利用活細胞系統方式探討Bβ2過度表現對細胞影響

Characterization of Bβ2 overexpression live cells

簡君宇(Jiun-Yu Jian)

指導教授 ： 謝秀梅

國立臺灣師範大學/理學院/生命科學系/碩士(2010年)

摘要

目前的研究指出脊髓小腦運動失調症12型(Spinocerebellar ataxia type 12, SCA12) 可能是PPP2R2B基因的5’端未轉譯區域的CAG三核苷酸擴充所致。PPP2R2B基因轉譯出具有腦特異性之Bβ調控次單元，這個Bβ調控次單元為蛋白去磷酸酶2A(PP2A)的其中一單元。由於5’端未轉譯區的不正常擴增，可能使得基因PPP2R2B產生選擇性剪切(alternative splicing)，而轉譯出其中的一個Bβ調節次單元Bβ2。過去的文獻提出Bβ2-PP2A會有粒腺體專一性，使Bβ2-PP2A停留在粒腺體外膜造成類神經細胞株PC-12凋亡。在細胞模式上，我們利用脂質體攜帶Bβ2基因送入神經母細胞瘤細胞株SH-SY5Y，並處理視網酸(retinoic acid)使其分化為似神經的成熟細胞來進行觀察過量Bβ2表現是否位於粒腺體上，及Bβ2過度表現對細胞所造成的氧化壓力。在活細胞系統的長時間連續性觀察中，我們可以發現過度表現Bβ2的SH-SY5Y細胞有較對照組不正常的細胞型態。在了解到Bβ2在細胞中所參與的角色，期能在未來提供一個藥物篩選的平台。

關鍵字

脊髓小腦運動失調症12型(SCA12) ； 粒線體 ； PP2A ； Bβ2過度表現

並列摘要

Spinocerebellar ataxia type 12 (SCA12) is associated with an expansion of a CAG repeats in the 5’region of the gene PPP2R2B, which encodes a brain-specific regulatory subunit (Bβ) of the protein phosphatase 2A (PP2A). One of the splice variants of PPP2R2B, Bβ2, has been reported to target PP2A to mitochondria to promote apoptosis in PC12 cells. In our cell model, we used liposome based transient transfection overexpressing Bβ2. The cellular localization of Bβ2 and oxidative stress stimulation by Bβ2 was characterized in retinoic acid differentiated SH-SY5Y neuroblastoma cell line. These tranfected SH-SY5Y cells were characterized on a time-lapsed live image recorded system. Further analysis using Western blot, and immunocytochemistry will be conducted to study the molecular significance. Hoping this system could provide a neurodegenerative platforms for cell model based pathological analysis and potential compound screening.

並列關鍵字

Spinocerebellar ataxia type 12 (SCA12) ； mitochondria ； PP2A holoenzyme ； Bβ2 overexpression

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