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研究生:陳佑安
研究生(外文):Yu-An Chen
論文名稱:探討HCN-4在妥瑞氏症病理機轉中扮演的角色
論文名稱(外文):A study of the role of HCN-4 in the pathogenesis of Tourette’s syndrome
指導教授:徐佳福葉啟斌葉啟斌引用關係
指導教授(外文):Jia-Fwu ShyuChin-Bin Yeh
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:49
中文關鍵詞:妥瑞氏症
外文關鍵詞:HCN-4Tourette syndrome
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中 文 摘 要
妥瑞氏症是一種幼兒時期好發的神經精神疾病。臨床症狀主要是患者會出現非自主的動作或聲語型的抽搐(Tic)。Tic主要是一種快速、短暫、突然、重複、不規律、無意義、無法預測、無節奏、呆板的動作或聲語。就目前為止,妥瑞氏症的致病機轉仍然不十分清楚;目前有證據指出,妥瑞氏症似乎是在患者的基底核以及大腦皮質-紋狀體-丘腦-紋狀體的迴路(cortico -striato -thalamo -cortical circuits)出現了功能上的缺損。有許多的研究都曾報告過,在妥瑞氏症及其他的類似性疾病,如薛登漢氏舞蹈症(Sydenham chorea),患者的血清中存在著抗神經元抗體,這種抗體的存在可能導致神經元功能的缺損。本實驗室之前的研究已經證實了,妥瑞症的患者血清中的確存在著這種抗神經元抗體,這抗體會與老鼠腦部組織中的蛋白質有免疫結合反應,在之前的實驗中利用免疫沉澱法結合西方點墨法,發現特異性蛋白質結合出現在分子量約120至130 KD的位置,進一步利用質譜儀進一步分析這個特殊的蛋白質後,認為此蛋白質最有可能是 hyperpolarization –activated cyclic nucleotide channel 4 (HCN-4)。本次實驗是利用免疫沈澱法結合西方點墨法確認妥瑞氏症病人的血清確實是與鼠腦組織有特異性的結合;利用免疫沈澱法結合西方點墨法去分析抗HCN-4抗體以及妥瑞氏症患者的血清與鼠腦蛋白質結合;以及利用免疫螢光法觀察妥瑞氏症患者血清與抗HCN-4抗體在GH3細胞株中免疫結和的位置,來證實妥瑞氏症患者血清中存在抗HCN-4的抗體;利用酵素免疫沈澱分析(ELISA)測量妥瑞氏患者血清中的C反應蛋白(C-reactive protein);利用將病人的血清以及抗HCN-4的抗體打入老鼠的鼠腦中,來觀察其行為是否有所改變。由實驗的結果可以得知妥瑞氏症患者的血清中確實存在了抗神經元的抗體,根據我們所得到的結果認為其抗體很可能即為抗HCN-4的抗體。此外利用酵素免疫沈澱分析所得到的結果也確切觀察到妥瑞氏症患者中的發炎指數相較於對照組有明顯上升的狀況,在利用病人的血清打入鼠腦中可以觀察到老鼠出現有出現類似tic的動作。這樣的結果可以證實妥瑞氏症患者確實存在發炎反應與抗神經元的抗體,同時也提供了在對於妥瑞氏症治療和研究的方向的。
Tourette’s syndrome (TS) is a neuropsychiatric disorder characterized by the presence of both motor and phonic tic. Tic is a rapid, sudden, recurrent, non-rhythmic, stereotyped motor movement or vocalization. At present, the pathogenesis of TS is still poorly understood. Furthermore, according to circumstantial evidence from imaging studies stresses the importance of basal ganglia and cortico-striato-thalamo-cortical (CSTC) circuits. Several studies have been reported the anti-neuronal antibodies that exist in sera of TS patients and its similar disorder, Sydenham chorea (SC), may cause neuron dysfunction. We had demonstrated the presence of specific anti-neuronal antibodies which recognized high molecular weight proteins in rat brain tissue. We used immunoprecipitation with Western blot to identify the specific protein, and the result showed the molecular weight of the protein between 120 to 130 KD. The results of mass spectrometry showed the protein was arrowed hyperpolarization – activated UcUyclic UnUucleotide channel 4 (HCN-4). In this study, in order to establish the role of HCN-4 in the pathogenesis of Tourette syndrome, the following experiments were performed: 1) increase the case number, 2) using immunoprecipitation with Western blot analysis to determine the molecular of anti-HCN4 and sera from TS, 3) using immunofluorescence analysis and then determine the labeling location in GH3 cell line, 4) using ELISA method to assay the CRP. The results demonstrated the presence of anti-neuron antibody in the sera of TS, 5) infusing TS sera/anti-HCN4 antibody into stratium then observing the difference of locomotion. The antigens recognized by the anti-neutron antibody showed immunoreactive with anti-HCN4 antibody. Colocalizaion of proteins bind to anti-neuron and anti-HCN4 anti-HCN4 was found. Elevated CRP indicated hyperimmunoreaction in TS patient. After infusing of TS sera/anti-HCN4 antibody, we could observe the presence of tic in the rat. Take together, our result indicated that the protein attacked by antineutron antibody in TS patient could be the HCN-4.
目 錄
目錄………………………………………..……………………Ⅰ
圖目錄………………………………………..…………………Ⅳ
中文摘要………………………………………..………………Ⅵ
英文摘要…………………………………………..……………Ⅷ
第一章:緒言……………………………………………………1
第一節、妥瑞氏症的臨床症狀及診斷:……………………1
第二節、妥瑞氏症的病理生理機轉:………………………3
第三節、妥瑞氏症的流行病學及遺傳學:…………………4
第四節、抗神經元抗體及自體免疫機轉:…………………6
第五節、實驗目的……………………………………………8
第二章、材料與方法……………………………………..…11
第一節、實驗材料……………………………………………11
壹、血清……………………………………………………11
貳、實驗動物………………………………………………11
参、細胞株…………………………………………………11
肆、實驗藥品及試劑…………………………………..…12
第二節、實驗方法…………………………………………..15
壹、血清的分離………………………………………..…15
貳、老鼠腦部組織蛋白質的萃取……………………...…15
参、細胞株蛋白質之萃取…………………………………15
肆、西方點墨法……………………………………………15
伍、免疫螢光化學染色分析………………………………17
陸、反轉錄-聚合酵素連鎖反應 …………………………18
柒、酵素免疫沈澱分析……………………………………19
捌、腦部抗體注射與動作分析……………………………20
第三章、結 果…………………………………………………..22
弟四章、討 論…………………………………………………..26
第五章、結 論…………………………………………………..31
參考文獻…………………………………………………………40
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