跳到主要內容

臺灣博碩士論文加值系統

(3.236.50.201) 您好!臺灣時間:2021/08/05 19:39
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:李萱葒
研究生(外文):Hsuan-Hung Lee
論文名稱:Irisin對人類神經膠質瘤細胞株U-87 MG之影響
論文名稱(外文):Study the Effect of Irisin on Human Glioblastoma Cell Line, U-87 MG
指導教授:黃楓婷黃楓婷引用關係
口試委員:周綠蘋許益瑞廖憶純
口試日期:2015-07-16
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:63
中文關鍵詞:irisin脂肪細胞browning細胞遷移細胞侵襲細胞共培養
外文關鍵詞:irisinadipocyte browningcell migrationcell invasionco-culture
相關次數:
  • 被引用被引用:0
  • 點閱點閱:230
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
臨床統計指出運動能有效降低罹癌風險及延緩癌症進程,並且具備無副作用之特性,然而,運動抗癌之生理機制尚未闡明。先前研究發現,運動使肌肉細胞釋放大量irisin,最後作用於白色脂肪細胞促進其browning成為類棕色脂肪細胞,這兩種脂肪細胞的能量代謝及激素分泌皆不同,其中最主要的差異在於類棕色脂肪會快速消耗能量,由此可知irisin具有調控脂肪代謝之功能。另外,研究證實腫瘤中含有白色脂肪細胞,白色脂肪細胞幫助癌細胞變得更具侵略性 (aggressive)。綜合以上所述,我們推測運動後產生之irisin能使腫瘤中白色脂肪細胞browning成為類棕色脂肪,進一步抵銷白色脂肪細胞對癌細胞之幫助,藉此抑制癌症進程。我的論文主要探討irisin對腫瘤細胞之影響,包含兩個方向:irisin是否直接影響癌細胞?或者先將白色脂肪細胞browning後,間接影響鄰近之癌細胞?
實驗主要分成三個階段:首先,於E.coli系統表現irisin,並且確認我們製備之irisin具有結合白色脂肪細胞及促進browning的能力。第二階段探討irisin對人類神經膠質瘤細胞株U-87 MG之影響,若直接以irisin處理U-87 MG,發現其細胞存活 (cell viability) 及遷移 (cell migration) 不受影響,而侵襲能力 (cell invasion) 則被抑制。若將irisin刺激產生的類棕色脂肪與U-87 MG共同培養,可觀察到U-87 MG的遷移和侵襲能力皆下降,且抑制效果比irisin直接處理癌細胞更顯著,證實irisin能透過將癌細胞周圍之白色脂肪細胞 browning 間接抑制癌細胞之遷移與侵襲。第三階段透過放射線標定irisin及小鼠PET影像系統,我們觀察到irisin聚集在皮下腫瘤,此現象說明irisin勢必在癌症中扮演重要角色。藉由第二及第三階段的實驗結果,本論文提議irisin很可能就是運動能延緩癌症進程的關鍵。


Clinical research indicated exercise could prevent cancer and delay cancer progression. Exercise has no side effect, therefore, the mechanism of how exercise affects cancer should be clarified for promoting development of cancer therapy. Previous studies found that exercise induces secretion of irisin from myocytes. Exercise-induced irisin finally targets to white adipocytes, and induces white adipocytes browning to brown-like adipocytes. Browning changes adipokine secretion and increases energy expenditure. Moreover, previous studies indicated white adipocytes also present in tumors and make tumor cells more aggressive. Based on above information, we speculated that exercise-induced irisin may affect cancer cells through inducing browning of cancer-associated adipocytes.
First, recombinant irisin protein was produced in E.coli and purified by the Ni-NTA column. The white adipocyte binding and browning ability of the recombinant irisin were confirmed. Next, we investigated the effect of irisin on the human glioblastoma cell line, U-87 MG. After irisin treatment, the cell viability and migration of U-87 MG remained unchanged, but the cell invasion was decreasing. Moreover, the cell migration and invasion of U-87 MG were both decreasing by co-culture with irisin-induced-brown-like adipocytes. Furthermore, with the injection of radio-isotope-labeled irisin into BALB/c nude mice, we found irisin had significant tumor targeting ability by PET imaging.
Our results indicated that irisin could significantly inhibit the cell invasion of U-87 MG in both direct and adipocytes-related indirect way. Cell invasion was important for metastasis and the indicator of aggressive cancers. Therefore, our results proposed irisin play the key role of how exercise delay cancer progression.


