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研究生:張方瑜
研究生(外文):Fang-Yu Chang
論文名稱:糖化終產物誘導骨骼肌細胞衰老及對肌肉再生之影響
論文名稱(外文):Advanced glycation end-products induce muscle cell senescence and affect muscle regeneration in aging mice
指導教授:劉興華劉興華引用關係
口試委員:楊榮森蕭水銀許美鈴姜至剛
口試日期:2018-07-27
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:59
中文關鍵詞:糖化終產物骨骼肌肌肉分化肌纖維母細胞細胞衰老老化
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世界人口正迅速邁向高齡化,近年內與年齡有關的疾病將大大增加,並對老年人的生活品質產生重大影響。肌肉功能障礙在老年人身上是一種相當普遍的慢性病,並且引起許多公共衛生問題。雖然年齡被認為是肌肉功能障礙的主要危險因素,但老化所涉及的機制眾說紛紜。肌肉中與年齡相關的主要變化之一是糖化終產物(AGEs)的累積,其由還原糖與蛋白質或脂質經梅納反應產生。先前的研究指出,人體內的AGEs與糖尿病併發之肌肉功能障礙高度相關。然而對於肌肉老化與AGEs的關係以及其中的機制尚待釐清,故本研究欲透過細胞及老化動物進行實驗,探討AGEs對肌肉修復功能造成的影響。細胞實驗部分,首先經由senescence-associated beta-galactosidase及hematoxylin and eosin染色結果觀察到,在未分化之肌纖維母細胞(C2C12 myoblast)或在其分化過程中暴露AGEs (50 μg/mL),皆會造成肌纖維母細胞老化並使其分化程度下降。接著使用西方墨點法發現,細胞老化指標蛋白(p16, SIRT6)經AGEs影響分別顯著上升及下降,而骨骼肌分化指標蛋白(myosin heavy chain, myogenin, phosphorylation of AMPK)則顯著下降。然而在抑制細胞膜上AGEs受體後,肌纖維母細胞便恢復其分化能力。動物部分,本研究使用D-galactose誘導C57BL/6小鼠老化之動物模式進行實驗,在皮下施打D-galactose (1 g/kg)並同時給予aminoguanidine (0.1 g/kg)達4週後,藉由滾輪跑步儀器測試小鼠肌肉強度,此外亦在小鼠脛前肌注射甘油誘導其肌肉損傷及再生。結果顯示,注射D-galactose (1 g/kg) 4週並未造成骨骼肌強度之變化,然而在脛前肌受損五日後經H&E染色觀察到,D-galactose誘導老化之小鼠肌肉修復功能顯著較差,然而餵食aminoguanidine後則恢復與控制組相同之修復能力。接著,為了解老化動物體內AGEs於肌肉累積之情形,使用immunohistochemistry (IHC)染色發現D-galactose組肌肉有大量AGEs堆積,而控制組及餵食aminoguanidine之組別則僅有極少量AGEs。綜合上述,本研究指出AGEs促使肌纖維母細胞老化及抑制其分化,並降低老化動物肌肉修復能力,然其詳細機制仍需未來更多探討。
The world population is aging rapidly so that the age‐related diseases will increase greatly in the next few decades and will have a great impact on the life quality of the elderly. Muscle dysfunction is a prevalent and disabling chronic condition affecting the elderly and causes many public health problems. Although age is identified as the main risk factor for muscle dysfunction, the mechanism by which aging is involved has various perspective. One of the major age‐related changes in muscle is the accumulation of advanced glycation end products (AGEs), which result from the spontaneous Maillard reaction of reducing sugars with proteins. Previous studies have reported that AGEs in the human body would accelerate in diabetes mellitus and highly associated with muscle dysfunction. Hence, the aim of this study is to investigate the role of AGEs on skeletal muscle differentiation in aging. First, a non-cytotoxic dose of AGEs (50 μg/mL) induced cell senescence and repressed myogenic differentiation in C2C12 myoblasts by senescence-associated beta-galactosidase (SA-β-gal) staining and hematoxylin and eosin (H&E) staining. The protein levels (myosin heavy chain, myogenin, phosphorylation of AMPK, p16, SIRT6) were also changed with AGEs exposure. After RAGE neutralized antibody treatment, all of the effects induced by AGEs were reversed. Moreover, skeletal muscle regeneration capacity in murine TA muscles after glycerol injury is retarded in D-galactose induced aging mice, which have AGEs accumulation in muscle. Taken together, these results suggest that AGEs may be a risk factor for myogenesis due to premature cell senescence. The further mechanism should be investigated in the future.
