留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

小鼠羊水间充质干细胞的分离、培养及鉴定

吴航飞 王康淳 潘崎 程颖

吴航飞, 王康淳, 潘崎, 等. 小鼠羊水间充质干细胞的分离、培养及鉴定[J]. 器官移植, 2022, 13(1): 67-73. doi: 10.3969/j.issn.1674-7445.2022.01.011
引用本文: 吴航飞, 王康淳, 潘崎, 等. 小鼠羊水间充质干细胞的分离、培养及鉴定[J]. 器官移植, 2022, 13(1): 67-73. doi: 10.3969/j.issn.1674-7445.2022.01.011
Wu Hangfei, Wang Kangchun, Pan Qi, et al. Isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cells[J]. ORGAN TRANSPLANTATION, 2022, 13(1): 67-73. doi: 10.3969/j.issn.1674-7445.2022.01.011
Citation: Wu Hangfei, Wang Kangchun, Pan Qi, et al. Isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cells[J]. ORGAN TRANSPLANTATION, 2022, 13(1): 67-73. doi: 10.3969/j.issn.1674-7445.2022.01.011

小鼠羊水间充质干细胞的分离、培养及鉴定

doi: 10.3969/j.issn.1674-7445.2022.01.011
基金项目: 

辽宁省教育厅科学研究项目 FWZR2020002

详细信息
    作者简介:

    吴航飞,男,1996年生,硕士,研究方向为器官移植,Email:wu452678240@163.com

    通讯作者:

    程颖,女,1975年生,博士,研究员,研究方向为器官移植,Email:chengying75@sina.com

  • 中图分类号: R617, R392.2

Isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cells

More Information
  • 摘要:   目的  探讨小鼠羊水间充质干细胞(AF-MSC)的分离、培养及鉴定。  方法  在无菌条件下获取孕鼠子宫,收集羊水后进行过滤和离心,对沉淀细胞团进行培养并传代。观察AF-MSC的形态,分析AF-MSC的增殖特点,采用流式细胞术鉴定AF-MSC的表面标志物,检测AF-MSC的三系分化能力及冷冻复苏后的细胞活力。  结果  小鼠AF-MSC呈典型的梭形,融合度 > 80%时会出现典型的漩涡状结构。小鼠AF-MSC传代培养无明显潜伏期,培养2~3 d进入对数增长期,增长速度最快,之后增殖速度减慢,进入平台期。AF-MSC表达干细胞抗原(Sca)-1、CD29、CD44,不表达CD34、CD45。小鼠AF-MSC成骨分化后,矿化结晶被茜素红染成深红色的点状;成软骨分化后,分泌的酸性粘多糖被阿利新蓝染成淡蓝色;成脂分化后,胞质脂滴被油红O染成红色。细胞冷冻复苏后存活率 > 95%,生长状态良好,6 d时增殖能力高于冻存前(P < 0.05),其他时间增殖能力与冻存前比较,差异无统计学意义(均为P > 0.05)。  结论  本实验成功分离了小鼠AF-MSC,过程简便、成本低,且分离的细胞可随传代次数的增加而纯化,冻存不影响其增殖能力。

     

