神經膠纖維酸性蛋白在C6細胞株過量表達之研究

Abstract

在神經膠細胞發育的過程中，有許多中間絲蛋白的表現已經被確認，包括nestin、vimentin以及glial fibrillary acidic protein (GFAP)。而GFAP則是成熟的星狀膠細胞中最主要的中間絲蛋白。在本研究中，主要探討GFAP在神經膠細胞中的型態排列，因此我們將大鼠的GFAP互補核酸序列(cDNA)在其前端接上綠色螢光蛋白的互補核酸序列，再轉殖進入大鼠神經膠細胞瘤C6細胞株中表現。在經過neomycin的相似藥物G418的篩選之後，我們建立了兩株穩定表達GFAP前端接有綠色螢光之融合蛋白(EGFP-GFAP)的細胞株，並命名為C6-EGFP-GFAP細胞。本實驗中先利用熱休克對於C6-EGFP-GFAP細胞進行處理，之後再利用免疫染色、穿透式電子顯微鏡術以及西方點墨法進行進一步的分析。 在暫時性的轉殖實驗中，EGFP-GFAP在轉殖後不久即會形成小型點狀聚集散佈於細胞質中，經過一段時間這些具有綠色螢光的蛋白會逐漸形成絲狀的結構。根據西方點墨法之實驗結果，nestin在C6-EGFP-GFAP細胞中的表現量與在穩定表達只有綠色螢光蛋白的C6-EGFP細胞，以及無轉殖的C6控制組細胞中相比，並沒有明顯的差異。但是vimentin的蛋白質表現量在C6-EGFP-GFAP細胞中卻有降低的情形。有趣的是，小熱休克蛋白αB-crystallin之蛋白質表現量在C6-EGFP-GFAP細胞中確有明顯增加的趨勢。另外，從細胞免疫染色的結果看來，C6-EGFP-GFAP細胞中的GFAP主要是分佈成分散而帶有細微的絲狀結構。 而在以熱休克處理之後，C6-EGFP-GFAP細胞中的GFAP會形成束狀的中間絲纖維結構；同時αB-crystallin蛋白也會和這些束狀的中間絲纖維呈現共同分佈的情況。 在C6細胞中的轉殖實驗看來，我們可以推測在神經膠細胞中GFAP的排列方式是動態的，而且可能被許多不同的機制所調控著。而由熱休克引發的GFAP重新排列之結果推論，小熱休克蛋白aB-crystallin在調控GFAP在細胞中的架構與排列扮演了重要的角色。

Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). Yet GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, the rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into rat C6 glioma cell line. After selection of neomycin analogue G418, two stable C6-EGFP-GFAP cell lines were established under. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually the filamentous structure of EGFP-GFAP was observed. Comparing the C6-EGFP-GFAP stable clone with pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, the protein level of nestin in C6-EGFP-GFAP was similar to others; where as the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that the distribution of GFAP was dispersed, as a pattern of little fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells significantly. Meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. From our observations in this study, it could be suggested that the organization of GFAP in glial cells was dynamic and regulated by several different mechanisms. The heat-induced GFAP reorganization we found suggested that small heat shock protein aB-crystallin may play a functional role to regulate the cytoarchitecture of GFAP.