中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (33): 5292-5296.doi: 10.3969/j.issn.2095-4344.0644

• 胚胎干细胞 embryonic stem cells • 上一篇    下一篇

两种方法培养人胎盘间充质干细胞的生物特性比较

张 飞1，王 武2，李慧娟3，刘亚飞4，张宝刚1，裴富强1，邵青伟1，武忠炎2   

  1. 1西安航天总医院，陕西省西安市 710100；新疆医科大学第五附属医院，2骨科，4修复重建外科，新疆维吾尔自治区乌鲁木齐市 830001；3西电集团医院，陕西省西安市 710100
  • 修回日期:2018-07-05 出版日期:2018-11-28 发布日期:2018-11-28
  • 通讯作者: 王武，硕士，主治医师，新疆医科大学第五附属医院骨科，新疆维吾尔自治区乌鲁木齐市 830001
  • 作者简介:张飞，男，1988年生，陕西省延安市人，汉族，2015年新疆医科大学毕业，硕士，医师，主要从事创伤骨科研究。现工作于西安航天总医院关节外科。 并列第一作者：王武，硕士，主治医师，新疆医科大学第五附属医院骨科，新疆维吾尔自治区乌鲁木齐市 830001
  • 基金资助:

    新疆维吾尔自治区自然科学基金项目(2016D01C239)

Biological characteristics of human placental mesenchymal stem cells isolated using two culture methods: a comparative study

Zhang Fei1, Wang Wu2, Li Hui-juan3, Liu Ya-fei4, Zhang Bao-gang1, Pei Fu-qiang1, Shao Qing-wei1, Wu Zhong-yan2   

  1. 1Xi’an Aerospace General Hospital, Xi’an 710100, Shaanxi Province, China; 2Department of Orthopedics, 4Department of Bone Repair and Remodeling, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830001, Xinjiang Uygur Autonomous Region, China; 3Xi’an XD Group Hospital, Xi’an 710100, Shaanxi Province, China
  • Revised:2018-07-05 Online:2018-11-28 Published:2018-11-28
  • Contact: Wang Wu, Department of Orthopedics, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830001, Xinjiang Uygur Autonomous Region, China
  • About author:Zhang Fei, Master, Physician, Xi'an Aerospace General Hospital, Xi’an 710100, Shaanxi Province, China; Wang Wu, Master, Attending physician, Department of Orthopedics, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830001, Xinjiang Uygur Autonomous Region, China; Zhang Fei and Wang Wu contributed equally to this work.
  • Supported by:

    the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2016D01C239

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摘要：

文章快速阅读：

文题释义：
胎盘间充质干细胞的优势：胎盘源性间充质干细胞可大量获得，具有增殖和分化能力，可与胚胎干细胞相媲美，因此胎盘间充质干细胞已逐渐成为应用前景广阔的种子细胞。根据课题组以往经验，胎盘间充质干细胞的原代培养较骨髓间充质干细胞更为困难，需要保证严格的无菌条件及胎盘组织的新鲜度。
胰酶冷消化法的优势：胰酶具有解离细胞之间糖蛋白和黏蛋白的功能，从而破坏细胞骨架，达到分离细胞的目的，故对细胞膜蛋白有很强的破坏性，容易导致细胞损伤，从而影响细胞的传代和培养。而胰酶冷消化法(4 ℃)具有实验条件简单、胰蛋白酶用量少、节约人力物力等优点，而且对细胞损伤相对较轻。

 

摘要
背景：胎盘间充质干细胞能够为细胞治疗提供理想的种子细胞，但其培养未曾使用过胰酶冷消化法，故对此方法进行探索，并与组织块法进行比较，从中筛选出一种方便易行、培养成功率更高的方法。
目的：观察胰酶冷消化法与组织块法分离培养的人胎盘间充质干细胞的生物学特性。
方法：采用胰酶冷消化法和组织块法从人胎盘中分离、纯化和传代培养人胎盘间充质干细胞(n=6)。记录胎盘间充质干细胞首次出现时间及原代培养周期，绘制第3代细胞生长曲线，流式细胞仪分析第3代细胞表面标志，检测其向成神经及成脂方向诱导分化能力。
结果与结论：①使用上述2种培养方法均可获得胎盘间充质干细胞，胰酶冷消化法首次出现贴壁细胞时间早于组织块法(P < 0.05)，原代培养周期短于组织块法(P < 0.05)；②生长曲线提示在进入平台期后的各个时间点胰酶冷消化法培养的细胞数量明显多于组织块法(P < 0.05)；③2种细胞具有均一的细胞表型，均表达CD29，CD105，不表达CD34，CD45；④2种方法培养的胎盘间充质干细胞经诱导后均具有成神经及成脂分化潜能；⑤上述结果表明，对于胎盘间充质干细胞的培养而言，胰酶冷消化法具有明显优势。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0003-2830-309X(张飞)

关键词: 胎盘间充质干细胞, 组织块法, 胰酶冷消化法, 生物学特性, 干细胞, 新疆维吾尔自治区自然科学基金

Abstract:

BACKGROUND: Placental mesenchymal stem cells can provide ideal seed cells for cell therapy, but trypsin cold digestion has not been used for cell culture. Therefore, we explored this method and compared it with the tissue explant method to screen a more convenient and more successful culture method.
OBJECTIVE: To observe the biological features of human placental mesenchymal stem cells isolated by trypsin cold digestion and tissue explant method.
METHODS: We isolated, purified, and subcultured mesenchymal stem cells (n=6) from human placenta by using trypsin cold digestion method and tissue explant method. The first appearance time and primary culture period of placental mesenchymal stem cells isolated using two methods were recorded. The growth curves of passage 3 cells from two culture methods were generated. Flow cytometry was used to analyze the expression of surface markers of passage 3 cells isolated using the two culture methods, and to study the neural and adipogenic differentiation of the cells.
RESULTS AND CONCLUSION: Placental mesenchymal stem cells could be obtained using both culture methods. Adherent cells in the trypsin cold digestion group appeared significantly earlier than those in the tissue explant group (P < 0.05), and the primary culture period was also significantly shorter in the trypsin cold digestion method (P < 0.05). The growth curve of passage 3 cells showed a higher cell number at plateau growth using trypsin cold digestion than tissue explant method (P < 0.05). The cells isolated using two culture methods were positive for CD29 and CD105, but negative for CD34 and CD45, indicating the same cell phenotypes. After the induction, placental mesenchymal stem cells cultured by both methods had the potential to differentiate into nerve cells and adipocytes. Therefore, both culture methods can produce placental mesenchymal stem cells, but trypsin cold digestion method is more suitable for the culture of this kind of cells as compared with the tissue explant method.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Placenta, Mesenchymal Stem Cells, Cell Culture Techniques, Tissue Engineering

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