本文選題：BTBR　切入點(diǎn)：雙光子活體成像　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:自閉癥是一種復(fù)雜的神經(jīng)系統(tǒng)失調(diào)導(dǎo)致的神經(jīng)發(fā)育障礙性疾病,以嚴(yán)重的溝通技能損害、重復(fù)刻板行為、興趣和活動減少為主要特征。近年來自閉癥的發(fā)病率越來越高,嚴(yán)重影響病患家庭的生活質(zhì)量。但是目前自閉癥的發(fā)病機(jī)制仍然不明,臨床上缺乏有針對性的治療方法和手段。腦神經(jīng)炎癥反應(yīng)與中樞神經(jīng)系統(tǒng)疾病密切相關(guān)。許多研究亦表明自閉癥患者的腦中存在持續(xù)性炎癥病變。粒細(xì)胞-內(nèi)皮細(xì)胞黏附是炎癥的關(guān)鍵步驟之一,參與介導(dǎo)神經(jīng)血管單元組分的炎性損傷級聯(lián)反應(yīng)。研究目的:本課題采用自閉癥模型BTBR小鼠,從腦血管粒細(xì)胞-內(nèi)皮細(xì)胞黏附相關(guān)的炎性信號出發(fā),探尋自閉癥中炎癥的發(fā)生、發(fā)展機(jī)制,并開展藥理學(xué)調(diào)控研究。研究方法:1)采用雙光子活體成像技術(shù)檢測腦血管內(nèi)粒細(xì)胞-內(nèi)皮細(xì)胞黏附現(xiàn)象;2)流式細(xì)胞術(shù)考察BTBR小鼠中性粒細(xì)胞數(shù)量及腦血管內(nèi)皮細(xì)胞黏附因子表達(dá)變化;3)免疫印跡、免疫熒光及實(shí)時熒光定量PCR考察BTBR小鼠腦內(nèi)相關(guān)炎癥因子的表達(dá)變化,尋找參與BTBR小鼠腦內(nèi)炎癥反應(yīng)的關(guān)鍵分子;4)小鼠腦內(nèi)植入式滲透壓泵勻速持續(xù)給藥驗證關(guān)鍵分子的靶標(biāo)作用及藥物對神經(jīng)炎癥的保護(hù)作用。研究結(jié)果:1)雙光子活體成像分析發(fā)現(xiàn)BTBR小鼠腦血管內(nèi)存在粒細(xì)胞黏附現(xiàn)象;2)流式細(xì)胞術(shù)檢測發(fā)現(xiàn)BTBR小鼠的血液中性粒細(xì)胞數(shù)量較野生型C57小鼠顯著增多,且中性粒細(xì)胞黏附因子CD11b、腦血管內(nèi)皮細(xì)胞黏附因子ICAM-1在BTBR小鼠中表達(dá)均顯著高于野生型C57小鼠;3)免疫印記結(jié)果顯示,BTBR小鼠大腦皮層炎癥因子Caspase-1、IL-1β、CleavedIL-1β的表達(dá)均明顯上調(diào);4)實(shí)時熒光定量PCR考察腦血管內(nèi)皮細(xì)胞多種趨化因子mRNA水平,發(fā)現(xiàn)CXCL7的mRNA水平顯著升高;5)免疫印跡尋找BTBR小鼠腦內(nèi)致炎關(guān)鍵分子,發(fā)現(xiàn)溶酶體水解酶CathepsinB表達(dá)量顯著上升;6)使用植入式滲透壓泵勻速持續(xù)給予CathepsinB活性抑制劑CA-074 Me,發(fā)現(xiàn)給藥后BTBR小鼠腦內(nèi)粒細(xì)胞-內(nèi)皮細(xì)胞黏附現(xiàn)象減弱,黏附因子表達(dá)減少,趨化因子CXCL7的mRNA水平顯著降低;7)埋珠實(shí)驗發(fā)現(xiàn)Cathepsin B抑制劑CA-074 Me可有效改善BTBR小鼠低試探性、興趣減少的自閉癥行為。結(jié)論:BTBR小鼠腦內(nèi)存在明顯血管炎性反應(yīng);血管內(nèi)皮細(xì)胞上的趨化因子CXCL7大量增多,招募粒細(xì)胞并促使其與血管內(nèi)皮細(xì)胞發(fā)生黏附。BTBR小鼠大腦皮層及血液中性粒細(xì)胞均發(fā)現(xiàn)CathepsinB的大量表達(dá),給予CathepsinB抑制劑CA-074 Me可顯著改善BTBR小鼠腦內(nèi)血管炎癥反應(yīng)。我們發(fā)現(xiàn)BTBR小鼠CathepsinB酶活性異常增加可誘導(dǎo)黏附因子CD11b、ICAM-1表達(dá)升高,趨化因子CXCL7水平增高,增加粒細(xì)胞-血管內(nèi)皮細(xì)胞黏附。綜上所述,藥物靶向Cathepsin B酶活性異常及其誘導(dǎo)的粒細(xì)胞-血管內(nèi)皮細(xì)胞炎性黏附分子事件可能是治療自閉癥的策略之一。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.94

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