与中枢神经系统疾病密切相关。许多研究亦表明自闭症患者的脑中存在持续性炎症病变。粒细胞-内皮细胞黏附是炎症的关键步骤之一,参与介导神经血管单元组分的炎性损伤级联反应。研究目的:本课题采用自闭症模型BTBR小鼠,从脑血管粒细胞-内皮细胞黏附相关的炎性信号出发,探寻自闭症中炎症的发生、发展机制,并开展药理学调控研究。研究方法:1)采用双光子活体成像技术检测脑血管内粒细胞-内皮细胞黏附现象;2)流式细胞术考察BTBR小鼠中性粒细胞数量及脑血管内皮细胞黏附因子表达变化;3)免疫印迹、免疫荧光及实时荧光定量PCR考察BTBR小鼠脑内相关炎症因子的表达变化,寻找参与BTBR小鼠脑内炎症反应的关键分子;4)小鼠脑内植入式渗透压泵匀速持续给药验证关键分子的靶标作用及药物对神经炎症的保护作用。研究结果:1)双光子活体成像分析发现BTBR小鼠脑血管内存在粒细胞黏附现象;2)流式细胞术检测发现BTBR小鼠的血液中性粒细胞数量较野生型C57小鼠显著增多,且中性粒细胞黏附因子CD11b、脑血管内皮细胞黏附因子ICAM-1在BTBR小鼠中表达均显著高于野生型C57小鼠;3)免疫印记结果显示,BTBR小鼠大脑皮层炎症因子Caspase-1、IL-1β、CleavedIL-1β的表达均明显上调;4)实时荧光定量PCR考察脑血管内皮细胞多种趋化因子mRNA水平,发现CXCL7的mRNA水平显著升高;5)免疫印迹寻找BTBR小鼠脑内致炎关键分子,发现溶酶体水解酶CathepsinB表达量显著上升;6)使用植入式渗透压泵匀速持续给予CathepsinB活性抑制剂CA-074 Me,发现给药后BTBR小鼠脑内粒细胞-内皮细胞黏附现象减弱,黏附因子表达减少,趋化因子CXCL7的mRNA水平显著降低;7)埋珠实验发现Cathepsin B抑制剂CA-074 Me可有效改善BTBR小鼠低试探性、兴趣减少的自闭症行为。结论:BTBR小鼠脑内存在明显血管炎性反应;血管内皮细胞上的趋化因子CXCL7大量增多,招募粒细胞并促使其与血管内皮细胞发生黏附。BTBR小鼠大脑皮层及血液中性粒细胞均发现CathepsinB的大量表达,给予CathepsinB抑制剂CA-074 Me可显著改善BTBR小鼠脑内血管炎症反应。我们发现BTBR小鼠CathepsinB酶活性异常增加可诱导黏附因子CD11b、ICAM-1表达升高,趋化因子CXCL7水平增高,增加粒细胞-血管内皮细胞黏附。综上所述,药物靶向Cathepsin B酶活性异常及其诱导的粒细胞-血管内皮细胞炎性黏附分子事件可能是治疗自闭症的策略之一。
[Abstract]:Background: autism is a neurodevelopmental disorder caused by complex neurological disorders, with severe communication skills impairment and repetitive stereotyping. The decrease of interest and activity is the main characteristic. The incidence of autism is increasing in recent years, which seriously affects the quality of life of the patient's family. However, the pathogenesis of autism is still unknown. Clinical lack of targeted treatment methods and means. Cerebral neuroinflammation and central nervous system diseases are closely related. Many studies also show that autistic patients have persistent inflammatory lesions in the brain, granulocyte-endothelium. Cell adhesion is one of the key steps in inflammation. Objective: to investigate the pathogenesis of inflammation in autistic BTBR mice from the inflammatory signals associated with adhesion of cerebrovascular granulocyte-endothelial cells. Development mechanisms, Methods: Two-photon imaging technique was used to detect the adhesion of granulocyte to endothelial cells in cerebral vessels. Flow cytometry was used to investigate the number of neutrophils and the fine endothelium of cerebral vessels in BTBR mice. The expression of cell adhesion factor was changed by Western blotting. Immunofluorescence and real-time fluorescence quantitative PCR were used to investigate the expression of related inflammatory factors in the brain of BTBR mice. To find the key molecule involved in BTBR mouse brain inflammatory response) mice brain implanted osmotic pump uniform continuous administration to verify the target role of key molecules and the protective effect of drugs on neuroinflammation. Results: 1) Two-photon activity. The blood neutrophil count of BTBR mice was significantly higher than that of wild-type C57 mice by flow cytometry. The expression of neutrophil adhesion factor CD11b and cerebrovascular endothelial cell adhesion factor ICAM-1 in BTBR mice was significantly higher than that in wild-type C57 mice. 4) Real-time fluorescence quantitative PCR was used to investigate the mRNA level of various chemokines in cerebrovascular endothelial cells. It was found that the level of mRNA in CXCL7 increased significantly (P < 0.05). Western blot was used to search for the key molecules of inflammation in the brain of BTBR mice. It was found that the expression of lysosomal hydrolase (CathepsinB) was significantly increased. (6) continuous administration of CathepsinB activity inhibitor CA-074 Meusing implanted osmotic pump at a constant rate showed that the adhesion of granulocyte-endothelial cells in the brain of BTBR mice was decreased and the expression of adhesion factor was decreased after administration of lysosomal hydrolase. The level of mRNA of chemokine CXCL7 was significantly decreased. The bead burying test showed that CA-074 me, a inhibitor of Cathepsin B, could effectively improve the low exploratory and decreased autism behavior of BTBR mice. Conclusion there is a significant vascular inflammatory reaction in the brain of BTBR mice. The chemokine CXCL7 on vascular endothelial cells increased significantly, and granulocytes were recruited and induced to bind to vascular endothelial cells. The expression of CathepsinB was found in cerebral cortex and neutrophil of mice. CA-074 me, a CathepsinB inhibitor, could significantly improve the vascular inflammation in the brain of BTBR mice. We found that the abnormal increase of CathepsinB activity in BTBR mice induced an increase in the expression of adhesion factor CD11bnICAM-1 and the level of chemokine CXCL7. To increase the adhesion of granulocyte-vascular endothelial cells. In conclusion, the abnormal activity of drug-targeted Cathepsin B and the induced inflammatory adhesion molecule events of granulocyte-vascular endothelial cells may be one of the strategies for the treatment of autism.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.94

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