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研究生：	梁敏聖 Liang, Vincent Roderick
論文名稱：	細胞骨架相關蛋白質 Palladin 在肌肉生成之功能研究 Characterization of cytoskeleton-associated protein,palladin, in myogenesis
指導教授：	王浩文 Wang, Hao-Ven
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	56
中文關鍵詞：	細胞骨架、肌肉生成
外文關鍵詞：	myogenesis, palladin, apoptosis
相關次數：	點閱：81 下載：2
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細胞骨架相關蛋白質palladin 在調控細胞貼附與細胞張力絲(stress fibers)的形成中扮演重要的角色。Palladin 主要含有三種isoforms，分別是200kDa、140kDa與90~92kDa 的isoforms，三種不同的isoforms 在小鼠個體內會表現在特定的組織當中，這也暗示著每種isoform 的功能與特性有所不同。小鼠在胚胎發育的過程中palladin 的存在極為重要，先前研究顯示，palladin 基因剔除的老鼠會在胚胎時期死亡。然而palladin 在骨骼肌發育的過程中所扮演的角色至今仍尚未明瞭。因此本研究利用小干擾RNA (siRNA) 抑制全部palladin isoforms 在C2C12 老鼠肌原母細胞(myoblast) 的表現，欲探討palladin 在肌肉生成時所扮演的功能。實驗首先將 siRNA 轉染入C2C12 肌原母細胞，結果顯示palladin 的表現成功地被抑制，此外並觀察到細胞生長的密度有降低的現象。進一步將palladin knockdown 的肌原母細胞誘導進行分化，發現其分化成為肌小管(myotube)的時間會延後。從結果可以推論palladin knockdown 可能會影響細胞增生的速率或造成細胞死亡。接著利用MTT assay 以及BrdU incorporation assay 的實驗分析發現，palladin knockdown 的C2C12 細胞會造成細胞存活率與細胞增生速率下降。而細胞凋亡實驗 (Caspase 3/7 assay) 結果也顯示palladin knockdown 的C2C12 細胞之細胞凋亡情形有顯著的增加。利用即時定量聚合酶連鎖反應(real-time PCR)分析基因表現，結果發現轉染入anti-pan-palladin siRNA#1 至細胞確實能抑制各種palladin isoforms 的表現。此外，將palladin knockdown 的C2C12 細胞誘導進行分化後，可看到myogenin 的表現量有下降的情形，而細胞增生的marker : Ki67，則有顯著的增加。從以上結果推論: 將C2C12 肌原細胞的palladin knockdown 會造成細胞增生速率的降低並增加細胞凋亡程度進而影響肌原細胞的分化。

Palladin, a cytoskeleton-associated protein, plays an important role in regulating cell adhesion and the formation of stress fibers. Palladin has three major isoforms: 200kDa, 140kDa, 90~92 kDa, and the expression of different isoforms has a tissue specific feature,suggesting that different palladin isoforms may be specialized for different functions.Palladin is important in embryonic development, palladin knockout mice caused embryonic lethal, but its role in skeletal muscle development is still unclear. In my dissertation, I tried to futher analyze the role of palladin in myoblast differentiation by using small interfering RNA (siRNA) to knockdown all palladin isoforms in C2C12 myoblasts. The transfection of palladin siRNAs successfully decreased the palladin expression in the C2C12 cells. I also found knockdown of palladin decreased cell density and delayed the formation of myotubes during myoblast differentiation. The result indicated that knockdown of palladin may induce cell death or decrease cell proliferation. The MTT assay and the BrdU incorporation assay results showed that cell proliferation rate was decreased by knockdown of palladin in C2C12. The down- regulation of palladin also induced cell apoptosis by measuring caspase3/7 activity in C2C12 cells. The real-time PCR results showed that all palladin isoforms were down-regulated after transfecting with anti-pan-palladin siRNA#1. The down-regulation of myogenin in palladin knockdown C2C12 cells indicated the myoblast differentiation was affected. The expression of the proliferation marker, Ki67, was increased in palladin-knockdown C2C12 cells after inducing to differentiate. In conclusion, this study demonstrated that knockdown of all palladin isoforms in C2C12 myoblast cells can increase the apoptosis and decrease cell proliferation rate.

中文摘要	         2
Abstract	3
Acknowlegement	4
Contents	5
List of Tables	7
List of Figures	8
1.   Introduction	9
1.1  Palladin and cytoskeleton	9
1.2  The palladin/myotilin/myopalladin protein family	11
1.3  Palladin-associated proteins	14
1.4  Disease studies	15
1.5  The Palladin-knockout mice	15
1.6  Aim of this study	16
2.   Materials and Methods	17
2.1  Cell line	17
2.2  Bacteria	17
2.3  Chemicals	17
2.4  Cell culture	17
2.5  RNA interference	18
2.6  Protein extraction	18
2.7  Western blot	19
2.8  Immunofluorescence	20
2.9  RNA extraction	21
2.10 Reverse transcription-polymerase chain reaction(RT-PCR)	21
2.11 Quantitative polymerase chain reaction (Q-PCR)	22
2.12 MTT assay	22
2.13 BrdU cell proliferation assay	22
2.14 Caspase 3/7 cell apoptosis assay	23
3    Results	24
3.1  Knockdown palladin in C2C12 myoblast affected cell    
     density	24
3.2  The effect of palladin knockdown was recovered during 
     myoblast differentiation	25
3.3  The palladin 200kDa and 140kDa isoforms expression 
     patterns were affected by pan-palladin knockdown 
     during myoblast differentiation	25
3.4  The palladin knockdown could interfere C2C12 cells 
     growth and delayed the formation of myotubes in 
     differentiation	26
3.5  Deficiency of palladin in myoblast decreased the cell 
     viability	27
4    Discussion	28
5    References	33
List of Tables
     Table 1. siRNA used in this experiment	42
     Table 2. Primers used for Q-PCR in this experiment	43
List of Figures
     Figure 1. Schematic presentation of the murine  
     palladin transcripts of all isoforms..	44
     Figure 2. Knockdown of palladin in C2C12 cells by 
     siRNA transfection	45
     Figure 3. Knockdown of palladin in C2C12 cells 
     decreasd cell density	46
     Figure 4. The palladin- knockdown effect was recovered 
     during C2C12 differentiation	47
     Figure 5. The expression of 200kDa- and 140kDa  
     palladin isoform was decreased in the pan-palladin 
     knockdown C2C12 after induction of differentiation.48
     Figure 6. Knockdown of palladin in C2C12 cells dalayed 
     myotubes formation after induction of differentiation
     49
     Figure 7. The expression of myogenin was decreased 
     after differentiation for three days in palladin-
     knockdown C2C12 cells	50
     Figure 8. knockdown of palladin did not change actin 
     expression in C2C12.	51
     Figure 9. The expression of α-actinin did not have 
     obviously change after differentiation for three days 
     in palladin-knockdown C2C12 cells	52
     Figure 10. MTT assay to measure cell proliferation and 
     viability in palladin-knockdown C2C12 during 
     differentiation.	53
     Figure 11. Cell proliferation was decreased by   
     knockdown of palladin in C2C12, but it was recovered 
     after the induction of differentiation.	54
     Figure 12. The expression of Ki67 was increased after 
     differentiation for three days in palladin-knockdown 
     C2C12 cells.	55
     Figure 13. Knockdown of palladin in C2C12 increased 
     apoptosis.	56

                                    

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校內：2017-05-09公開
校外：2017-05-09公開

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