利用可穿越細胞膜的MyoD重組蛋白將體細胞重新編寫成肌肉前驅細胞以治療杜顯氏肌肉萎縮症; Using cell-penetrating recombinant MyoD protein to reprogram somatic cells into myogenic progenitor cells for treating Duchenne Muscular Dystrophy

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Title:	利用可穿越細胞膜的MyoD重組蛋白將體細胞重新編寫成肌肉前驅細胞以治療杜顯氏肌肉萎縮症;Using cell-penetrating recombinant MyoD protein to reprogram somatic cells into myogenic progenitor cells for treating Duchenne Muscular Dystrophy
Authors:	張雲婷;Chang,Yun-timg
Contributors:	生命科學系
Keywords:	可穿越細胞膜的MyoD重組蛋白;杜顯氏肌肉萎縮症;肌肉前驅細胞
Date:	2013-09-27
Issue Date:	2013-11-27 11:22:49 (UTC+8)
Publisher:	國立中央大學
Abstract:	針對杜顯氏肌肉萎縮症Duchenne muscular dystrophy (DMD)疾病延伸出許多治療方式，由於dystrophy基因突變缺陷導致肌肉細胞不能正常產生的Dystrophin蛋白質，Dystrophin缺陷會造成心肌和骨骼肌不穩定，並且大量消耗satellite cell pool，最終導致死亡(Wallace and McNally, 2009b)，從利用病毒(virus)感染細胞有外來的基因序列嵌入到基因體的疑慮到帶有穿膜序列的蛋白質，一直以來成功的效率和安全性問題備受關注；於是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PTD，為了增加成功比率，會搭配Streptolysin O (SLO)技術產生可回復的短暫通透性來誘導出myogenic progenitor cells；利用reporter assay確認所純化的protein具有功能性後，在本實驗中成功將fibroblast轉變成myoblast，成功誘導MyoD mRNA的表現，但搭配SLO似乎對於MyoD protein進入到cell比例沒明顯幫助，此外從mdx mice所取下的tail tip fibroblast一樣能將primary cell誘導走向myogenic lineage，可惜的是停止加MyoD protein會讓cell的myogenic marker下降；為了讓trans-differentiated走向myogenic cell具有增生能力，結合 vitamin C、VPA、bFGF在不同情況下，利用RT-PCR方式檢測myogenic和satellite cell 的marker，意外中發現將myogenic cell養在有collagen I coating plate中，對於keep myogenic cell是有幫助的，進一步確認所誘導的myogenic cell具有分化成myotube能力，將所誘導的myogenic cell和C2C12 co-cultured 成功分化成myotube；未來可以加入CRP-Pax3、CRP-Pax7、CRP-Oct4、CRP-Sox2幫助誘導的myogenic cell更能維持在未分化狀態和具有增生能力。 Duchenne muscular dystrophy (DMD), one of the most prevalent pediatric genetic disorders , is caused by point mutations or deletions in the gene Dystrophin (Dys). Currently, there is no cure for this disease and patients generally pass away at their 2nd to 3rd decade of life due to serious dystrophy of cardiac or diaphragm muscle. We used cell-penetrating peptide-fused MyoD (CRP-MyoD) proteins to reprogram fibroblasts into myoblasts. Current results show that CRP-MyoD is functional and can activate reporters driven by targeted gene promoters. Furthermore, trans-differentiated myotubes are observed and endogeneous MyoD mRNA expression is induced by CRP-MyoD, suggesting the success of this inducing system, Unfortunately, treatment with streptolysin-O (SLO) failed to promote penetration of CRP-MyoD into cells, and MyoD mRNA expression declined over time after reprogrammed cells were cultured in medium without CRP-MyoD. We are combining CRP-MyoD, recombinant basic fibroblast growth factor, and small compounds, such as valproic acid, vitamin C to determine the optimal condition for reprograming fibroblasts into myogenic progenitor cells. In our experiment, myogenic cells cultured on collagen-coated plate maintain myogenic lineage better than those on non-coated plate. Further, induced myogenic cell has the ability to differentiate into myotube；The co-cultured myogenic cell and C2C12 cell successfully differentiated into myotube.In the future, CRP-Pax3, CRP-Pax7 ,CRP-Oct4 and CRP-Sox2 will be included in the induction system to generate satellite cell-like myogenic stem cells for long-term stem cell source.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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