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

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張雲婷(Yun-timg Chang) 查詢紙本館藏

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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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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 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

關鍵字(英)

論文目次

中文摘要 i
Abstract ii
目錄 iii
一、緒論 1
I. 肌肉的起源 1
II. MRFs (Muscle Regulatory Factors)家族及MRFs家族對肌肉發展重要性 2
III. MyoD對於肌肉發育的重要性 3
IV.纖維母細胞生長因子(basic fibroblast growth factor, bFGF) 3
V. 肌肉衛星細胞（Satellite cell） 4
VI. Duchenne muscular dystrophy (DMD) 5
VII. PTD (protein transduction domains)或CPPs(cell penetrating peptides) 7
VIII. 細胞膜的短暫通透性 (transient permeabilization and protein treatment) 7
IX. 研究動機與目的 8
二、實驗材料與方法 9
2-1. 細胞株 9
穩定表現細胞株： 9
2-2質體構築 (Cloning) 與轉型作用 (Transformation) 10
2-2-1 質體構築 10
2-2-2 菌株 11
2-2-3 聚合酶鏈鎖反應 (Polymerase Chain Reaction ,PCR) 11
2-2-4 插入 (Insert) DNA的純化 12
2-2-5 載體 DNA的 5’端去磷酸根反應(Calf Intestinal Alkaline Phosphatase, NEB C.I.P)與純化 12
2-2-6 Klenow (DNA polymerase I, Large Fragment, NEB) 12
2-2-7接合反應 (Ligation) 13
2-2-8 大腸桿菌的轉型作用 (Transformation) 13
2-3 RT-PCR 13
2-3-1 Total RNA 製備： 13
2-3-2 反轉錄酶反應 (Reverse Transcriptase, RT) 14
2-4 Real Time PCR 定量實驗 15
2-5 轉染實驗 16
2-5-1 轉染作用 (Transfection) 16
2-5-2 螢火蟲冷光活性方法 (Luciferase Activity Assay) 16
2-5-3 免疫染色 (Immunohistochemistry) 16
2-6 蛋白質純化 17
2-6-1 轉型作用 17
2-6-2 蛋白質表現及純化 17
2-7西方墨點實驗 (Western blot) 20
2-7-1 Total Protein Lysate 的製備 20
2-7-2 SDS-polyacrylamide Gel Electropheresis 20
2-7-3 Blocking 以及 Antibody 辨識 20
2-10-4 蛋白質脫附 (Striping) 21
2-8 細胞膜的短暫通透性 21
2-9 人工染色體純化 22
2-10 Chicken embryo extract 製備 23
三、實驗結果 24
3-1架構與純化帶有蛋白質穿透序列 (cell penetrating peptides, CPPs) 的MyoD重組蛋白 24
3-2-1 Recombination MyoD protein 在C3H10T1/2 cells 中活化MyoD 6.0-promoter-enhancer driven Luciferase reporter 24
3-2-2 Recombination MyoD protein 在C3H10T1/2中活化Myogenin promoter driven Luciferase reporter 25
3-2-3 Recombination MyoD protein 在C2C12中活化Myogenin promoter driven Luciferase reporter 25
3-3 架構與純化纖維母細胞生長因子(basic fibroblast growth factor, bFGF) 26
3-4 確認所純化的bFGF recombination protein是否具有功能 26
3-5 利用SLO作用使C3H10T1/2細胞膜產生短暫通透性 27
3-6 將所純化的cell-penetrating recombinant MyoD (CRP-MyoD) protein 27
3-7 將所純化的cell-penetrating recombinant MyoD (CRP-MyoD) protein直接加入pStable 10T PME Luc+細胞中，並且combined Vitamin C、VPA、bFGF增加reprograming 效率 28
3-8 將10T fibroblast trans-differentiate into myogenic cell進行分化 29
3-9 C3H10T1/2 PyCAGIP eGFP和C2C12 PyCAGIP co-cultured 方式觀察C3H10T1/2 PyCAGIP eGFP處理MyoD protein 後的cell是否具有differentiation成myotube的能力 30
四、實驗討論 31
4-1 重組蛋白的純化條件 31
4-2 SLO在C3H10T1/2 fibroblasts產生通透性 31
4-3 cell-penetrating MyoD protein結合cytokine 、compounds增加 32
4-4 將10T fibroblast trans-differentiate into myogenic cell進一步誘導分化 33
4-5 以co-cultured方式觀察C3H10T1/2 PyCAGIP eGFP處理MyoD protein 後cell是否具有differentiation 成myotube的能力 33
五、參考文獻 35
六、圖表 41
圖一、cell-penetrating recombinant MyoD (CRP-MyoD) protein純化過程 43
圖二、利用在C3H10T1/2 cells 中活化MyoD 6.0-promoter-enhancer driven Luciferase reporter測試所純化CRP-MyoD protein是否具有正常function 44
圖三、利用在C3H10T1/2 cells 中活化Myogenin promoter driven Luciferase reporter測試所純化CRP-MyoD protein是否具有正常function 45
圖四、利用C2C12中活化Myogenin promoter driven Luciferase reporter 測試所純化CRP-MyoD protein是否具有正常function 46
圖五、建構並純化pGEX-4T-bFGF 47
圖六、利用在C3H10T1/2 cells 中活化Nanog promoter-4828~+190 driven Luciferase reporter測試所純化bFGF protein是否具有正常function 48
圖七、trypan blue uptake測試細胞通透性 49
圖八、將純化後的CRP-MyoD protein加入具有通透性的穩定細胞株 51
圖九、觀察處理過SLO細胞株型態的改變 52
圖十、利用RT-PCR的方式，檢測有關myogenesis /satellite cell mRNA表現量變化 53
圖十一、利用pStable 10T PME Luc+ 細胞株檢測哪個組合可以activate promoter的活性較高 54
圖十二、利用RT-PCR的方式探討CRP-MyoD protein combined cytokine、compounds對於有關myogenesis /satellite cell mRNA表現量變化和分化情形及其MyoD mRNA表現量和C2C12 myoblast cell之間的差異 56
圖十三、利用RT-PCR的方式探討Mdx tail tip fibroblast 處理CRP-MyoD protein對於有關myogenesis /satellite cell mRNA表現量變化 57
圖十四、利用RT-PCR的方式探討在沒有MyoD protein存在下myogenic cell可持續表現MyoD多久和MyoD protein花多少時間進入cell中以及collagen coating plate有無幫助myoblast cell 的貼附能力 59
圖十五、 C3H10T1/2 PyCAGIP eGFP和C2C12 PyCAGIP co-cultured 方式觀察C3H10T1/2 PyCAGIP eGFP處理MyoD protein 後的cell是否具有differentiation成myotube能力 61
附錄一 62
質體構築 62
附錄二 63
Primer 對照表 63
附錄三 65
溶液及試劑配方 65
縮寫與全名對照表-藥品及材料 68

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指導教授

陳盛良(Shen-liang Chen)

審核日期

2013-9-27

推文