楊西苑 教授臺灣大學：分子與細胞生物學研究所馬瑪宣Ma, Ma-HsuanMa-HsuanMa2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49932中樞神經系統受傷時，會引發一連串的細胞性反應，以進行長達數週的修復程序，一般稱之為膠細胞增殖反應，其中星形膠細胞為最主要之反應細胞類別。Nestin是胚胎時期表現之中間絲蛋白質，主要出現於移行及進行細胞分裂中的細胞，包括神經幹細胞、星形膠細胞的祖元細胞，以及未成熟的肌細胞。近來研究顯示，nestin常用以標定不同時期的分化細胞，以及發育階段的細胞。據前人研究，未成熟的星形膠細胞會表現nestin，隨著星形膠細胞的發育，nestin 的表現量會逐漸下降。在成熟的星形膠細胞中並不會表現nestin，然而，它會重新表現在神經受傷後引發的反應型星形膠細胞中。但引起星形膠細胞再表現nestin的確切因子仍無定論。前人研究指出，凝血酶除了凝血作用之外，亦為一種良好的促細胞分裂素，會引起星形膠細胞增生以及形態改變。因此於受傷反應周圍自血液中釋出之凝血酶，很可能為引起nestin之再表現之反應因子。為了證實凝血酶為促使星形膠細胞再表現nestin的反應因子，本實驗係以初級培養的星形膠細胞為模組，以凝血酶及其促效劑（SFLLRN）、 PAR-1拮抗劑（YFLLRNP）分別刺激之。西方點墨法的結果顯示，凝血酶的確能夠促進反應型星形膠細胞再表現nestin，並且有依劑量增加而增加的效果。其中，以0.5 U/ml凝血酶處理的組別nestin的回復量最高。另外，凝血酶促效劑也能夠使反應型星形膠細胞之nestin再表現，西方點墨法的結果指出，不同濃度的促效劑對於nestin再表現的影響亦有依劑量增加而增加的效果，證實凝血酶確實為促使反應型星形膠細胞再表現nestin的反應因子。然而，凝血酶PAR-1拮抗劑並不能抑制凝血酶的作用，換言之，凝血酶對於反應型星形膠細胞nestin再表現的作用並非透過PAR-1，可能為經由PAR-3或PAR-4。除了上述西方點墨法之外，本研究亦以雙重免疫螢光染色觀察GFAP及nestin在反應型星形膠細胞中之表現。觀察發現，在細胞飢餓兩天後，以凝血酶或其促效劑處理的反應型星形膠細胞nestin表現量有回復。然而，以凝血酶及其PAR-1拮抗劑同時處理的組別，nestin的再表現並無受到抑制。總結免疫螢光染色的結果與西方點墨法的結果，證實凝血酶能夠促進星形膠細胞再表現nestin。由於nestin的再表現為反應型星形膠細胞的特徵之一，是故，由實驗結果可進一步推論，在中樞神經受傷時，凝血酶很可能為引起膠細胞增殖反應之重要反應因子，於影響神經細胞修復及再生之作用上扮演重要的角色。It is well known that traumatic injuries of the CNS induce a gliotic reaction characterized by reactive astrocytes, which are among the most abundant cells in brain and are involved in multiple brain functions. During embryonic development, nestin is present mainly in migrating and mitotic cells, such as neural stem cell, progenitors of astrocytes and immature myocytes. Recently, nestin has been frequently used as a marker of multipotent neuroectodermal precursor cells. It is expressed in a cell-cycle-dependent manner and is down-regulated as neuroepithelial stem cells cease division and differentiate along their respective neural or glial lineages like astrocytes. It has been reported that immature astrocytes exhibit nestin, but not mature astrocytes. However, nestin is re-expressed in reactive asytrocytes induced by CNS injuries. As for the factor stimulating the re-expression of nestin fallowing brain injuries remains unclear. According to the previous studies, thrombin plays essential roles in the coagulation cascade, and also acts as an effective mitogen for primary cultured astrocytes, which leads to cell proliferation and shape alterations. Therefore, we hypothesize that it might be probably thrombin released from blood around injured cells that induces the re-expression of nestin in reactive astrocytes. To examine if thrombin is the potent factor that evokes nestin re-expression, neonatal rat astrocytes in primary cultures were stimulated with thrombin, thrombin agonist（SFLLRN）or PAR-1 antagonist（YFLLRNP）. Western blot results showed that nestin re-expression can be induced by thrombin in a dose-dependent manner