• 1 解放軍總醫(yī)院老年心血管病研究所（北京，100853）;;
  • 2 解放軍總醫(yī)院第一附屬醫(yī)院心血管內(nèi)科;

【摘 要】 目的 體外分離培養(yǎng)人羊水間充質(zhì)干細胞（human amniotic fluid-derived mesenchymal stem cells，
HAFMSCs），觀察低溫凍存復蘇后HAFMSCs 生物學特征，為進一步研究奠定理論基礎。 方法 取12 份自愿捐贈的孕
16 ～ 20 周羊水標本，采用改良兩步法分離培養(yǎng)HAFMSCs，用含量不同的FBS、DMSO 凍存液凍存細胞，液氮凍存12 周
后42℃水浴復蘇，錐蟲藍染色檢測細胞存活率，MTT 法檢測細胞增殖速度并繪制生長曲線，流式細胞儀檢測凍存復蘇后
HAFMSCs 表型。對凍存復蘇后的HAFMSCs 進行成脂、成骨誘導分化培養(yǎng)，并分別采用油紅O、von Kossa 染色進行鑒定；
實時熒光定量PCR 分析細胞凍存前后Oct-4、Nanog mRNA 表達差異。 結(jié)果 細胞凍存12 周后，不同的凍存方案對細
胞存活率影響有差異，優(yōu)化的凍存方案為DMEM/FBS/DMSO=50%/40%/10%。凍存復蘇后的HAFMSCs 呈漩渦狀排列，
生長曲線呈S 形，與凍存前細胞生長曲線相似。流式細胞儀檢測示凍存復蘇后細胞的MSCs 表型CD29、CD44、CD73、
CD90 為陽性，造血干細胞表型CD34、CD45 為陰性。成脂、成骨誘導21 d，油紅O、von Kossa 染色均呈陽性。實時熒光
定量PCR 檢測示凍存前后Oct-4、Nanog mRNA 表達水平差異無統(tǒng)計學意義（P  gt; 0.05）。 結(jié)論 HAFMSCs 具有體外
增殖快、分化能力強的優(yōu)勢；并可耐受短期凍存，復蘇后細胞存活率高，生物學特征及分化潛能未發(fā)生明顯變化，凍存液
DMEM/FBS/DMSO=50%/40%/10% 是較好凍存方案。

引用本文： 王一茹 ,白靜,陳杰,劉麗鳳,王禹. 人羊水來源間充質(zhì)干細胞凍存后生物學特征研究. 中國修復重建外科雜志, 2012, 26(2): 141-145. doi: 復制

