利用帶有穿膜序列的MyoD重組蛋白誘導體細胞表現內生性肌肉基因;Using cell-penetrating recombinant MyoD protein to induce endogenous myogenic genes in somatic cells

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題名:	利用帶有穿膜序列的MyoD重組蛋白誘導體細胞表現內生性肌肉基因;Using cell-penetrating recombinant MyoD protein to induce endogenous myogenic genes in somatic cells
作者:	蘇峻賢;Su, Chun-Hsien
貢獻者:	生命科學系
關鍵詞:	肌肉;發育;肌肉萎縮症;MyoD;DMD;Myogenesis
日期:	2018-01-23
上傳時間:	2018-04-13 10:50:52 (UTC+8)
出版者:	國立中央大學
摘要:	杜顯氏肌肉萎縮症Duchenne muscular dystrophy (DMD)是目前號發率最高的一種肌肉萎縮症，其發生的原因為dystrophy基因突變缺陷導致肌肉細胞不能正常產生的Dystrophin蛋白質進而導致心肌和骨骼肌結構不穩定變得極度容易受損，在修補的過程中耗盡satellite cell pool，最終肌肉被脂肪和結締組織取代導致死亡(Wallace and McNally, 2009)；此病沒有根治的方法，有許多正在進行研究的治療方式，這些方式多需要利用病毒(virus)感染細胞有外來的基因序列嵌入到基因體的疑慮，所以我們利用安全性疑慮較低的蛋白質，將肌肉重要決定因子MyoD基因建構並純化出MyoD protein讓其帶有穿膜序列稱為CPP-MyoD，試圖用此蛋白質將fibroblast cell誘導轉化成myogenic progenitor cells；在利reporter assay確認純化的蛋白質有功能性後我們成功的在C3H10T1/2 cells中誘導內生性的MyoD mRNA並且使之出現細胞型態上的變化和少量的多核細胞，由於並沒有更進一步的分化加上western blot結果顯示沒有內生性MyoD protein的表現，我們利用加入RISC inhibitor aurintricarboxylic acid (ATA)和PKR inhibitor C16來幫助MyoD protein表現，結果並沒有太大的差異，但是在確認加入CPP-MyoD是否會導致內生性MyoD protein表現下降的實驗中發現加入帶有穿膜序列的蛋白質確實使MyoD protein表現下降，由RT-PCR方式檢測發現MyoD mRNA表現也有下降，推測加入的高量蛋白質會造成細胞環境壓力造成後續的分化無法進行，未來可以嘗試立用長期處理低量蛋白質的方式以達到降低環境壓力。;Duchenne muscular dystrophy (DMD) is a kind of muscular dystrophy , which affects about one in 5,000 males at birth. It is caused by mutation in dystrophy gene, patients’ muscle will repeat degeneration and regeneration and finally most of the skeletal muscle is replaced with adipose and fibrotic connective tissue. Currently, there is no cure for this disease. By using virus transfected a muscle specific transcription factor MyoD we can reprogram fibroblast into myogenic cell. Due to the viral vectors used for efficient gene transfection, it is impossible to use this myogenic cell in a clinical application. In our research we tried to use cell-penetrating peptide-fused MyoD ( CPP-MyoD ) protein to reprogram fibroblast into myoblast. We used reporter assay to test whether the CPP-MyoD has function, and found that CPP-MyoD is functional, and we also succeed to induce endogenous MyoD mRNA by treated fibroblast with CPP-MyoD, these results suggest the inducing system is workable. Unfortunately, although we can induce fibroblast’s endogenous MyoD mRNA, the efficiency of the myogenic conversion is still very low, we also found that there was no endogenous MyoD was detected in western blot test. We combine CPP-MyoD, RISC inhibitor aurintricarboxylic acid (ATA) and PKR inhibitor C16 to help fibroblast cell express endogenous MyoD protein, but the result shows that there is no difference. In another experiment, we use 10T sense MyoD cells which always express high level MyoD protein to test whether the level of MyoD protein in 10T sense MyoD cells will decrease after treat with cell-penetrating peptide-fused protein, and we found both of the level MyoD protein and MyoD mRNA become lower, these result shows that adding high concentration protein in medium will cause the increasing of environment stress and make some of the mRNA won’t translate into protein we think this is the reason why there is no endogenous MyoD protein detected in previous experiment.
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