謝辭 i
中文摘要 ii
Abstract iii
Table of contents v
Chapter 1 Introduction 1
1.1 Exercise induced myokine-Irisin 2
1.2 “Browning” of white adipocyte 3
1.3 Cancer associated adipocytes 4
1.4 Research purpose 5
Chapter 2 Materials and Methods 7
2.1 Recombinant protein preparation 7
2.1.1 Irisin gene expression plasmid (TA-irisin) construction 7
2.1.2 Irisin gene with His-tag plasmid (pET28b-irisin) construction 7
2.1.3 Transformation of the pET28b-irisin plasmid into BL21(DE3) 8
2.1.4 IPTG induction and irisin purification 9
2.1.5 Desalting and concentration 10
2.1.6 Endotoxin detection and removal 11
2.2 Protein analysis 12
2.2.1 Gel electrophoresis 12
2.2.2 CBR staining 12
2.3 Cell culture 13
2.3.1 Cell culture (2D) 13
2.3.2 Three-dimensional (3D) cell culture of U-87 MG by magnetic levitation 13
2.4 Induction of adipocyte differentiation and browning 14
2.4.1 Differentiation of 3T3-L1 pre-adipocyte into white adipocyte 14
2.4.2 Oil red O staining for confirming differentiation level 15
2.4.3 Induction of 3T3-L1 adipocyte browning 15
2.5 Irisin binding assay 16
2.5.1 FITC labeling 16
2.5.2 Cell staining and detection 16
2.6 mRNA quantitation 17
2.6.1 Total RNA extraction 17
2.6.2 DNase treatment 17
2.6.3 cDNA synthesis 18
2.6.4 Reverse-transcription quantitative PCR (RT-qPCR) 18
2.6.5 Relative quantitation 19
2.7 Immunocytochemistry (ICC) 19
2.8 Co-culture of U-87 MG and 3T3-L1 adipocyte 20
2.8.1 Co-culture using 0.4 m transwell (2D) 20
2.8.2 Co-culture using magnetic levitation (3D) 20
2.9 Cell behavior analysis of U-87 MG 21
2.9.1 Cytotoxicity assay (cell viability) 21
2.9.2 Migration assay 21
2.9.3 Invasion assay using transwell (2D) 22
2.9.4 Three-dimensional (3D) spheroid BME invasion assay 23
2.9.5 MMP-2 activity assay 24
2.10 Positron emission tomography (PET) imaging 24
Chapter 3 Results 25
3.1 Recombinant irisin expression 25
3.2 Browning of 3T3-L1 adipocytes with irisin stimulation 25
3.2.1 Differentiation of 3T3-L1 adipocytes 25
3.2.2 Binding of irisin to 3T3-L1 adipocytes 26
3.2.3 The browning of irisin treated adipocyte 26
3.3 Direct effects of irisin on U-87 MG cells 27
3.3.1 Cell viability of irisin-treated U-87 MG 27
3.3.2 The cell migration ability of irisin-stimulated U-87 MG 28
3.3.3 The cell invasion ability of irisin-stimulated U-87 MG 28
3.4 Indirect effects of irisin on U-87 MG through browning adjacent adipocytes 29
3.4.1 The cell migration ability of U-87 MG which co-cultured with white or irisin-induced-brown-like adipocytes 29
3.4.2 The cell invasion ability of U-87 MG which co-cultured with white or irisin-induced-brown-like adipocytes 30
3.5 The cell invasion ability of U-87 MG in 3D culture 31
3.6 The released MMP-2 level of U-87 MG with irisin treatment or co-culture with adipocytes 32
3.7 Tumor targeting ability of irisin in vivo 33
Chapter 4 Discussion 34
4.1 The browning efficiency of irisin produced in E.coli 34
4.2 The effect of white adipocytes to U-87 MG cells 34
4.3 Three-dimensional invasion level of U-87 MG co-cultured with white adipocytes 35
4.4 The adipocyte-targeting ability of irisin in vivo 36
Chapter 5 Summary and future prospects 37
Chapter 6 Reference list 39
Figures 42
Figure 1. The purity of recombinant irisin was checked by SDS-PAGE and CBR staining 43
Figure 2. Differentiation of 3T3-L1 44
Figure 3. The binding of irisin to adipocytes 45
Figure 4. Browning of white adipocytes induced by the irisin treatment 46
Figure 5. Cell Viability of irisin-stimulated U-87 MG cells 47
Figure 6. Cell migration of irisin-stimulated U-87 MG cells 48
Figure 7. Cell invasion of irisin-stimulated U-87 MG cells 50
Figure 8. Cell migration of U-87 MG co-cultured with white or irisin-induced-brown-like adipocytes 52
Figure 9. Cell invasion of U-87 MG co-cultured with white or irisin-induced-brown-like adipocytes 55
Figure 10. Three-dimensional cell invasion of U-87 MG 57
Figure 11. MMP-2 level of the cell culture supernatant of U-87 MG 59
Figure 12. The tumor targeting ability of irisin 61
Appendixes 62
A. Details of primers 63
B. PCR conditions 63