  • 图  1  AF-MSC的形态

    注:A、C、E图分别为P0、P2、P4 AF-MSC形态(×100);B、D、F图为P0、P2、P4 AF-MSC形态(×200)。

    Figure  1.  The morphology of AF-MSC

    图  2  AF-MSC的增殖曲线

    注:与P3比较,aP<0.05/3。

    Figure  2.  Proliferation curves of AF-MSC

    图  3  AF-MSC表面标志物的流式细胞图

    Figure  3.  Flow cytometry of surface markers of AF-MSC

    图  4  AF-MSC的三系分化染色图

    注:A图为AF-MSC茜素红染色图(×200);B图为AF-MSC阿利新蓝染色图(×100);C图为AF-MSC油红O染色图(×200)。

    Figure  4.  The trilineage differentiation figures of AF-MSC

  • [1] FU X, LIU G, HALIM A, et al. Mesenchymal stem cell migration and tissue repair[J]. Cells, 2019, 8(8): 784. DOI: 10.3390/cells8080784.
    [2] ARAÚJO AB, SALTON GD, FURLAN JM, et al. Comparison of human mesenchymal stromal cells from four neonatal tissues: amniotic membrane, chorionic membrane, placental decidua and umbilical cord[J]. Cytotherapy, 2017, 19(5): 577-585. DOI: 10.1016/j.jcyt.2017.03.001.
    [3] VALIULIENĖ G, ZENTELYTĖ A, BERŽANSKYTĖ E, et al. Metabolic profile and neurogenic potential of human amniotic fluid stem cells from normal vs. fetus-affected gestations[J]. Front Cell Dev Biol, 2021, 9: 700634. DOI: 10.3389/fcell.2021.700634.
    [4] FRIEDENSTEIN AJ, CHAILAKHYAN RK, LATSINIK NV, et al. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. cloning in vitro and retransplantation in vivo[J]. Transplantation, 1974, 17(4): 331-340. DOI: 10.1097/00007890-197404000-00001.
    [5] 薛玲玲, 陈锦阳, 庄盼, 等. 人羊膜上皮细胞和人羊膜间充质干细胞的研究进展[J/CD]. 中华细胞与干细胞杂志(电子版), 2021, 11(3): 184-188. DOI: 10.3877/cma.j.issn.2095-1221.2021.03.008.

    XUE LL, CHEN JY, ZHUANG P, et al. Updated progress of human amniotic epithelial cells and human amniotic mesenchymal stem cells[J/CD]. Chin J Cell Stem Cell, 2021, 11(3): 184-188. DOI: 10.3877/cma.j.issn.2095-1221.2021.03.008.
    [6] MUCIENTES A, HERRANZ E, MORO E, et al. Influence of mesenchymal stem cell sources on their regenerative capacities on different surfaces[J]. Cells, 2021, 10(2): 481. DOI: 10.3390/cells10020481.
    [7] 华天桢, 马雨诗, 房贺. 干细胞在肝损伤治疗中的应用与机制[J]. 实用医学杂志, 2021, 37(7): 839-844. DOI: 10.3969/j.issn.1006-5725.2021.07.003.