and that the maximal effect occurs at the concentration of 0.5 U/ml. Moreover, thrombin agonist mimics the effect of thrombin, and nestin re-expression is also elevated with increasing concentrations of agonist. These results suggest that thrombin induces nestin re-expression in reactive astrocytes. However, it is interesting that thrombin antagonist did not attenuate the thrombin response significantly in this assay. In other words, the effect of thrombin on nestin re-expression in reactive astrocytes might not mediated through the activation of PAR-1, which regulates cell proliferation and shape alterations, but rather mediated via PAR-3 and/or PAR-4.目次 頁數 誌謝…………………………………………………………………………………i 中文摘要……………………………………………………………………………ii 英文摘要……………………………………………………………………………iv 目次…………………………………………………………………………………vi 緒論 一、前言……………………………………………………………………….1 二、星形膠細胞……………………………………………………………….2 三、膠細胞增殖反應………………………………………………………….3 四、反應型星形膠細胞……………………………………………………….4 五、中間絲…………………………………………………………………….6 六、Nestin……………………………………………………………………...8 七、凝血酶為促成反應型星形膠細胞的可能因子………………………….10 八、凝血酶…………………………………………………………………….11 九、凝血酶對於細胞的影響………………………………………………….12 十、凝血酶蛋白結構………………………………………………………….13 十一、凝血酶的受體（Protease-activated receptors, PARs）………………..14 十二、星形膠細胞中所表現的PARs…………………………………………15 十三、PAR-1……………………………………………………………………16 十四、PAR-2……………………………………………………………………17 十五、PAR-3……………………………………………………………………19 十六、PAR-4……………………………………………………………………19 十七、凝血酶受體PARs活化的訊息傳導路徑………………………………20 十八、實驗目的…………………………………………………………………23 實驗材料與方法 一、實驗材料……………………………………………………………………24 二、細胞處理所需藥品…………………………………………………………24 三、Intermediate Filament-enriched Preparation 所需藥品…………………….25 四、西方點墨法所需藥品及抗體……………………………………………….25 五、免疫螢光染色所需抗體…………………………………………………….25 實驗方法 一、星形膠細胞初級細胞培養………………………………………………….27 二、細胞處理…………………………………………………………………….27 三、中間絲製備（IF-enriched preparation）……………………………………28 四、西方點墨法………………………………………………………………….28 五、免疫螢光顯微鏡技術……………………………………………………...29 實驗結果 一、初級培養星形膠細胞之形態觀察…………………………………………...30 二、所培養的反應型星形膠細胞所佔比例……………………………………...30 三、Starvation使星形膠細胞nestin表現下降..………………………………….31 四、不同濃度凝血酶對於星形膠細胞nestin再表現的影響：西方點墨法……31 五、不同濃度凝血酶促效劑對於星形膠細胞nestin再表現的影響 ：西方點墨法………………………………………………………………..32 六、不同濃度凝血酶 PAR-1 結抗劑對於星形膠細胞nestin再表現的影響 ：西方點墨法………………………………………..………………………..32 七、凝血酶處理的星形膠細胞之nestin表現情形：免疫螢光染色…………….33 八、凝血酶促效劑處理的星形膠細胞之nestin表現情形：免疫螢光染色…….34 九、凝血酶結抗劑處理的星形膠細胞之nestin表現情形：免疫螢光染色…….34 討論 一、凝血酶對於反應型星形膠細胞nestin再表現的影響……………………….36 二、凝血酶的非凝血生理作用…………………………………………….……38 三、以凝血酶刺激反應型星形膠細胞再表現nestin的可能原因……………….39 四、反應型星形膠細胞再表現nestin之可能功能……………………………….41 五、以凝血酶刺激反應型星形膠細胞再表現nestin之訊息傳導路徑 與PARs的關係………………………………………………………...……43 參考資料……………………………………………………………………………..47 附圖…………………………………………………………………………………...61851623 bytesapplication/pdfen-US反應型星形膠細胞中間絲星形膠細胞凝血酶nestinthrombinGFAPastrocytesreactive astrocytesPARsotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49932/1/ntu-94-R90225013-1.pdf