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

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研究生：	蘇峻賢 Chun-Hsien Su
論文名稱：	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
指導教授：	陳盛良 Shen-Liang Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	75
中文關鍵詞：	肌肉、發育、肌肉萎縮症
外文關鍵詞：	MyoD, DMD, Myogenesis
相關次數：	點閱：15 下載：0
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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.

目錄 
目錄 ........................................................................................................................................... i 
中文摘要.................................................................................................................................... v
Abstract.................................................................................................................................... vi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 .......................................................................................... 2 
IV. 肌肉衛星細胞（Satellite cell）........................................................................................... 3 
V. Duchenne muscular dystrophy (DMD) ................................................................................. 3 
VI. PTD (protein transduction domains)或CPPs(cell penetrating peptides).............................. 4 
VII. 研究動機與目的 ............................................................................................................. 5 
二、實驗材料與方法 ................................................................................................................ 6 
2-1. 細胞株 .............................................................................................................................. 6 
穩定表現細胞株： .................................................................................................................... 6 
2-2質體構築 (Cloning) 與轉型作用 (Transformation) ......................................................... 6 
2-2-1 質體構築 ....................................................................................................................... 6 
2-2-2 菌株 ............................................................................................................................... 7 
2-2-3 聚合酶鏈鎖反應 (Polymerase Chain Reaction ,PCR) .................................................. 7 
2-2-4 插入 (Insert) DNA的純化 ............................................................................................ 8 
2-2-5 載體 DNA的限制酶修飾與 5’端去磷酸根反應(Calf Intestinal Alkaline Phosphatase, NEB C.I.P)與純化...................................................................................................................... 8 
2-2-6接合反應 (Ligation) ........................................................................................................ 8
2-2-7 大腸桿菌的轉型作用 (Transformation) ....................................................................... 9 
2-3 RT-PCR ............................................................................................................................... 9 
2-3-1 Total RNA 製備：............................................................................................................ 9 
2-3-2 反轉錄酶反應 (Reverse Transcriptase, RT) .................................................................. 9 
2-4 Real Time PCR 定量實驗................................................................................................. 11 
2-5 轉染實驗 ......................................................................................................................... 11 
2-5-1 轉染作用 (Transfection) .............................................................................................. 11 
2-5-2 螢火蟲冷光活性方法 (Luciferase Activity Assay) ..................................................... 12 
2-6 蛋白質純化 ..................................................................................................................... 12 
2-6-1 轉型作用 ..................................................................................................................... 12 
2-6-2 蛋白質表現及純化 ..................................................................................................... 12 
2-7 西方墨點實驗 (Western blot) ......................................................................................... 13 
2-7-1 Total Protein Lysate 的製備 ........................................................................................ 13 
2-7-2 SDS-polyacrylamide Gel Electropheresis ...................................................................... 14 
2-7-3 Blocking 以及 Antibody 辨識 ................................................................................... 14 
2-7-4 蛋白質脫附 (Striping) ................................................................................................. 15 
三、實驗結果 .......................................................................................................................... 19 
3-1純化帶有蛋白質穿透序列 (cell penetrating peptides, CPPs) 的MyoD重組蛋白.............................................................................................................................................. 16 
3-2確認所純化的MyoD recombination protein是否具有功能.............................................. 16 