    HUA TZ, MA YS, FANG H. The mechanism and progress of stem cells in the treatment of hepatocyte injury[J]. J Pract Med, 2021, 37(7): 839-844. DOI: 10.3969/j.issn.1006-5725.2021.07.003.
    [8] SHEN C, YANG C, XU S, et al. Comparison of osteogenic differentiation capacity in mesenchymal stem cells derived from human amniotic membrane (AM), umbilical cord (UC), chorionic membrane (CM), and decidua (DC)[J]. Cell Biosci, 2019, 9: 17. DOI: 10.1186/s13578-019-0281-3.
    [9] MATHEW SA, NAIK C, CAHILL PA, et al. Placental mesenchymal stromal cells as an alternative tool for therapeutic angiogenesis[J]. Cell Mol Life Sci, 2020, 77(2): 253-265. DOI: 10.1007/s00018-019-03268-1.
    [10] CALCAT-I-CERVERA S, SANZ-NOGUÉS C, O'BRIEN T. When origin matters: properties of mesenchymal stromal cells from different sources for clinical translation in kidney disease[J]. Front Med (Lausanne), 2021, 8: 728496. DOI: 10.3389/fmed.2021.728496.
    [11] FU YX, JI J, SHAN F, et al. Human mesenchymal stem cell treatment of premature ovarian failure: new challenges and opportunities[J]. Stem Cell Res Ther, 2021, 12(1): 161. DOI: 10.1186/s13287-021-02212-0.
    [12] KUNISAKI SM. Amniotic fluid stem cells for the treatment of surgical disorders in the fetus and neonate[J]. Stem Cells Transl Med, 2018, 7(11): 767-773. DOI: 10.1002/sctm.18-0018.
    [13] DE COPPI P, BARTSCH G JR, SIDDIQUI MM, et al. Isolation of amniotic stem cell lines with potential for therapy[J]. Nat Biotechnol, 2007, 25(1): 100-106. DOI: 10.1038/nbt1274.
    [14] ALESSIO N, PIPINO C, MANDATORI D, et al. Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: an in vitro study[J]. J Cell Physiol, 2018, 233(11): 8996-9006. DOI: 10.1002/jcp.26845.
    [15] PRATHEESH MD, DUBEY PK, GADE NE, et al. Comparative study on characterization and wound healing potential of goat (Capra hircus) mesenchymal stem cells derived from fetal origin amniotic fluid and adult bone marrow[J]. Res Vet Sci, 2017, 112: 81-88. DOI: 10.1016/j.rvsc.2016.12.009.
    [16] PARK HJ, CHO HY, CHA DH. The amniotic fluid cell-free transcriptome provides novel information about fetal development and placental cellular dynamics[J]. Int J Mol Sci, 2021, 22(5): 2612. DOI: 10.3390/ijms22052612.
    [17] LOUKOGEORGAKIS SP, DE COPPI P. Stem cells from amniotic fluid--potential for regenerative medicine[J]. Best Pract Res Clin Obstet Gynaecol, 2016, 31: 45-57. DOI: 10.1016/j.bpobgyn.2015.08.009.
    [18] AZARGOON A, NEGAHDARI B. Lung regeneration using amniotic fluid mesenchymal stem cells[J]. Artif Cells Nanomed Biotechnol, 2018, 46(3): 447-451. DOI: 10.1080/21691401.2017.1337023.
    [19] PARK J, JUN EK, SON D, et al. Overexpression of Nanog in amniotic fluid-derived mesenchymal stem cells accelerates dermal papilla cell activity and promotes hair follicle regeneration[J]. Exp Mol Med, 2019, 51(7): 1-15. DOI: 10.1038/s12276-019-0266-7.
    [20] TAKOV K, HE Z, JOHNSTON HE, et al. Small extracellular vesicles secreted from human amniotic fluid mesenchymal stromal cells possess cardioprotective and promigratory potential[J]. Basic Res Cardiol, 2020, 115(3): 26. DOI: 10.1007/s00395-020-0785-3.
    [21] FEI X, CAI Y, LIN F, et al. Amniotic fluid mesenchymal stem cells repair mouse corneal cold injury by promoting mRNA N4-acetylcytidine modification and ETV4/JUN/CCND2 signal axis activation[J]. Hum Cell, 2021, 34(1): 86-98. DOI: 10.1007/s13577-020-00442-7.
    [22] HAWKINS KE, CORCELLI M, DOWDING K, et al. Embryonic stem cell-derived mesenchymal stem cells (MSCs) have a superior neuroprotective capacity over fetal MSCs in the hypoxic-ischemic mouse brain[J]. Stem Cells Transl Med, 2018, 7(5): 439-449. DOI: 10.1002/sctm.17-0260.
    [23] HUANG B, DING C, ZOU Q, et al. Human amniotic fluid mesenchymal stem cells improve ovarian function during physiological aging by resisting DNA damage[J]. Front Pharmacol, 2020, 11: 272. DOI: 10.3389/fphar.2020.00272.
    [24] SHENDE P, SUBEDI M. Pathophysiology, mechanisms and applications of mesenchymal stem cells for the treatment of spinal cord injury[J]. Biomed Pharmacother, 2017, 91: 693-706. DOI: 10.1016/j.biopha.2017.04.126.
    [25] KANGARI P, TALAEI-KHOZANI T, RAZEGHIAN-JAHROMI I, et al. Mesenchymal stem cells: amazing remedies for bone and cartilage defects[J]. Stem Cell Res Ther, 2020, 11(1): 492. DOI: 10.1186/s13287-020-02001-1.
    [26] TRACY SA, AHMED A, TIGGES JC, et al. A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: bone marrow and amniotic fluid[J]. J Pediatr Surg, 2019, 54(1): 86-90. DOI: 10.1016/j.jpedsurg.2018.10.020.
    [27] DOMINICI M, LE BLANC K, MUELLER I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[J]. Cytotherapy, 2006, 8(4): 315-317. DOI: 10.1080/14653240600855905.
    [28] GALIPEAU J, SENSÉBÉ L. Mesenchymal stromal cells: clinical challenges and therapeutic opportunities[J]. Cell Stem Cell, 2018, 22(6): 824-833. DOI: 10.1016/j.stem.2018.05.004.
  • 加载中
图(5)
计量
  • 文章访问数:  88
  • HTML全文浏览量:  79
  • PDF下载量:  20
  • 被引次数: 0
出版历程
  • 收稿日期:  2021-09-22
  • 网络出版日期:  2022-01-12
  • 刊出日期:  2022-01-15

目录

    /

    返回文章
    返回