肌動蛋白微絲(actin filament)為細胞骨架三大組成結構之一，參與了許多細胞活動，例如：細胞遷移、分裂和原質的流動、囊泡運輸、維持細胞結構及肌肉收縮。目前研究中已知微絲和許多微絲結合蛋白(ABP; Actin-binding protein)互相結合共同調節微絲的動態，如組裝及拆解，進而影響細胞的機械性。Palladin是微絲結合蛋白之一，也是一種鷹架蛋白，為Myotilin/Myopalladin/Palladin家族一員。人類Palladin基因稱為PALLD基因，擁有不同的起始位置及選擇性剪切(alternative splicing)的特性，使得人類palladin約有9種不同大小的異構物。目前主要研究的異購物有三種：200 kDa isoform、140 kDa isoform及90 kDa isoform，三種異構物在不同組織中的表現量級分佈情況會有所不同。本篇研究主要探討人類palladin三種異構物在骨骼肌分化過程中基因的調控、表現量的變化及palladin在分化過程蛋白的分佈。我們利用cloning的技術製作出三種異構物個片段的質體，再利用螢火蟲冷光酶系統(luciferase assay)檢測可能的啟動子活性，最後使用PROMO網站搜尋可能參與調控的轉錄因子。發現human palladin 200 kDa isoform轉錄因子調控片段位於第1個外顯子前-1644~-903片段；140 kDa isoform 位於第4個外顯子前-1010~+190片段；90 kDa isoform位於第15個外顯子前-948~-105片段。接著探討PALLD基因在人類骨骼肌(HSkM)分化過程RNA及蛋白質表現變化，我們利用了即時聚合酶連鎖反應(real-time PCR)及西方墨點法(Western blotting)兩種技術，發現90kDa isoform會隨著分化天數增加，表現量先增加在緩慢減少。最後利用免疫螢光染色法來觀察palladin在骨骼肌分化過程中表現位置分佈及與其他蛋白質交互作用的關係，由結果發現actin及α-actinin會與palladin有共區域化情況，與前人研究相符；vinculin會與palladin有部分重疊的情況；其它蛋白如desmin、dystrophin、myosin heavy chain、myosin light chain並無與palladin有共區域化現象。本篇結果得到palladin 90 kDa iosform參與人類骨骼肌分化過程，而詳細調控機制及內容，在未來還須透過更多研究來釐清，建構出更完整的機制。

Palladin is a microfilament associated protein, which belongs to palladin/myotilin/myopalladin family. Palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Previous study indicated palladin is recognized three distinct isoforms, the 200kDa isoform, 140kDa isoform and 90~92 kDa isoform. These three isoforms are generated via alternative splicing and alternative start-site. Then, Palladin is reported to interact with several actin associated proteins and further impact on cell movement and adhesion. With regard to cancer, the presence of an abnormal palladin gene and the over expression of palladin protein in pancreatic or breast cancer cause cytoskeletal changes and contribute to the tumor’s strong invasive and migratory abilities. The main aim in this study is to characterize the promoter region which controls the expression of three palladin isoforms during myogenesis. First, we utilized online database promoter prediction program to search the potential promoter region of palladin, and analysis the promoter core region reporter by luciferase reporter assay and find out the putative transcription factor binding sites. The potential promoter regions located between -1589 and -903 upstream of 200 kDa isoform, and located between -1010 and +190 upstream of 140 kDa isoform, and located between -948 and -105 upstream of 90 kDa isoform. In addition, we investigated endogenous mRNA and protein expression level of three kinds of palladin by real-time PCR and Western blotting. From our results, we found 90 kDa isoform may play an important role during human skeletal myoblast differentiation. Furthermore, we utilized the immunofluoresence staining to study palladin expression pattern. We found palladin may colocalize with actin, α-actinin, nebulin, and partially overlap with vinculin during myogenesis. Taken together, we offered some important information about human PALLD gene expression during myoblasts differentiation. We hope our research will provide more information for physiological development and disease studies in the future.

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