目的 分析hBMSCs 經(jīng)5- 氮雜胞苷(5-azacytidine,5-aza)誘導(dǎo)向心肌樣細胞分化過程中基因表達譜
的改變。 方法 取胸外科非血液病患者手術(shù)中廢棄的肋骨骨髓分離培養(yǎng)hBMSCs,5-aza 誘導(dǎo)第2 代hBMSCs。取誘導(dǎo)
前及誘導(dǎo)后的細胞,免疫細胞化學(xué)法檢測α-actin、肌鈣蛋白T(cardiac troponin T,cTnT)和連接蛋白43(connexin 43)表
達,流式細胞儀檢測cTnT 陽性細胞百分率;利用人表達譜基因芯片技術(shù)篩選分化過程中的差異表達基因,對部分基因進
行功能分類和分層聚類分析。 結(jié)果 hBMSCs 經(jīng)5-aza 誘導(dǎo)后,部分呈肌細胞樣形態(tài)。免疫細胞化學(xué)染色檢測示誘導(dǎo)
前細胞α-actin、cTnT 染色呈弱陽性,connexin 43 染色呈陰性;誘導(dǎo)后3 周細胞α-actin、cTnT、connexin 43 染色均呈陽性。
流式細胞儀檢測示誘導(dǎo)前cTnT 陽性細胞百分率為7.43% ± 0.02%,誘導(dǎo)后3 周為49.64% ± 0.05%。分化過程中共檢測到
1 814 個顯著差異表達基因,對其中647 個基因分層聚類,聚為5 類,生物功能包括信號傳導(dǎo)、細胞代謝、增殖分化、發(fā)育以
及形態(tài)發(fā)生等。 結(jié)論 hBMSCs 經(jīng)5-aza 誘導(dǎo)向心肌樣細胞分化過程受信號傳導(dǎo)通路、轉(zhuǎn)錄基因、生長因子等多種因素
在不同時間點上的共同調(diào)控。
引用本文: 楊琳 ,沈悌,陳連鳳,曹欣欣,賴晉智. BMSCs 向心肌分化的基因表達譜分析. 中國修復(fù)重建外科雜志, 2012, 26(5): 607-611. doi: 復(fù)制
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2. | Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet, 2004, 364(9429): 141-148. |
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5. | Ling SK, Wang R, Dai ZQ, et al. Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5-azacytidine enhances therapeutic efficacy for myocardial infarction. Biotechnol Prog, 2011, 27(2): 473-482. |
6. | Xu W, Zhang X, Qian H, et al. Mesenchymal stem cells from adult human bone marrow differentiates into a cardiomyocyte phenotype in vitro. Exp Biol Medi (Maywood), 2004, 229(7): 623-631. |
7. | Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest, 1999, 103(5): 697-705. |
8. | Cohen ED, Tian Y, Morrisey EE. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal. Development, 2008, 135(5): 789-798. |
9. | Flaherty MP, Abdel-Latif A, Li Q, et al. Noncanonical Wnt11 signaling is sufficient to induce cardiomyogenic differentiation in unfractionated bone marrow mononuclear cells. Circulation, 2008, 117(17): 2241-2252. |
10. | Qyang Y, Martin-Puig S, Chiravuri M, et al. The renewal and differentiation of isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway. Cell Stem Cell, 2007, 1(2): 165-179. |
11. | Zhu W, Shiojima I, Ito Y, et al. IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis. Nature, 2008, 454(7202): 345-349. |
12. | Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis in Xenopus laevis. Genes Dev, 2001, 15(3): 304-315. |
13. | Tzahor E, Lassar AB. Wnt signals from the neural tube block ectopic cardiogenesis. Genes Dev, 2001, 15(3): 255-260. |
14. | Li Y, Hiroi Y, Ngoy S, et al. Notch1 in bone marrow-derived cells mediates cardiac repair after myocardial infarction. Circulation, 2011, 123(8): 866-876. |
15. | Crispino JD, Lodish MB, Thurberg BL, et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes Dev, 2001, 15(7): 839-844. |
16. | Zhao R, Watt AJ, Battle MA, et al. Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice. Dev Biol, 2008, 317(2): 614-619. |
17. | Sachinidis A, Kolossov E, Fleischmann BK, et al. Generation of cardiomyocytes from embryonic stem cells experimental studies. Herz, 2002, 27(7): 589-597. |
18. | Yang HS, Bhang SH, Kim IK, et al. In situ cardiomyogenic differentiation of implanted bone marrow mononuclear cells by local delivery of transforming growth factor-β1. Cell Transplant, 2011. [Epub ahead of print]. |
19. | Wan CR, Chung S, Kamm RD. Differentiation of embryonic stem cells into cardiomyocytes in a compliant microfluidic system. Ann Biomed Eng, 2011, 39(6): 1840-1847. |
20. | Afouda BA, Hoppler S. Different requirements for GATA factors in cardiogenesis are mediated by non-canonical Wnt signaling. Dev Dyn, 2011, 240(3): 649-662. |
- 1. Passier R, van Laake LW, Mummery CL. Stem-cell-based therapy and lessons from the heart. Nature, 2008, 453(7193): 322-329.
