中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (13): 2068-2074.doi: 10.3969/j.issn.2095-4344.0508

• 造血干细胞 hematopoietic stem cells • 上一篇    下一篇

不同共培养模式下间充质干细胞对造血干细胞增殖的作用

汪姝玥1，林凡莉1，钱 怡1，陈小青1，刘 洋1，李书坛1，程 艳1，熊 皓1，黄纯兰1，2   

  1. 1西南医科大学附属医院血液内科，四川省泸州市 646000；2成都市第三人民医院血液内科，四川省成都市 610031
  • 修回日期:2018-03-19 出版日期:2018-05-08 发布日期:2018-05-08
  • 通讯作者: 黄纯兰，博士，主任医师，教授，硕士生导师，西南医科大学附属医院血液内科，四川省泸州市 646000；成都市第三人民医院血液内科，四川省成都市 610031
  • 作者简介:汪姝玥，女，1992年生，四川省攀枝花市人，汉族，西南医科大学血液内科在读硕士，主要从事骨髓微环境及干细胞相关研究。
  • 基金资助:

    国家自然科学基金资助项目(81450030)；四川省教育厅重大培育项目(14CZ0017)

Coculture with mesenchymal stem cells facilitates the proliferation of hematopoietic stem cells under different coculture modes

Wang Shu-yue1, Lin Fan-li1, Qian Yi1, Chen Xiao-qing1, Liu Yang1, Li Shu-tan1, Cheng Yan1, Xiong Hao1, Huang Chun-lan1, 2   

  1. 1Department of Hematology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Department of Hematology, Third People’s Hospital of Chengdu, Chengdu 610031, Sichuan Province, China
  • Revised:2018-03-19 Online:2018-05-08 Published:2018-05-08
  • Contact: Huang Chun-lan, M.D., Chief physician, Professor, Master’s supervisor, Department of Hematology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Department of Hematology, Third People’s Hospital of Chengdu, Chengdu 610031, Sichuan Province, China
  • About author:Wang Shu-yue, Master candidate, Department of Hematology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81450030; the Major Culture Program of Sichuan Provincial Education Department, No. 14CZ0017

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摘要：

文章快速阅读：

文题释义：
骨髓微环境：在体内，造血干细胞的增殖分化等生理活动受周围环境的控制。骨髓微环境或造血龛由特定的空间结构和细胞组成，包括间质细胞、细胞外的基质和可溶性的能与膜结合的细胞因子。间质细胞由不同的细胞群组成，如间充质干细胞(MSC)、成纤维细胞、平滑肌细胞、脂肪细胞、成骨前体细胞、内皮细胞。造血龛被认为是提供复杂的分子信号确定干细胞自我更新和分化成为成熟的功能细胞。因此，一个体外模拟体内造血微环境的模型，需要一个能够支持干、祖、前体和成熟细胞的复杂的多向组成成分。
共培养：将间充质干细胞与造血干细胞通过各种方式进行共同体外培养，已建立的培养模式包括：非接触共培养、分泌物共培养、接触共培养(2D)、借助各种基质胶的3D共培养、细胞团包裹法的3D共培养等。

 

摘要
背景：虽然进行了大量相关研究，但目前仍缺乏切实可行的体外扩增造血干细胞的方法。间充质干细胞能分泌多种促进造血干细胞增殖并抑制其分化的细胞因子，在维持造血微环境及调控造血干细胞功能中发挥着重要的作用。
目的：探讨不同共培养模式下骨髓间充质干细胞在体外对造血干细胞增殖的影响。
方法：采用全骨髓贴壁法体外培养C57BL/6小鼠间充质干细胞至P3代，采用miniMACS磁珠分选仪分选GFP小鼠(C57系)骨髓CD117+细胞(造血干细胞)，采用不同共培养模式将2种细胞进行共培养：对照组为造血干细胞单独培养组；实验组为Transwell共培养组(上室接种造血干细胞、下室接种间充质干细胞)；2D接触共培养组(24孔板共同接种造血干细胞与间充质干细胞)。分别于共培养1，3，5，7 d于倒置相差显微镜、荧光显微镜下观察造血干细胞的形态，并检测造血干细胞的活性细胞数。
结果与结论：共培养1-7 d，各组造血干细胞数量均随着培养时间的增加而增加(P < 0.05)。各组细胞自3 d开始进入对数生长期，5 d时部分造血干细胞开始出现形态变化。比较第7天造血干细胞活性细胞数，可知与间充质干细胞共培养的实验组及2D接触共培养组均明显高于对照组(P < 0.05)：而2D接触共培养组造血干细胞数明显高于非接触培养的实验组(P < 0.05)。结果提示：间充质干细胞在体外能够有效促进造血干细胞增殖，接触共培养条件下其促进作用更明显。

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: 0000-0002-5273-5872(汪姝玥)

关键词: 造血干细胞, 间充质干细胞, 共培养, 接触共培养组, 3D共培养, Transwell共培养, 饲养层

Abstract:

BACKGROUND: Although a large number of related studies have been carried out, there is still a lack of practical methods to amplify hematopoietic stem cells (HSCs) in vitro. Mesenchymal stem cells (MSCs) secrete a variety of cytokines that promote the HSCs proliferation and inhibit their differentiation. These cytokines play an important role in maintaining the hematopoietic microenvironment and regulating HSCs function.
OBJECTIVE: To investigate the effect of bone marrow MSCs on the proliferation of HSCs in vitro under different coculture modes.
METHODS: Mesenchymal stem cells from the bone marrow of C57BL/6 mice were cultured in vitro using the whole bone marrow adherent culture. CD117+ cells (HSCs) were sorted from passage 3 cells by using miniMACS magnetic beads sorting. Then, CD117+ cells were co-cultured with MSCs under different coculture models, including single culture of HSCs (control group), Transwell coculture (upper chamber, HSCs; lower chamber, MSCs) and two-dimensional contact coculture (coculturing HSCs and MSCs in 24-well plates). The morphology of HSCs was observed under phase contrast microscope and fluorescence microscope, and the number of active cells of HSCs was counted at 1, 3, 5, and 7 days after coculture.
RESULTS AND CONCLUSION: During the coculture of 1-7 days, the number of HSCs in the two groups was increased with culture time (P < 0.05). After 3 days of coculture, HSCs in each group was grown into the logarithmic growth phase, and morphological changes in some HSCs were detected at 5 days of coculture. At 7 days of coculture, the viabilities of HSCs in different culture models were ranked as follows: single culture model < Transwell coculture model < two-dimensional contact coculture model (P < 0.05). These findings suggest that MSCs can effectively promote the proliferation of HSCs in vitro, and the promotion effect is increased under contact coculture conditions.

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: Hematopoietic Stem Cells, Mesenchymal Stem Cells, Stem Cells, Cell Culture Techniques, Coculture Techniques

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