致謝………………………………………………………………………….……….III
中文摘要..…………………………………………………………………………….V
Abstract……………………………………………………………………………...VII
Abbreviation Summary…………………………………..……………………….….IX
Part 1: Introduction……………………………………………...………………….….1
1.1 Age-related changes in muscle structure and function……………………….1
1.2 Age-related molecular changes in muscle…………………………………....1
1.3 Advanced glycation end products formation and the toxic effects…………..3
1.4 AGEs in elderly………………………………………………………………4
1.5 The pathological role of AGEs in muscle………………………………..…..5
1.6 Aminoguanidine…………………………………………………..………….6
1.7 D-galactose induced aging………………………………………….………..7
Part 2: Aim………………………………………………...…………………………..9
Part 3: Materials and methods………………………………………………………..10
3.1 Cell culture………………………………………...…………………..……10
3.2 Advanced glycation end‐products (AGEs) treatment…………………...….10
3.3 Estimation of cell viability………………………………………………….11
3.4 Estimation of cell proliferation……………………………………...…..….11
3.5 Morphological myotube analysis………………………………………..….12
3.6 Senescence-associated beta-galactosidase activity analysis…………….….12
3.7 Immunoblotting…………………………………………….………………13
3.8 Animals and treatment………………………………………………...……14
3.8.1 Normal aging model………………………………………………….14
3.8.2 D-galactose induced aging model………………………………….…15
3.9 Muscle fatigue task………………………………………………………....16
3.10 Muscle regeneration study…………………………………….……..……16
3.11 Histological assessments………………………………….……….………17
3.12 Statistics…………………………………………………...…………..…..18
Part 4: Results…………………………………………...………………..………….19
4.1 AGEs inhibit myogenic differentiation in C2C12 myoblasts………………19
4.2 AGEs induce cell senescence in C2C12 myoblasts……………...…………19
4.3 Inhibition of RAGE rescues the inhibitory effects of AGEs in C2C12 cells.20
4.4 Effects of AGEs on body weight, muscle weights, and muscle function in D-galactose induced aging mice………………………………………………..21
4.5 Effects of AGEs on the muscle regeneration capacity after injury in D-galactose induced aging mice. …………………………………………..…..22
4.6 AGEs accumulation in muscles and lead to increased expression of the senescence markers. ……………………………………………………………22
4.7 The effects of AGEs on muscle satellite cells in muscles of D-galactose induced aging mice. ………………………………………………………....….23
Part 5: Discussion…………………………………………………………….….…...25
Part 6: Conclusion. …………………………………………………………….….…28
Part 7: Reference………………………………………………….……………….…29
Part 8: Figures and Figure legends…………………………………………….……..38
Figure 1. Effect of AGEs on cell viability and cell proliferation in C2C12 myoblasts. …………………………...……………………………………….…38
Figure 2. Effect of AGEs on myogenic differentiation in C2C12 myoblasts. .....39
Figure 3. Time course of the protein expression of myogenesis markers after AGEs treatment for 48 hours in C2C12 myoblast and during differentiation process. …………………………………………...…………………………….41
Figure 4. Effect of AGEs on senescence-associated beta-galactosidase activity in C2C12 myoblast and during differentiation process. ……………………….….43
Figure 5. Time course of the protein expression of cell senescence markers after AGEs treatment for 48 hours in C2C12 myoblast and during differentiation process. ………………………………………………………………..……….45
Figure 6. Inhibition of RAGE reduces the inhibitory effect of AGEs in C2C12 cells. ……………………………………………………………………..……..47
Figure 7. Effects of AGEs on body weight, muscle weights, and muscle function in D-galactose induced aging mice. ……………………………………………49
Figure 8. Effects of AGEs on the muscle regeneration capacity after glycerol injection in D-galactose induced aging mice. ………………………………….51
Figure 9. The expression of p21 in muscles of D-galactose induced aging mice. ………………………….………………………………………………..53
Figure 10. The accumulation of AGEs in muscles of normal aging mice. ……54
Figure 11. The blank control of muscles of normal aging mice. ……………...55
Figure 12. The accumulation of AGEs in muscles of D-galactose induced aging mice. ………..………………………………………………………………….56
Figure 13. The blank control of muscles of D-galactose induced aging mice. ..57
Figure 14. The effects of AGEs on cell senescence in muscles of D-galactose induced aging mice. ……………………………………..………………….….58
Figure 15. The effects of AGEs on muscle satellite cells in muscles of D-galactose induced aging mice. ……………………………..………….…….59
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