人脐带沃顿胶间充质干细胞体外分离条件的优化及传代效应的流式细胞学检测
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摘要
目的观察不同胶原酶消化方法对人脐带沃顿胶间充质干细胞(mesenchymal stem cell,MSC)分离和培养结果的影响,并鉴定其分化潜能;探讨传代效应对其免疫表型的影响。方法将制备好的脐带标本分别加入Ⅰ型、Ⅱ型及Ⅳ型胶原酶,持续消化4~18 h,筛网过滤,离心收集细胞,用DMEM/F12培养基重悬细胞,调整细胞密度4.8×103~1×104/cm2,接种培养,比较不同消化法分离人脐带沃顿胶MSC的效果。Von kossa钙结节染色、四环素荧光标记鉴定人脐带沃顿胶MSC向成骨方向分化的能力,RT-PCR鉴定其向心肌细胞分化的能力。应用流式细胞仪检测连续传代后MSC的免疫表型变化。结果Ⅰ型胶原酶消化法能够从人脐带沃顿胶获取了数量较多、活力较高的MSC,而且细胞出现伸展的时间及原代培养时间均短于Ⅱ型胶原酶及Ⅳ型胶原酶消化法。表面标记分析显示:随着传代次数的增加,阳性标记CD29、CD44、CD73、CD90、CD105的表达百分率没有变化,而阴性标记CD31、CD34和HLA-DR的表达率增加明显。体外诱导实验表明:来源于人脐带沃顿胶的MSC具有体外成骨和成心肌样细胞分化的能力。结论Ⅰ型胶原酶消化法简单易行,对细胞损伤小,能稳定、高效地从人脐带沃顿胶中分离出MSC。
Objective To observe the effects of different collagenase digestions on isolating human umbilical cordmesenchymal stromal cells(MSC) from Wharton's jelly, to exam their differentiation ability and to investigate their passageeffect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠ or Ⅱor Ⅳ for4-18 hours then were passed through sieves. Cells were collected by centrifugation then inoculated in DMEM/F12 mediumat concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa stainingand tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employedto identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected byflow cytometry after subculture. Results Using collagenaseⅠ digestion, the number of MSCs isolated from human umbilicalcord in Wharton's jelly and their vitality were much higher while the period to show cell extension and primary culture timewere shorter than those using collagenaseⅡ or Ⅳ digestions. The analysis of surface marker revealed that the expression ofpositive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markerssuch as CD31, CD34 and HLA-DR increased significantly with passages; Differential experiments induced in vitro show thathuman umbilical cord MSC in wharton's jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Conclusion The human umbilical cord MSC in Wharton's jelly was successfully isolated by collagenaseⅠ digestion. This meth-od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possible. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.
Objective To observe the effects of different collagenase digestions on isolating human umbilical cordmesenchymal stromal cells(MSC) from Wharton's jelly, to exam their differentiation ability and to investigate their passageeffect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠ or Ⅱor Ⅳ for4-18 hours then were passed through sieves. Cells were collected by centrifugation then inoculated in DMEM/F12 mediumat concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa stainingand tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employedto identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected byflow cytometry after subculture. Results Using collagenaseⅠ digestion, the number of MSCs isolated from human umbilicalcord in Wharton's jelly and their vitality were much higher while the period to show cell extension and primary culture timewere shorter than those using collagenaseⅡ or Ⅳ digestions. The analysis of surface marker revealed that the expression ofpositive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markerssuch as CD31, CD34 and HLA-DR increased significantly with passages; Differential experiments induced in vitro show thathuman umbilical cord MSC in wharton's jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Conclusion The human umbilical cord MSC in Wharton's jelly was successfully isolated by collagenaseⅠ digestion. This meth-od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possible. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.
引文
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[14]Pang RQ,He J,Li FB,et al.A simple method for isolation of humanumbilical cord mesenchymal stem cell[J].Chin J Cell Stem Cell(Electronic Edition),2011,1(2):30-33.[庞荣清,何洁,李福兵,等.一种简单的人脐带间充质干细胞分离培养方法[J].中华细胞与干细胞杂志(电子版),2011,1(2):30-33].