3-2-1 Recombination MyoD protein 在C3H10T1/2 cells 中活化MyoD 6.0-promoter-enhancer driven Luciferase reporter ......................................................................................... 16 
3-2-2 Recombination MyoD protein 在C3H10T1/2 cells 中誘導內生性MyoD mRNA的表現…………………………….................................................................................................. 17
3-3將不同濃度的cell-penetrating recombinant MyoD (CPP-MyoD) protein加入C3H10T1/2細胞中觀察細胞型態之變化.................................................................................................. 17 
3-4將不同濃度的RISC inhibitor aurintricarboxylic acid (ATA)同時加入處理了CPP-MyoD protein的C3H10T1/2細胞中觀察細胞型態之變化............................................................... 18 
3-5將不同濃度的PKR inhibitor C16同時加入處理了CPP-MyoD protein的C3H10T1/2細胞中觀察細胞型態之變化.......................................................................................................... 19 
3-6對10T sense MyoD穩定細胞株處理CPP-MyoD protein和CPP-EGFP protein來確認Tat basic region會影響細胞內MyoD protein的表現.................................................................... 19 
3-7利用不同位置之primer進行PCR以確認細胞處理CPP-MyoD protein後表現之MyoD mRNA是否只是純化protein過程時的plasmid汙染.............................................................. 20 
3-8去除外部加入之CPP-MyoD以確認細胞內生性MyoD protein是否有表現………………………………………………………………………………………..….... 21
3-9以長時間處理低濃度CPP-MyoD取代一次性高濃度的處理以降低細胞環境壓力看是否有助於C3H10T1/2細胞進一步分化………………...……………………………..…….. 21
3-10架構並純化另一個重要的肌肉決定因子Myf5 protein.................................................. 22
3-11架構pStable-Dystrophin promoter並以此測試CPP-MyoD是否具有活化Dystrophin promoter之能力....................................................................................................................... 23 
四、 實驗討論 ........................................................................................................................ 24 
4-1重組蛋白的純化條件......................................................................................................... 24 
4-2CPP-MyoD結合不同的抑制劑是否可以幫助C3H10T1/2細胞進一步分化.............................................................................................................................................. 24 
4-3長時間處理低濃度CPP-MyoD取代一次性高濃度的處理以降低細胞環境壓力以達到幫助C3H10T1/2進行分化……………………………………………………………........... 25 
五、參考文獻 .......................................................................................................................... 26 
六、圖表 .................................................................................................................................. 29 
圖一、cell-penetrating recombinant MyoD (CPP-MyoD) protein純化過程............................ 29 
圖二、利用在C3H10T1/2 cells中活化MyoD 6.0-promoter-enhancer driven Luciferase reporter測試所純化CPP-MyoD protein是否具有正常function…………………………………...... 31 
圖三、利用對C3H10T1/2 cells處理CPP-MyoD protein測試所純化CPP-MyoD protein是否能進入並誘導出C3H10T1/2 cells的MyoD mRNA................................................................ 32 
圖四、將不同濃度的CPP-MyoD protein加入C3H10T1/2細胞中觀察細胞型態之變化……………………………………………………………..…………………………..….. 34 
圖五、將不同濃度的RISC inhibitor aurintricarboxylic acid (ATA)同時加入處理了CPP-MyoD protein的C3H10T1/2細胞中觀察細胞型態之變化.................................................... 36 
圖六、將不同濃度的PKR inhibitor C16同時加入處理了CPP-MyoD protein的C3H10T1/2細胞中觀察細胞型態之變化.................................................................................................. 39
圖七、對10T sense MyoD穩定細胞株處理CPP-MyoD protein和CPP-EGFP protein來確認Tat basic region會影響細胞內MyoD protein的表現.............................................................. 42 
圖八、利用不同位置之primer進行PCR以確認細胞處理CPP-MyoD protein後表現之MyoD mRNA是否只是純化protein過程時的plasmid汙染.............................................................. 45 
圖九、長時間處理低濃度CPP-MyoD取代一次性高濃度的處理以降低細胞環境壓力觀察C3H10T1/2細胞是否進一步分化........................................................................................... 47 
圖十、建構並純化pET32a-Myf5-LPRH................................................................................. 50 
圖十一、利用在C3H10T1/2 cells中測試CPP-MyoD protein是否具有活化Dystrophin promoter -3000~+513 driven Luciferase reporter的能力......................................................... 52  
附錄一 .................................................................................................................................... 54 
質體構築.................................................................................................................................. 54 
附錄二 .................................................................................................................................... 55 
Primer 對照表 ...................................................................................................................... 55 
附錄三 .................................................................................................................................... 56 
溶液及試劑配方 .................................................................................................................... 56 
附錄四 .................................................................................................................................... 60

                                

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