臺灣博碩士論文加值系統

MBNL3屬於Muscleblind-like(MBNL) 基因家族，與神經肌肉疾病-強直性肌肉萎縮症第一型(DM1) 之病理機制有密切關係。哺乳動物Mbnl3主要表現在高度增生的組織並在肌肉分化過程表現量大幅下降。先前實驗室發現表現shMbnl3 RNA的C2C12老鼠肌纖維母細胞(shMbnl3 C2C12)的細胞週期會受到cyclin D1相關路徑調控減緩細胞增生速率下降且提前進入老化。因此本論文第一部分的研究，我進一步探討Mbnl3在shMbnl3 C2C12調控細胞週期的路徑，我發現(1)shMbnl3 C2C12細胞的分化能力明顯受到抑制 (2)Mbnl3藉由調控Sp1表現量進一步影響cyclin D1的轉錄速率 (3)將cyclin D1下游目標基因Rb弱化後，可明顯回復shMbnl3 C2C12的生長速率及減少老化細胞 (4)將老化主要調控分子p16弱化後，亦會回復shMbnl3 C2C12生長速率及老化程度。這些結果顯示Mbnl3會透過Sp1/cyclin D1/Rb途徑調節C2C12的生長及老化，且p16亦可能會去影響cyclin D1下游路徑。第二部分的研究主要針對MBNL3對癌症細胞增生所造成的影響。在MBNL3過度表現的肝癌細胞株(Huh7、HepG2)及MBNL3無過度表現得乳癌細胞株(MCF-7、MDA-MB-231)中，將MBNL3弱化會明顯減緩細胞增生及增加老化比例；而進一步弱化Rb，則會回復增生速率及減少老化。此外，為了證實MBNL3對正常細胞的影響，我將乳房表皮細胞株(MCF-10A)弱化MBNL3，發現亦會減緩細胞增生。以上結果顯示MBNL3對細胞生長是非常重要的，但是MBNL3是否能成為癌症的標靶基因還需更進一步的研究。

Muscleblind-like 3 (MBNL3) belongs to the MBNL family which has been implicated in the pathogenesis of neuromuscular disease myotonic dystrophy. While MBNL3 is barely detectable in adult muscle, it is expressed in proliferative myoblasts and transiently during muscle regeneration. Our previous study found that Mbnl3 knockdown interferes with cell cycle progression and induces senescence through cyclin D1-mediated pathway in C2C12 myoblasts. The first aim of my study is to confirm the effect of Mbnl3 knockdown on C2C12 cells. The result indicated that the effect of shMbnl3 on cyclin D1 expression occurs at the transcriptional level through down-regulation of Sp1 expression. In addition, down-regulation of pRb as well as p16 expression greatly reversed the effect of shMbnl3 on C2C12 cells, and finally Mbnl3 depletion interferes with C2C12 differentiation. These findings provide the first experimental evidence to suggest that Mbnl3 is required for the proliferation and survival of regenerating myoblasts through cyclin D1/pRB pathway. The second aim of my study is to unravel the role of MBNL3 in cancer cells. Previous study showed that MBNL3 was specifically up-regulated in hepatoma cell lines Huh7 and HepG2, and knockdown of MBNL3 severely affected the survival of these cells. In addition, MBNL3 is not overexpression in breast cancer cell lines MCF-7、MDA-MB-231 MBNL3 but knockdown of MBNL3 also severely affects the survival of these cells. Furthermore, down-regulation of pRb expression reversed the effect of Mbnl3 knockdown on hepatoma cells. Finally, non-tumorigenic epithelial cell line MCF-10A depleted with MBNL3 also showed decreased proliferation rate. These results suggest that MBNL3 plays an important role in cell proliferation.

致謝 I
目錄 III
圖表目錄 VI
中文摘要 1
Abstract 3
序論 5
•強直型肌肉萎縮症(myotonic dystrophy；DM) 5
•DM致病分子機制 6
•Muscleblind-like (MBNL)基因家族 8
•肌肉再生(muscle regeneration) 10
•細胞週期 (cell cycle ) 12
•細胞老化 (Cellular senescence) 13
•腫瘤生成(tumorigenesis) 15
材料與方法 17
一、細胞培養(Cell Culture)： 17
二、慢病毒製備(Production of VSVG-pseudotyped lentiviral particles)： 18
三、慢病毒感染(Lentiviral infection)： 19
四、Sp1 3’UTR選殖 (Cloning of Sp1 3’UTR)： 20
五、Sp1 3’UTR活性測試 (Sp1 3’UTR activity analysis)： 21
六、細菌轉型(Transformation)： 22
七、及時定量之反轉錄聚合酵素連鎖反應(Real-time RT-PCR)： 23
八、蛋白質萃取與西方點墨法(Protein extraction and Western blotting)： 24
九、MTT細胞存活檢測（MTT cell viability assay）： 27
十、老化指標-Galactosidase染色 (Senescence-associated -Galactosidase (S.A--gal) staining)： 28
十一、統計分析(Statistical analysis) 28
結果 29
一、Mbnl3透過轉錄機制調控Sp1的基因表現 29
二、Rb參與Mbnl3調控細胞生長之路徑 30
三、p16參與Mbnl3調控細胞生長及老化途徑 32
四、弱化Mbnl3會抑制肌肉細胞分化及再生 33
五、Mbnl3在癌細胞中所扮演的角色 35
六、建立弱化MBNL3的癌細胞株 36
七、Mbnl3在肝癌細胞中所扮演的角色 36
八、Mbnl3對乳房細胞及乳癌細胞所造成的影響 38
討論 41
未來展望 48
圖表 49
附錄 66
參考文獻 77

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