• 1 解放軍總醫(yī)院第一附屬醫(yī)院心血管內(nèi)科（北京，100048）;;
  • 2 解放軍總醫(yī)院老年心血管病研究所;

【摘 要】 目的 探討人羊水來源c-kit+ 間充質(zhì)干細(xì)胞（c-kit+ human amniotic fluid-derived mesenchymal stem
cells，c-kit+ HAFMSCs）的生物學(xué)特征及心肌誘導(dǎo)分化能力。 方法 通過產(chǎn)前診斷或自愿引產(chǎn)獲取50 份孕中期羊水標(biāo)
本，經(jīng)流式細(xì)胞儀分選c-kit+ HAFMSCs，MTT 法觀察細(xì)胞增殖情況，并通過流式細(xì)胞儀、細(xì)胞免疫化學(xué)染色觀察行細(xì)胞表
型鑒定。在體外經(jīng)成骨及成脂誘導(dǎo)分化，于誘導(dǎo)培養(yǎng)后21 d 采用von Kossa 染色觀察鈣沉積顆粒，油紅O 染色觀察脂滴
形成情況。實(shí)時(shí)熒光定量PCR 檢測(cè)細(xì)胞經(jīng)5 氮-2’ 脫氧胞苷心肌誘導(dǎo)前后，NKx2.5、Tbx5、GATA-4、α-MHC 4 種心肌特
異性基因表達(dá)情況。 結(jié)果 經(jīng)流式細(xì)胞儀分選，人孕中期羊水中c-kit+ HAFMSCs 含量約為貼壁細(xì)胞的3.07% ± 1.03%；
體外增殖迅速，表達(dá)MSCs 表面標(biāo)志CD29、CD44、CD73、CD90、CD105，不表達(dá)造血干細(xì)胞表面標(biāo)志CD34、CD45；表
達(dá)人類白細(xì)胞抗原（human leukocyte antigen，HLA）-ABC，不表達(dá)HLA-DR。細(xì)胞免疫化學(xué)染色示CD29、CD44、CD73、CD90、CD105、Oct-4 染色呈陽性，CD34、CD45 染色呈陰性。MTT 檢測(cè)示第5、10、15 代c-kit+ HAFMSCs 具有相似的生長(zhǎng)曲線。成骨和成脂誘導(dǎo)培養(yǎng)后21 d，細(xì)胞內(nèi)有鈣鹽沉積和脂滴形成。實(shí)時(shí)熒光定量PCR 檢測(cè)示，心肌誘導(dǎo)后14 d， NKx2.5、Tbx5、GATA-4、α-MHC 4 種心肌特異性基因表達(dá)均較誘導(dǎo)前明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。 結(jié)論 通過流式細(xì)胞儀分選的c-kit+ HAFMSCs 在體外可以誘導(dǎo)分化為心肌樣細(xì)胞，可能成為心肌再生性治療的種子細(xì)胞。

引用本文： 白靜 ,王一茹,劉麗鳳,陳杰,王禹. 人羊水來源c-kit+ 間充質(zhì)干細(xì)胞的生物學(xué)特征和心肌誘導(dǎo)分化. 中國(guó)修復(fù)重建外科雜志, 2012, 26(2): 152-157. doi: 復(fù)制

