楊西苑Yang, Hsi-Yuan臺灣大學:分子與細胞生物學研究所張凱為Chang, Kai-WeiKai-WeiChang2010-06-022018-07-062010-06-022018-07-062008U0001-0907200816572500http://ntur.lib.ntu.edu.tw//handle/246246/184675中樞神經受傷時，會引發一連串的細胞性反應，一般稱之為膠細胞增殖反應，其中星形膠細胞是最主要之反應細胞種類，在這些反應中伴隨著神經膠疤的生成，並且此時的星形膠細胞能再度具有增生能力，此星狀膠細胞被稱之為反應型星形膠細胞。先前的研究指出，在未成熟的星形膠細胞中會表現一種中間絲蛋白nestin，隨著星形膠細胞的發育，nestin的表現量會逐漸下降，最終，成熟的星形膠細胞並不會表現nestin，然而，它會重新表現於神經受傷後引發的反應型星形膠細胞中。於我們的先前研究也指出，反應型星形膠細胞只有在nestin再度表現的情形下才會移動到受傷的區域，近期的研究更指出，nestin具有抗細胞凋亡的功能，這些結果暗示了nestin可能參與CNS受傷產生的反應型星形膠細胞反應，但當前的研究對於nestin的調控機制與因子尚無結論。FGF-2為一常見的促細胞分裂素，在中樞神經系統的發育與生理反應扮演相當重要的角色，其表現於發育中的神經，且具有刺激神經母細胞、神經膠前趨細胞還有平滑肌細胞的增生與分化，而這些地方也正好是nestin表現之處。為此，以C6神經膠瘤細胞作為模型，研究nestin的調控機制，結果指出，在C6神經膠瘤細胞中的nestin會受到FGF-2的刺激而使得表現量提升，且此nestin的表現是經由蛋白質新生作用生成的，更進一步的研究顯示此現象是經由FGFR-Ras-ERK這條訊息傳遞路徑調控。本結果對nestin在反應型星形膠細胞之調控提供的新的見解，並且提供了nestin在膠細胞疤生成之功能研究的基礎。CNS traumatic injuries induce a specific gliotic reaction in which astrocytes are involved. In the process of gliotic scar formation, astrocytes regain the ability of proliferation and are called reactive astrocytes. Previous studies have demonstrated that nestin, an intermediate filament proteins, is a differentiation antigen that is presented in radial glia/immature astrocytes and is re-expressed by reactive astrocytes. Our previous study has also shown that reactive astrocytes migrate to the wound area only after the re-expression of nestin. In addition, recent report showed that nestin functions as a survival determinant by sequestering Cdk5/p35 complex and prevent Cdk5 induced apoptosis in neural stem cells. These results indicate that nestin may participate in the pathological responses of reactive astrocytes upon CNS injury. Although substantial researches have been focused on the cellular characteristics of reactive astrocytes, little is known about the factors that induce and regulate the re-expression of nestin. Fibroblast growth factor-2 (FGF-2) is an effective mitogen and is expressed in the developing nervous tissue. It stimulates the proliferation and differentiation of neuroblasts, glial precursor cells and smooth muscle cells, in which nestin are expressed. To examine if FGF-2 is a potent factor that evokes the re-expression of nestin, C6 glioma cells were used as the model system. The results showed that nestin expression is regulated by FGF-2 via de novo protein synthesis. Further signaling analyses also revealed that the FGF-2-induced nestin re-expression is mediated through the activation of the FGFR-MAPK-ERK signaling axis. These findings provide new insights into the regulation of nestin in reactive astrocytes and enable the further studies on the function of nestin in glial scar formation.誌謝……………………………………………………………………………………i文摘要……………………………………………………………………………..i i文摘要……………………………………………………………………………...iv 次…………………………………………………………………………………....v 論、前言………………………………………………………………………….1、星形膠細胞………………………………………………………………….2、膠細胞增殖反應、膠細胞疤與反應型星形膠細胞……………………….4、細胞骨骼.…………………………………………………………………...6、中間絲………………………………………………………………………9、Nestin…………………………………………………………………….....11、纖維母細胞生長因子…………………………. ……………………….....13、第二類纖維母細胞生長因子………………………………………………16、纖維母細胞生長因子受體…………………………………………………17、纖維母細胞生長因子的訊息傳遞路徑……………………………………19一、實驗目的……………………………….. ………………………………21驗材料與方法、實驗材料……………………………………………………………………22 二、細胞培養材料………………………………………………………………22 三、電泳及西方墨點法材料……………………………………………………22、抗體……………………………………………... …………………………23、藥品…………………………………………………………………………23驗方法、C6細胞株培養…………………………………………………………. .…24、單一細胞株之純化…………………………………………………. …….24、免疫螢光顯微鏡技術………………………………………………………25、細胞處理………………………………………………………………..….26、蛋白質之粹取……………………………………………………………...26、SDS膠體電泳及西方墨點法……………………………………………..26驗結果、C6神經膠瘤母細胞型態之觀察……………………………………..…...28、單一細胞之選殖:細胞株之純化….……………………………………....28、Starvation對C6-D6 nestin表現之影響..………………………………….29、不同growth factors對nestin表現之影響…………………………..……29、FGF-2引發C6-D6 nestin表現之型態觀察………………………………30、FGF-2對C6-D6 nestin表現之濃度效應…………………………………..30、FGF-2對C6-D6 nestin表現之時間效應……………………..…………...31、FGF-2促進C6-D6 nestin表現之來源…………………………………….31、FGF-2影響C6-D6 nestin表現之受質(Ligand)層級……………………..32、FGF-2影響C6-D6 nestin表現之受體(Receptor)層級……………………32一、FGFR訊息傳遞路徑Ι……………………………………………….…33二、FGFR訊息傳遞路徑ΙΙ…………………………………………….…….33論、C6 之巢蛋白調控………………………………..……………………….35、第二類纖維母細胞生長因子與nestin關係的相關證據…….………….36、第二類纖維母細胞生長因子的來源……………………………………...38、反應型星形膠細胞再表現nestin之可能功能…………………….……...39、未來展望………....……………………………………………...…….......41考資料…………………………………………………………………………...43圖............................................................................................................................53application/pdf1563092 bytesapplication/pdfen-US巢蛋白第二類纖維母細胞生長因子神經膠疤星形膠細胞反應型星形膠細胞nestinfibroblast growth factor-2glial scarastrocytesreactive astrocyteshttp://ntur.lib.ntu.edu.tw/bitstream/246246/184675/1/ntu-97-R95b43031-1.pdf