中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (45): 8455-8459.doi: 10.3969/j.issn.1673-8225.2010.45.022

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

小鼠骨髓源性树突状细胞培养与冻存　

朱国辉，翁小东，刘修恒，匡幼林   

  1. 武汉大学人民医院泌尿外科，湖北省武汉市 430060
  • 出版日期:2010-11-05 发布日期:2010-11-05
  • 通讯作者: 刘修恒，教授，主任医师，博士生导师，武汉大学人民医院泌尿外科，湖北省武汉市430060 drliuxh@hotmail.com
  • 作者简介:朱国辉★，男，1983年生，河北省围场县人，满族，武汉大学第一临床学院在读硕士，主要从事树突状细胞与肿瘤疫苗的研究。 drzhugh@hotmail.com
  • 基金资助:

    国家自然科学基金(30672107)，课题名称：新型树突状细胞疫苗治疗前列腺癌及其机制的研究。

Culture and cryopreservation of mouse bone marrow-derived dendritic cells

Zhu Guo-hui, Weng Xiao-dong, Liu Xiu-heng, Kuang You-lin　   

  1. Department of Urinary Surgery, Renmin Hospital of Wuhan University, Wuhan  430060, Hubei Province, China
  • Online:2010-11-05 Published:2010-11-05
  • Contact: Liu Xiu-heng, Professor, Chief physician, Doctoral supervisor, Department of Urinary Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China drliuxh@hotmail.com
  • About author:Zhu Guo-hui★, Studying for master’s degree, Department of Urinary Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China drzhugh@hotmail.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30672107*

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摘要：

背景：树突状细胞因其良好的抗原提呈功能及促T细胞增殖功能，成为肿瘤抗原的良好载体。获得大量的树突状细胞，对于肿瘤疫苗的研制具有重要作用。
目的：对小鼠骨髓树突状细胞进行诱导、培养、扩增及冻存，并对比冻存后复苏细胞与未冻存细胞的细胞特性，寻找一种能够大量获得树突状细胞及有效储存的方法。
方法：取小鼠骨髓细胞，在含重组小鼠粒-巨噬细胞集落刺激因子(10 μg/L)和重组小鼠白细胞介素4(5 μg/L)的完全培养基中对其进行诱导、培养及扩增。培养第6天，对培养获得的树突状细胞在加有冷冻保护剂DMSO的完全培养液中冻存。复苏后，在培养基中加入脂多糖、对细胞诱导，获得大量的成熟树突状细胞，最终获得的树突状细胞与未进行冻存的细胞在细胞活力、形态学、细胞表型、混合淋巴细胞反应等方面对比。
结果与结论：复苏后，(82.2±4.73)%细胞存活，存活的细胞经脂多糖诱导后发育为成熟树突状细胞，在形态学、细胞表型及混合淋巴细胞反应等方面与未经冻存的树突状细胞差异无显著性意义。提示小鼠骨髓源性树突状细胞冻存复苏后，细胞生物学特性与未冻存的细胞无明显差别，用冻存的方法储存树突状细胞，能够避免在不同时期应用细胞时反复进行培养，可以获得大量的同质细胞。

关键词: 树突状细胞, 骨髓细胞, 培养, 冻存, 细胞保存

Abstract:

BACKGROUND: Dendritic cell is an ideal carrier of tumor antigen due to the presenting function and the promotion of T cell proliferation. How to obtain large numbers of dendritic cells is important for producing tumor vaccine.
OBJECTIVE: To induce, culture, amplify and cryopreserve dendritic cells derived from mouse bone marrow, then compare the biological characters of cryopreserved dendritic cells with the fresh dendritic cells, than find an effective method to obtain and store large numbers of dendritic cells.
METHODS: Dendritic cells from mouse bone marrow cells were induced, cultured and amplified in complete medium with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (10 μg/L) and recombinant mouse interleukin 4 (rmIL-4) (5 μg/L). On day 6, the dendritic cells were frozen in complete medium with dimethyl sulphoxide. After thawing, lipopolysaccharide was added to induce the maturation. At last, large number of mature dendritic cells was obtained. Then the viability, morphology, dendritic cells markers and mixed lymphocyte reaction were compared between the thawed cells and fresh cells.
RESULTS AND CONCLUSION: After thawing, the recovery rate of cells was (82.2±4.73)%. The survival cells became mature dendritic cells induced by lipopolysaccharide. Compared with fresh dendritic cells, there was no significant difference in morphology, dendritic cells markers and mixed lymphocyte reaction. Results suggest that there was no significant difference in biological characters between frozen-thawed dendritic cells and fresh dendritic cells. To store dendritic cells through cryopreservation can avoid repeating culture in different times and can obtain large numbers of dendritic cells.

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