1. Jeremy L, Herrmann, Aaron M, et al. Cell-based therapy for ischemic heart disease: a clinical update. Ann Thorac Surg, 2009, 88(5): 1714-1722.
2. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Eng J Med, 2001, 344(23): 1750-1757.
3. Kajstura J, Ubannek K, Perl S, et al. Cardiomyogenesis in the adult human heart. Circ Res, 2010, 107(2): 305-315.
4. Lloyd-Jones D, Adams RJ, Brown TM, et al. Executive summary: heart disease and stroke statistics——2010 update: a report from the American Heart Association. Circulation, 2010, 121(7): 948-954.
5. 顧東風(fēng), 黃廣勇, 河江, 等. 中國(guó)心力衰竭流行病學(xué)調(diào)查及其患病率. 中華心血管病雜志, 2003, 31(1): 3-6.
6. Prusa AR, Marton E, Rosner M, et al. OCT-4 expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod, 2003, 18(7): 1489-1493.
7. Wolbank S, Stadler G, Peterbauer A, et al. Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: maintenance of differentiation and immunomodulatory characteristics. Tissue Eng Part A, 2009, 15(7): 1843-1854.
8. De Coppi P, Bartsch G Ir, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
9. Cabral AL, Anqelo PC, Leite HV, et al. Isolation, differentiation and biochemical aspects of amniotic fluid stem cell. Rev Assoc Med Bras, 2008, 54(6): 489-493.
10. Prusa AR, Hengstschlager M. Amniotic fluid cells and human stem cell research: a new connection. Med Sci Monit, 2003, 8(11): RA253-257.
11. Da Sacco S, Sedrakyan S, Boldrin F, et al. Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications. J Urol, 2010, 183(3): 1193-1200.
12. Kim J, Lee Y, Kim H, et al. Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells. Cell Prolif, 2007, 40(1): 75-90.
13. Delo DM, De Coppi P, Bartsch G Jr, et al. Amniotic fluid and placental stem cells. Methods Enzymol, 2006, 419: 426-438.
14. Fleischman RA. From white spots to stem cells: the role of the Kit receptor in mammalian development. Trends Genet, 1993, 9(8): 285-290.
15. Perin L, Sedrakyan S, Giuliani S, et al. Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis. PLoS One, 2010, 5(2): e9357.
16. Arnhold S, Glüer S, Hartmann K, et al. Amniotic-fluid stem cells: growth dynamics and differentiation potential after a CD-117-based selection procedure. Stem Cells Int, 2011, 2011: 715341.
17. Phermthai T, Odglun Y, Julavijitphong S, et al. A novel method to derive amniotic fluid stem cells for therapeutic purposes. BMC Cell Biol, 2010, 11: 79.
18. Hauser PV, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol, 2011, 177(4): 2011-2021.
19. Sieqel N, Rosner M, Unbekandt M, et al. Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet, 2010, 19(17): 3320-3331.
20. Yeh YH, Wei HJ, Lee WY, et al. Cellular cardiomyoplasty with human amniotic fluid stem cells: in vitro and in vivo studies. Tissue Eng Part A, 2010, 16(6): 1925-1936.
  1. 1. Jeremy L, Herrmann, Aaron M, et al. Cell-based therapy for ischemic heart disease: a clinical update. Ann Thorac Surg, 2009, 88(5): 1714-1722.
  2. 2. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Eng J Med, 2001, 344(23): 1750-1757.
  3. 3. Kajstura J, Ubannek K, Perl S, et al. Cardiomyogenesis in the adult human heart. Circ Res, 2010, 107(2): 305-315.
  4. 4. Lloyd-Jones D, Adams RJ, Brown TM, et al. Executive summary: heart disease and stroke statistics——2010 update: a report from the American Heart Association. Circulation, 2010, 121(7): 948-954.
  5. 5. 顧東風(fēng), 黃廣勇, 河江, 等. 中國(guó)心力衰竭流行病學(xué)調(diào)查及其患病率. 中華心血管病雜志, 2003, 31(1): 3-6.
  6. 6. Prusa AR, Marton E, Rosner M, et al. OCT-4 expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod, 2003, 18(7): 1489-1493.
  7. 7. Wolbank S, Stadler G, Peterbauer A, et al. Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: maintenance of differentiation and immunomodulatory characteristics. Tissue Eng Part A, 2009, 15(7): 1843-1854.
  8. 8. De Coppi P, Bartsch G Ir, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
  9. 9. Cabral AL, Anqelo PC, Leite HV, et al. Isolation, differentiation and biochemical aspects of amniotic fluid stem cell. Rev Assoc Med Bras, 2008, 54(6): 489-493.
  10. 10. Prusa AR, Hengstschlager M. Amniotic fluid cells and human stem cell research: a new connection. Med Sci Monit, 2003, 8(11): RA253-257.
  11. 11. Da Sacco S, Sedrakyan S, Boldrin F, et al. Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications. J Urol, 2010, 183(3): 1193-1200.
  12. 12. Kim J, Lee Y, Kim H, et al. Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells. Cell Prolif, 2007, 40(1): 75-90.
  13. 13. Delo DM, De Coppi P, Bartsch G Jr, et al. Amniotic fluid and placental stem cells. Methods Enzymol, 2006, 419: 426-438.
  14. 14. Fleischman RA. From white spots to stem cells: the role of the Kit receptor in mammalian development. Trends Genet, 1993, 9(8): 285-290.
  15. 15. Perin L, Sedrakyan S, Giuliani S, et al. Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis. PLoS One, 2010, 5(2): e9357.
  16. 16. Arnhold S, Glüer S, Hartmann K, et al. Amniotic-fluid stem cells: growth dynamics and differentiation potential after a CD-117-based selection procedure. Stem Cells Int, 2011, 2011: 715341.
  17. 17. Phermthai T, Odglun Y, Julavijitphong S, et al. A novel method to derive amniotic fluid stem cells for therapeutic purposes. BMC Cell Biol, 2010, 11: 79.
  18. 18. Hauser PV, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol, 2011, 177(4): 2011-2021.
  19. 19. Sieqel N, Rosner M, Unbekandt M, et al. Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet, 2010, 19(17): 3320-3331.
  20. 20. Yeh YH, Wei HJ, Lee WY, et al. Cellular cardiomyoplasty with human amniotic fluid stem cells: in vitro and in vivo studies. Tissue Eng Part A, 2010, 16(6): 1925-1936.