- 2. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet, 2004, 364(9429): 141-148.
- 3. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation, 2003, 108(18): 2212-2218.
- 4. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J Clin Invest, 2005, 115(3): 572-583.
- 5. Ling SK, Wang R, Dai ZQ, et al. Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5-azacytidine enhances therapeutic efficacy for myocardial infarction. Biotechnol Prog, 2011, 27(2): 473-482.
- 6. Xu W, Zhang X, Qian H, et al. Mesenchymal stem cells from adult human bone marrow differentiates into a cardiomyocyte phenotype in vitro. Exp Biol Medi (Maywood), 2004, 229(7): 623-631.
- 7. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest, 1999, 103(5): 697-705.
- 8. Cohen ED, Tian Y, Morrisey EE. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal. Development, 2008, 135(5): 789-798.
- 9. Flaherty MP, Abdel-Latif A, Li Q, et al. Noncanonical Wnt11 signaling is sufficient to induce cardiomyogenic differentiation in unfractionated bone marrow mononuclear cells. Circulation, 2008, 117(17): 2241-2252.
- 10. Qyang Y, Martin-Puig S, Chiravuri M, et al. The renewal and differentiation of isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway. Cell Stem Cell, 2007, 1(2): 165-179.
- 11. Zhu W, Shiojima I, Ito Y, et al. IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis. Nature, 2008, 454(7202): 345-349.
- 12. Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis in Xenopus laevis. Genes Dev, 2001, 15(3): 304-315.
- 13. Tzahor E, Lassar AB. Wnt signals from the neural tube block ectopic cardiogenesis. Genes Dev, 2001, 15(3): 255-260.
- 14. Li Y, Hiroi Y, Ngoy S, et al. Notch1 in bone marrow-derived cells mediates cardiac repair after myocardial infarction. Circulation, 2011, 123(8): 866-876.
- 15. Crispino JD, Lodish MB, Thurberg BL, et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes Dev, 2001, 15(7): 839-844.
- 16. Zhao R, Watt AJ, Battle MA, et al. Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice. Dev Biol, 2008, 317(2): 614-619.
- 17. Sachinidis A, Kolossov E, Fleischmann BK, et al. Generation of cardiomyocytes from embryonic stem cells experimental studies. Herz, 2002, 27(7): 589-597.
- 18. Yang HS, Bhang SH, Kim IK, et al. In situ cardiomyogenic differentiation of implanted bone marrow mononuclear cells by local delivery of transforming growth factor-β1. Cell Transplant, 2011. [Epub ahead of print].
- 19. Wan CR, Chung S, Kamm RD. Differentiation of embryonic stem cells into cardiomyocytes in a compliant microfluidic system. Ann Biomed Eng, 2011, 39(6): 1840-1847.
- 20. Afouda BA, Hoppler S. Different requirements for GATA factors in cardiogenesis are mediated by non-canonical Wnt signaling. Dev Dyn, 2011, 240(3): 649-662.