[15]Chen LM,Xu RN,Lv S,et al.Role of human umbilical cord mesen-chymal stem cells in the regulation of activation and apoptosis of he-patic stellate cell[J].Med J Chin PLA,2014,39(1):11-14.[陈黎明,徐若男,吕飒,等.脐带间充质干细胞对肝星状细胞活化、凋亡的调控作用研究[J].解放军医学杂志,2014,39(1):11-14].doi:10.11855/j.issn.0577-7402.2014.01.03.
[2]Wang HS,Hung SC,Peng ST,et al.Mesenchymal stem cells in theWharton's jelly of the human umbilical cord[J].Stem Cells,2004,22(7):1330-1337.
[3]Lu LL,Liu YJ,Yang SG,et al.Isolation and characterization of hu-man umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials[J].Haematologica,2006,91(8):1017-1026.
[4]Zhang YQ,Liang J,Tian QS,et al.Optimization of isolation and culture methods of human umbilical cord-derived mesenchymal stemcells[J].Journal of Mudanjiang Medical University,2013,34(5):7-9.[张亚倩,梁军,田沁森,等.人脐带间充质干细胞分离培养方法的优化[J].牡丹江医学院学报,2013,34(5):7-9].
[5]Yang WC,Dong YH.The research progress of human umbilicalcord mesenchymal stem cells[J].Journal of Tissue Engineering andReconstructive Surgery,2010,6(5):292-294.[杨文成,董有海.人脐带间充质干细胞的研究进展[J].组织工程与重建外科杂志,2010,6(5):292-294].
[6]Wang HS,Hung SC,Peng ST,et al.Mesenchymal stem cells in theWharton's jelly of the human umbilical cord[J].Stem Cells,2004,22(7):1330-1337.
[7]Romanov YA,Svintsitskaya VA,Smirnov VN.Searching for alterna-tive sources of postnatal human mesenchymal stem cells:candidateMSC-like cells from umbilical cord[J].Stem Cells,2003,21(1):105-110.
[8]Covas DT,Siufi JL,Silva AR,et al.Isolation and culture of umbili-cal vein mesenchymal stem cells[J].Braz J Med Biol Res,2003,36(9):1179-1183.
[9]Sarugaser R,Lickorish D,Baksh D,et al.Human umbilical cordperivascular(HUCPV)cells:a source of mesenchymal progenitors[J].Stem Cells,2005,23(2):220-229.
[10]Mitchell KE,Weiss ML,Mitchell BM,et al.Matrix cells from Whar-ton’s jelly form neurons and glia[J].Stem Cells,2003,21(1):50-60.
[11]Sun GD,Li ZZ.The research progress of umbilical cord derived-mesenchymal stem cells in tissue engineering[J].Progress in Mod-ern Biomedicine,2009,9(12):2389-2391.[孙国栋,李志忠.人脐带间充质干细胞在组织工程中的研究进展[J].现代生物医学进展,2009,9(12):2389-2391].
[12]Zheng ZJ,Zhuang WX,Fu WY.Advances of the umbilical cordMesenchymal stem cells[J].Progress of Anatomical Sciences,2008,14(1):100-104.[郑志娟,庄文欣,付文玉.人脐带间充质千细胞的研究进展[J].解剖科学进展,2008,14(1):100-104].
[13]Feng K,Xiao L,Ma XH,et al.Isolation,culture and surface markersdetection of human umbilical cord mesenchymal stem cells[J].Jour-nal of Leukemia&Lymphoma,2013,22(6):354-356.[冯凯,肖漓,马锡慧,等.人脐带间充质干细胞分离培养及表面标志检测[J].白血病·淋巴瘤,2013,22(6):354-356].
[14]Pang RQ,He J,Li FB,et al.A simple method for isolation of humanumbilical cord mesenchymal stem cell[J].Chin J Cell Stem Cell(Electronic Edition),2011,1(2):30-33.[庞荣清,何洁,李福兵,等.一种简单的人脐带间充质干细胞分离培养方法[J].中华细胞与干细胞杂志(电子版),2011,1(2):30-33].
[15]Chen LM,Xu RN,Lv S,et al.Role of human umbilical cord mesen-chymal stem cells in the regulation of activation and apoptosis of he-patic stellate cell[J].Med J Chin PLA,2014,39(1):11-14.[陈黎明,徐若男,吕飒,等.脐带间充质干细胞对肝星状细胞活化、凋亡的调控作用研究[J].解放军医学杂志,2014,39(1):11-14].doi:10.11855/j.issn.0577-7402.2014.01.03.