Bostrom, P., J. Wu, M. P. Jedrychowski, A. Korde, L. Ye, J. C. Lo, K. A. Rasbach, E. A. Bostrom, J. H. Choi, J. Z. Long, S. Kajimura, M. C. Zingaretti, B. F. Vind, H. Tu, S. Cinti, K. Hojlund, S. P. Gygi and B. M. Spiegelman (2012). "A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis." Nature 481(7382): 463-468.
Cao, L., X. Liu, E. J. Lin, C. Wang, E. Y. Choi, V. Riban, B. Lin and M. J. During (2010). "Environmental and genetic activation of a brain-adipocyte BDNF/leptin axis causes cancer remission and inhibition." Cell 142(1): 52-64.
Choe, G., J. K. Park, L. Jouben-Steele, T. J. Kremen, L. M. Liau, H. V. Vinters, T. F. Cloughesy and P. S. Mischel (2002). "Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype." Clin Cancer Res 8(9): 2894-2901.
Choi, Y. K., M. K. Kim, K. H. Bae, H. A. Seo, J. Y. Jeong, W. K. Lee, J. G. Kim, I. K. Lee and K. G. Park (2013). "Serum irisin levels in new-onset type 2 diabetes." Diabetes Res Clin Pract 100(1): 96-101.
Cristancho, A. G. and M. A. Lazar (2011). "Forming functional fat: a growing understanding of adipocyte differentiation." Nat Rev Mol Cell Biol 12(11): 722-734.
Dirat, B., L. Bochet, M. Dabek, D. Daviaud, S. Dauvillier, B. Majed, Y. Y. Wang, A. Meulle, B. Salles, S. Le Gonidec, I. Garrido, G. Escourrou, P. Valet and C. Muller (2011). "Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion." Cancer Res 71(7): 2455-2465.
Forsyth, P. A., H. Wong, T. D. Laing, N. B. Rewcastle, D. G. Morris, H. Muzik, K. J. Leco, R. N. Johnston, P. M. Brasher, G. Sutherland and D. R. Edwards (1999). "Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas." Br J Cancer 79(11-12): 1828-1835.
Friedenreich, C. M. and M. R. Orenstein (2002). "Physical activity and cancer prevention: etiologic evidence and biological mechanisms." J Nutr 132(11 Suppl): 3456-3464.
Gannon, N. P., R. A. Vaughan, R. Garcia-Smith, M. Bisoffi and K. A. Trujillo (2015). "Effects of the exercise-inducible myokine irisin on malignant and non-malignant breast epithelial cell behavior in vitro." Int J Cancer 136(4): E197-202.
Harms, M. and P. Seale (2013). "Brown and beige fat: development, function and therapeutic potential." Nat Med 19(10): 1252-1263.
Knols, R., N. K. Aaronson, D. Uebelhart, J. Fransen and G. Aufdemkampe (2005). "Physical exercise in cancer patients during and after medical treatment: a systematic review of randomized and controlled clinical trials." J Clin Oncol 23(16): 3830-3842.
Murphy, E. A., J. M. Davis, T. L. Barrilleaux, J. L. McClellan, J. L. Steiner, M. D. Carmichael, M. M. Pena, J. R. Hebert and J. E. Green (2011). "Benefits of exercise training on breast cancer progression and inflammation in C3(1)SV40Tag mice." Cytokine 55(2): 274-279.
Nieman, K. M., H. A. Kenny, C. V. Penicka, A. Ladanyi, R. Buell-Gutbrod, M. R. Zillhardt, I. L. Romero, M. S. Carey, G. B. Mills, G. S. Hotamisligil, S. D. Yamada, M. E. Peter, K. Gwin and E. Lengyel (2011). "Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth." Nat Med 17(11): 1498-1503.
Ornish, D., G. Weidner, W. R. Fair, R. Marlin, E. B. Pettengill, C. J. Raisin, S. Dunn-Emke, L. Crutchfield, F. N. Jacobs, R. J. Barnard, W. J. Aronson, P. McCormac, D. J. McKnight, J. D. Fein, A. M. Dnistrian, J. Weinstein, T. H. Ngo, N. R. Mendell and P. R. Carroll (2005). "Intensive lifestyle changes may affect the progression of prostate cancer." J Urol 174(3): 1065-1069; discussion 1069-1070.
Ruden, E., D. A. Reardon, A. D. Coan, J. E. Herndon, 2nd, W. E. Hornsby, M. West, D. R. Fels, A. Desjardins, J. J. Vredenburgh, E. Waner, A. H. Friedman, H. S. Friedman, K. B. Peters and L. W. Jones (2011). "Exercise behavior, functional capacity, and survival in adults with malignant recurrent glioma." J Clin Oncol 29(21): 2918-2923.
Stengel, A., T. Hofmann, M. Goebel-Stengel, U. Elbelt, P. Kobelt and B. F. Klapp (2013). "Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity--correlation with body mass index." Peptides 39: 125-130.
Zebisch, K., V. Voigt, M. Wabitsch and M. Brandsch (2012). "Protocol for effective differentiation of 3T3-L1 cells to adipocytes." Anal Biochem 425(1): 88-90.
Zhang, Y., A. Daquinag, D. O. Traktuev, F. Amaya-Manzanares, P. J. Simmons, K. L. March, R. Pasqualini, W. Arap and M. G. Kolonin (2009). "White adipose tissue cells are recruited by experimental tumors and promote cancer progression in mouse models." Cancer Res 69(12): 5259-5266.
Zhang, Y., L. R., Y. Meng, S. Li, W. Donelan, Y. Zhao, L. Qi, M. Zhang, X. Wang, T. Cui, L. Yang and D. Tang (2014). "Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling." Diabetes 63(2): 514-525.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top