基因轉殖血管螢光斑馬魚蛋白質體學分析

Proteomics analyses of Fli-1 transgenic zebrafish

呂學霖(Syue-Lin Lyu)

指導教授 ： 黃銓珍

國立臺灣大學/生命科學院/生化科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00548

摘要

血管內皮是一層緊密排列在各種血管內壁的表面細胞層，在血管空腔的循環血流與血管壁中形成一個交界面，內皮細胞在血管發育、凝血、免疫系統甚至在動脈硬化與癌症中都扮演著重要的角色。血管的正常發育明顯是胚胎發育的關鍵，然而在成人中血管發育的異常則容易導致疾病的產生，例如: 腫瘤的生長。另一方面，在人類疾病的診斷上生物標誌是一個被普遍運用的工具。Fli-1: EGFP基因轉殖斑馬魚在發育過程中，fli-1啟動子可以驅動EGFP表現在血管組織。在此研究中，我們希望能藉由Fli-1: EGFP基因轉殖斑馬魚胚胎來探索新的血管內皮細胞生物標誌。首先，我們個別收集並分離 4 hpf 與120 hpf 的Fli-1: EGFP基因轉殖斑馬魚胚胎，接著利用流式細胞分選儀 (FACSAria) 分選出帶有綠色螢光的血管內皮細胞。在進行分選前，先將受精後4小時Fli-1: EGFP的胚胎細胞與餵養細胞進行短暫共同培養來增加細胞數量。接著利用SDS-PAGE分離細胞萃取液後將蛋白質片段切下並進行膠內水解後再以 LC/MS/MS 分析，最後利用 MSCOT 資料庫(http://www.matrixscience.com ) 分析所得 MS 數據。血管內皮細胞在兩個不同發育階段中蛋白質型態呈現著不同的分布，比較實驗的數據，我們可以發現一些發育階段專一性蛋白的表現與可能的血管內皮細胞生物標誌，Thrombospondin-4A、4B。更進一步，我們製備針對斑馬魚Thrombospondin-4A、4B的抗體並透過免疫染色分析發現Thrombospondin-4A與已知的血管內皮細胞生物標誌 (VEGFR1、VEGFR2 與 Tie-2) 表達在相同的位置。Thrombospondin-4A具有可以在人類疾病管理上作為臨床診斷與預後生物標誌的潛能。

關鍵字

斑馬魚 ； 血管內皮細胞 ； 生物標誌

並列摘要

The vascular endothelium is a thin layer of cells that lines the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. Vascular endothelial cells play an important role in vascular development, blood coagulation, immune system and even in atherosclerosis and cancer. It has been reported that normal blood vessel formation is critical for embryonic development, whereas the aberrant blood vessel formation in the adult contributes to pathologies such as tumor growth. On the other hand, biomarker is a prevalent tool for early diagnosis of human diseases. Zebrafish fli-1 promoter is able to drive expression of EGFP in all blood vessels throughout embryogenesis. In this study, we intend to identify some novel biomarker of vascular endothelial cells by using Fli-1: EGFP transgenic zebrafish embryos. First, we collected and isolated cells of Fli-1: EGFP transgenic zebrafish embryo at 4hpf and 120hpf and then the EGFP positive vascular endothelial cells were sorted by Flow cytometry (FACSAria). The cells from 4hpf Fli-1 embryos were shortly co-cultured with feeder layer to increase the cell number (for expanding cell population) before sorting process. After resolving the total lysate from the sorted cells by SDS-PAGE, the separated proteins were subjected to trypsin in gel digestion followed by LC/MS/MS analysis. The data of proteomics analysis present the different protein patterns in these two stages. According to these data, we could identify some developmental stage-specific proteins and potential vascular endothelial cell biomarkers, Thrombospondin-4A and 4B. Furthermore, we clone the zebrafish TSP4A and TSP4B for the His-TSP4A and His-TSP4B fusion proteins and generate the antibodies against these antigens. By using immuno-staining, we could find out zebrafish Thrombospondin-4A respectively co-localize with VEGFR1、VEGFR2 and Tie-2. Thrombospondin-4A may be a biomarker for clinical diagnosis or prognosis in human disease management.

並列關鍵字

zebrafish ； vascular endothelial cells ； biomarker

參考文獻

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