1. 1 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. 2 Tsai MS, Lee JL, Chang YJ, et al. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod, 2004, 19(6): 1450-1456.
2. Roubelakis MG, Pappa KI, Bitsika V, et al. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev, 2007, 16(6): 931-952.
3. Sessarego N, Parodi A, Podesta M, et al. Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application. Haematologica, 2008, 93(3): 339-346.
4. Ghionzoli M, Cananzi M, Zani A, et al. Amniotic fluid stem cell migration after intraperitoneal injection in pup rats: implication for therapy. Pediatr Surg Int, 2010, 26(1): 79-84.
5. Baghaban Eslaminejad M, Jahangir S, Aghdami N. Mesenchymal stem cells from murine amniotic fluid as a model for preclinical investigation. Arch Iran Med, 2011, 14(2): 96-103.
6. Shaw SW, David AL, De Coppi P, et al. Clinical applications of prenatal and postnatal therapy using stem cells retrieved from amniotic fluid. Curr Opin Obstet Gynecol, 2011, 23(2): 109-116.
7. Gekas J, Walther G, Skuk D, et al. In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage. Clin Exp Med, 2010, 10(1): 1-6.
8. Tsai MS, Hwang SM, Tsai YL, et al. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod, 2006, 74(3): 545-551.
9. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood, 2003, 102(4): 1548-1549.
10. De Coppi P, Bartsch G Jr, Siddiqui MM, et al. Isolation of amniotic stem cells with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
11. Cananzi M, Atala A, De Coppi P. Stem cells derived from amniotic fluid: new potentials in regenerative medicine. Reprod Biomed Online, 2009, 18 Suppl 1: 17-27.
12. Decembrini S, Cananzi M, Gualdoni S, et al. Comparative analysis of the retinal potential of embryonic stem cells and amniotic fluid-derived stem cells. Stem Cells Dev, 2011, 20(5): 851-863.
13. Hilfiker A, Kasper C, Hass R, et al. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation. Langenbeck Arch Surg, 2011, 396(4): 489-497.
14. 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, 2010, 177(4): 2011-2021.
15. Teodelinda M, Michele C, Sebastiano C, et al. Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model. Biomaterials, 2011, 32(15): 3689-3699.
16. 趙春華. 干細胞原理、技術(shù)與臨床. 北京: 化學工業(yè)出版社, 2006: 309-310.
17. 王有為, 韓之波, 嚴淑琳, 等. 人臍帶間充質(zhì)干細胞凍存復蘇后的生物學特征. 中國組織工程研究與臨床康復, 2010, 14(10): 1729-1733.
18. Karlmark KR, Freilinger A, Marton E, et al. Activation of ectopic Oct- 4 and Rex-1 promoters in human amniotic fluid cells. Int J Mol Med, 2005, 16(6): 987-992.
  1. 1. 1 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. 2 Tsai MS, Lee JL, Chang YJ, et al. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod, 2004, 19(6): 1450-1456.
  2. 2. Roubelakis MG, Pappa KI, Bitsika V, et al. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev, 2007, 16(6): 931-952.
  3. 3. Sessarego N, Parodi A, Podesta M, et al. Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application. Haematologica, 2008, 93(3): 339-346.
  4. 4. Ghionzoli M, Cananzi M, Zani A, et al. Amniotic fluid stem cell migration after intraperitoneal injection in pup rats: implication for therapy. Pediatr Surg Int, 2010, 26(1): 79-84.
  5. 5. Baghaban Eslaminejad M, Jahangir S, Aghdami N. Mesenchymal stem cells from murine amniotic fluid as a model for preclinical investigation. Arch Iran Med, 2011, 14(2): 96-103.
  6. 6. Shaw SW, David AL, De Coppi P, et al. Clinical applications of prenatal and postnatal therapy using stem cells retrieved from amniotic fluid. Curr Opin Obstet Gynecol, 2011, 23(2): 109-116.
  7. 7. Gekas J, Walther G, Skuk D, et al. In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage. Clin Exp Med, 2010, 10(1): 1-6.
  8. 8. Tsai MS, Hwang SM, Tsai YL, et al. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod, 2006, 74(3): 545-551.
  9. 9. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood, 2003, 102(4): 1548-1549.
  10. 10. De Coppi P, Bartsch G Jr, Siddiqui MM, et al. Isolation of amniotic stem cells with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
  11. 11. Cananzi M, Atala A, De Coppi P. Stem cells derived from amniotic fluid: new potentials in regenerative medicine. Reprod Biomed Online, 2009, 18 Suppl 1: 17-27.
  12. 12. Decembrini S, Cananzi M, Gualdoni S, et al. Comparative analysis of the retinal potential of embryonic stem cells and amniotic fluid-derived stem cells. Stem Cells Dev, 2011, 20(5): 851-863.
  13. 13. Hilfiker A, Kasper C, Hass R, et al. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation. Langenbeck Arch Surg, 2011, 396(4): 489-497.
  14. 14. 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, 2010, 177(4): 2011-2021.
  15. 15. Teodelinda M, Michele C, Sebastiano C, et al. Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model. Biomaterials, 2011, 32(15): 3689-3699.
  16. 16. 趙春華. 干細胞原理、技術(shù)與臨床. 北京: 化學工業(yè)出版社, 2006: 309-310.
  17. 17. 王有為, 韓之波, 嚴淑琳, 等. 人臍帶間充質(zhì)干細胞凍存復蘇后的生物學特征. 中國組織工程研究與臨床康復, 2010, 14(10): 1729-1733.
  18. 18. Karlmark KR, Freilinger A, Marton E, et al. Activation of ectopic Oct- 4 and Rex-1 promoters in human amniotic fluid cells. Int J Mol Med, 2005, 16(6): 987-992.