Taipei Medical University Institutional Repository:Item 987654321/5074
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    Please use this identifier to cite or link to this item: http://libir.tmu.edu.tw/handle/987654321/5074


    Title: 粒線體生合成調控對胰島β-細胞功能的影響
    Effects of mitochondrial biogenesis regulation on pancreatic beta–cell function
    Authors: 蘇湘雯
    Hsiang-Wen Su
    Contributors: 保健營養學研究所
    Keywords: 胰島素
    RNA interference
    Date: 2005
    Issue Date: 2009-09-10 18:03:17 (UTC+8)
    Abstract: 中文摘要

    為了瞭解胰島β-細胞中粒線體生合成對胰島素分泌的機轉，本研究探討粒線體功能異常是否會影響β-細胞胰島素的分泌。本實驗利用大鼠胰島β-細胞，RIN-m5F細胞，運用RNA干擾技術 (RNA interference，RNAi)，以短暫轉染(transient transfection)方式干擾粒線體轉錄因子A mRNA (mitochondrial transcription factor A, TFAM)，以期降低粒線體DNA複製與轉錄，進而影響粒線體呼吸傳遞鏈功能，使ATP生成降低，影響胰島素分泌。經過兩次轉染，以同步定量聚合?鏈鎖反應 (real-time PCR) 進行分析，轉染後第4小時起，開始抑制TFAM mRNA的表現，最高可抑制99 %，在第48 ~ 72小時，也觀察到TFAM mRNA下降53.3 %，並影響粒線體轉錄之ND6 mRNA（NADH-uibiquinone oxidoreductase subunit 6 , ND6）表現量下降，僅剩原本之15 %；同時也觀察到粒線體DNA拷貝數的下降。粒線體轉譯之呼吸傳遞鏈複合體四中次單位COX I (cytochrome c oxidase, subunit I)也顯著降低27 %，可能導致粒線體呼吸傳遞鏈功能缺失，進而使胰島素分泌降低，可用於模擬第二型糖尿病人粒線體功能不全的情形。本研究利用100 ng/mL溴化乙錠 (ethidium bromide, EtBr)破壞RIN-m5F細胞粒線體DNA作為另一研究模式，25天後觀察到粒線體DNA拷貝數僅剩0.13 %。以EtBr處理五個月後，明顯觀察到細胞因粒線體DNA被破壞後，生長速度趨於緩慢，進一步利用葡萄糖、白胺酸(Leucine)、精胺酸(Arginine)來刺激經EtBr處理的RIN-m5F細胞，ATP的生成明顯降低，且胰島素分泌趨於減少，推測應為粒線體功能異常，ATP無法正常生成進而影響胰島素分泌。本研究顯示粒線體DNA套數減少及粒線體基因的表現受到抑制皆會導致粒線體功能異常進而影響β-細胞胰島素分泌情形。
    Abstract

    To examine the impact of mitochondrial dysfunction on insulin secretion of pancreatic b–cell, multiple RIN-m5F cell clones with mitochondrial DNA (mtDNA) depletion by RNA interference (RNAi) or ethidium bromide (EtBr) treatment were created. The mitochondrial transcription factor A (TFAM) were presented to regulate mtDNA replication and transcription process. It could affect the ATP production from respiratory chain reaction, and insulin secretion in the pancreatic b–cell. In this study, related genes mRNA expression and mtDNA copy number were determined by real-time quantitative PCR analysis. The mRNA expression levels of TFAM and mtDNA-encoded NADH-uibiquinone oxidoreductase subunit 6 (ND6) were significantly decreased after transient transfection of siRNA 4 to 72 hours. TFAM gene expression was disrupted up to 99 % and decreased to 53.3 % of TFAM after twice siRNA transfection. Both of mtDNA copy number (10 % of control after 64 h transfection) and mtRNA expression (reduced 85 % of ND6 mRNA) were swayed by defective mitochondrial biogenesis. RIN-m5F cells characterized with slow growth rate, reduced nutrients (glucose, leucine, arginine)-induced insulin secretion and ATP synthesis after EtBr treatment over 5 months. We suggested that both mtDNA depletion and reduced mitochondrial genes expression may compromise mitochondrial function and disrupt insulin secretion by pancreatic b–cell.
    Data Type: thesis
    Appears in Collections:[School of Nutrition and Health Sciences] Dissertations/Theses

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