课题背景：本项目获得湖北省自然科学基金、湖北省教育厅基金、湖北省科技攻关项目基金的资助，文章是此项研究的一个组成部分，探索骨髓和外周血培养间充质干细胞的可靠的方法，进一步了解造血生长因子动员间充质干细胞的机制和效率。

应用要点：①实验结果提示粒细胞集落刺激因子具有动员间充质干细胞的能力，培养的外周血成纤维样细胞集落符合公认的间充质干细胞的特性。②提高培养环境的黏附性将提高外周血成纤维样细胞集落出现频率，为使用造血生长因子及其组合改善外周血造血干细胞移植物提供实验基础。

偏倚或不足：①本文使用了鼠尾胶包被培养板，以提高培养环境的黏附性，由于鼠尾胶成分复杂，究竟是鼠尾胶中的何种成份提高了环境的黏附性有待考察。②全血细胞接种法由于未去除大量红细胞而使观察细胞生长受到一定影响。

摘要

背景：动物和人外周血中分离出的间充质干细胞生长繁殖受分离方法、培养条件等因素的影响。
目的：采用重组人粒细胞集落刺激因子动员小鼠外周血间充质干细胞,体外培养条件中加入鼠尾胶包被，观察其对细胞分离、培养、增殖的影响。
设计、时间及地点：开放性实验，于2005-05/2007-12在郧阳医学院附属人民医院临床医学研究所完成。
材料：昆明小鼠20只随机分为鼠尾胶包被组和不包被对照组，每组10只。
方法：小鼠皮下注射重组人粒细胞集落刺激因子80 μg /(kg·d)连续动员5 d，最后一次注射后6 h取外周血。肝素抗凝后，采用全血细胞分离培养，用含体积分数为0.15胎牛血清DMEM培养基，分别接种于包被和未包被鼠尾胶的12孔板，接种密度为1×105/孔，48 h后半量换液以后每3天换液1次，培养10 d。
主要观察指标：①鼠尾胶包被的培养板对形成成纤维样细胞集落的影响。②成纤维样细胞集落中的间充质干细胞表面标记流式检测结果。③成纤维样细胞集落成骨、成脂肪诱导后嗜银法和油红O法染色鉴定。
结果：①使用鼠尾胶包被的培养板可增加重组人粒细胞集落刺激因子动员后外周血成纤维样细胞集落出现的频率（Ｐ < 0.05）。②经流式细胞术鉴定形成成纤维样细胞集落的细胞不表达CD34、CD133,表达CD90、CD105。③成骨、成脂肪诱导后细胞嗜银法和油红O法染色鉴定均呈阳性。
结论：培养的细胞符合公认间充质干细胞的特征，鼠尾胶可增加培养皿的黏附性促进间充质干细胞分离。
关键词：间充质干细胞；集落形成单位测定；粒细胞集落刺激因子

刘岐焕，陈龙，程范军，唐俊明，王家宁，高清平.鼠尾胶在小鼠外周血间充质干细胞分离培养中的作用[J].中国组织工程研究与临床康复，2008，12(21):4017-4020 [www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4017(ps).pdf]

Effect of rat tail collagen on the isolation and culture of mesenchymal stem cells from mouse peripheral blood

Abstract

BACKGROUND: Culture of mesenchymal stem cells (MSCs) derived from animals or human peripheral blood may be influenced by many factors, such as isolation method and culture condition.
OBJECTIVE: To investigate the effect on the isolation, culture and proliferation of mouse peripheral blood MSCs mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) after rat tail collagen (RTC) is added for in vitro culture.
DESIGN, TIME AND SETTING: An open trial was carried out at the Institute of Clinical Medicine in the Affiliated People’s Hospital of Yunyang Medical College between May 2005 and December 2007.
MATERIALS: Twenty Kunming mice were divided into RTC-coated group and uncoated control group (n=10).
METHODS: Mice were injected subcutaneously with rhG-CSF at a dose of 80 μg/kg per day, once daily for 5 days. The peripheral blood samples were obtained 6 hours after the final administration. The cells were anticoagulated by heparin, and then whole blood culture was used to isolate MSCs. Peripheral blood mononuclear cells were seeded into a 12-well plate at a concentration of 1×105 cells per well in DMEM containing 0.15 volume fraction of feral bovine serum, coated with RTC or without, respectively. Half medium was refreshed 48 hours later and then changed completely every 3 days. Cells were cultured for 10 days.
MAIN OUTCOME MEASURES: ①Evaluation of the colony-forming units fibroblast (CFU-F) on the culture plate coated with RTC.②Flow cytometry detection of MSCs surface label in CFU-F.③Differentiation of CFU-F into adipocyte and osteocytes identified by oil red O staining and von Kossa’s staining.
RESULTS: After mobilization with rhG-CSF, the CFU-F frequency of peripheral blood was increased on the culture plate coated with RTC compared with that without coating (P < 0.05). Flow cytometry detection showed that MSCs were positive for CD90, CD105 and negative for CD34 and CD133. MSCs were found to differentiate into adipocyte and osteocyte.
CONCLUSION: The cultured MSCs are consistent with the acknowledged MSC characteristics, RTC can increase the adhesive property of culture plate and promote the isolation of MSCs.

Liu QH, Chen L, Cheng FJ, Tang JM, Wang JN, Gao QP.Effect of rat tail collagen on the isolation and culture of mesenchymal stem cells from mouse peripheral blood.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(21):4017-4020(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4017(ps).pdf]