中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (14): 2601-2604.doi: 10.3969/j.issn.1673-8225.2011.14.028

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

逆向解剖法游离股骨/胫骨快捷制备小鼠骨髓细胞

付必莽1，苏莹珍2，何小文3，胡安斌4，何晓顺4，黄洁夫4   

  1. 1昆明医学院第二附属医院肝胆胰外科，云南省肝胆胰外科研究所，云南省昆明市650101
    2南方医科大学热带公共卫生学院流行病学系，广东省广州市  510000
    3中山大学附属第六医院结直肠外科，广东省广州市510655
    4中山大学附属第一医院，广东省广州市510080
  • 收稿日期:2010-12-29 修回日期:2011-02-10 出版日期:2011-04-02 发布日期:2013-11-02
  • 作者简介:付必莽☆，男，1972年生，云南省会泽县人，汉族，2009年中山大学外科学专业毕业，博士，主要从事肝脏外科疾病基础与临床研究。 bmangfu@msn.com
  • 基金资助:

    教育部高等学校博士学科点专项科研基金(新教师类)( 20105317120002)；云南省联合专项面上项目基金(2010ZC127)。

A rapid method for harvesting murine bone marrow cells using retrograde isolation of the femur and tibia

Fu Bi-mang1, Su Ying-zhen2, He Xiao-wen3, Hu An-bin4, He Xiao-shun4, Huang Jie-fu4   

  1. 1Department of Hepatobiliary Surgery, Second Affiliated Hospital of Kunming Medical College, Yunnan Institute of Hepatobiliary Surgery, Kunming  650101, Yunnan Province, China
    2Department of Epidemiology, Institute of Tropical Public Health, Southern Medical University, Guangzhou  510000, Guangdong Province, China
    3Department of Colorectal Surgery, Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou  510655, Guangdong Province, China
    4First Affiliated Hospital of Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China
  • Received:2010-12-29 Revised:2011-02-10 Online:2011-04-02 Published:2013-11-02
  • About author:He Bi-mang☆, Doctor, Department of Hepatobiliary Surgery, Second Affiliated Hospital of Kunming Medical College, Yunnan Institute of Hepatobiliary Surgery, Kunming 650101, Yunnan Province, China bmangfu@msn.com
  • Supported by:

    Science and Technology Foundation for Doctors, Ministry of Education, No. 20105317120002*; Yunan United Special Foundation, No. 2010ZC127*

PDF

914

被引次数

22

摘要：

背景：如何快速洁净地游离小鼠股骨和胫骨直接影响骨髓细胞缺血时间，虽文献报道可用手术刀片刮除、纱布和纸巾等器物协助去除肌肉组织。但对其具体操作技巧并无详细报道，也无统一简捷的方法。
目的：寻找制备小鼠骨髓细胞快捷实用的方法，最大限度减少骨髓细胞缺血时间。
方法：采用逆向肌肉剥离法由踝关节向髋关节逆行解剖，以镊子配合剪刀行“剪合-滑动-折转”3个动作，彻底去除附着于小鼠后肢股骨的肌肉组织，快捷制备骨髓细胞悬液。记录解剖骨骼和制备骨髓细胞的时间，所得骨髓细胞培养72 h后光学显微镜观察，评价该制备方法的效果。
结果与结论：骨骼解剖时间为(1.9±0.4) min，骨髓细胞悬液制备时间为(14.7±1.8)min；游离过程骨折断或髓腔破损率为0%；制备的骨髓细胞培养72 h，细胞数量[(8.1±2.0)×107个]较培养前[(4.7±0.7)×107]明显增加(P < 0.01)，而活细胞比率在培养前(97.5±1.1)%和培养后(95.4±1.0)%无明显差异(P > 0.05)；镜下微生物污染率为0%，未见肌肉细胞等污染。结果显示该方法快捷实用、不易污染、操作容易程序化，具有很好的参考价值。

关键词: 逆向解剖, 胫骨/股骨, 快捷, 小鼠, 骨髓细胞

Abstract:

BACKGROUND: There are no detailed reports about how to rapidly isolate muscle tissues from the femur and tibia which directly influences the ischemic period of bone marrow cells in mice.
OBJECTIVE: To investigate the rapidest and feasible method for harvesting murine bone marrow cells (BMCs) in order to decrease the ischemia injury of BMCs.
METHODS: Muscle and connective tissues surrounding the femur and tibia were entirely removed by using an initiated retrograde method by which dissection of bones were started at ankle joints and ended at hip joints. The key point, in the whole isolating procedure, was “crossing → slipping → replicating” with appropriate force with ophthalmic scissors worked in coordination with fine tissue forceps. Time of entirely isolating the femur and tibia as well as time of harvesting BMCs were recorded. The obtained BMCs were cultured in 25 cm2 corning flasks using RPMI1640 with 10% fetal bovine serum, and after 72 hours, the cells were stained with trypan blue and the survival rate was detected with cell counter.
RESULTS AND CONCLUSION: Time of isolating the femurs and tibia and time of harvesting BMCs were (1.9±0.4) minutes and (14.7±1.8) minutes, respectively. No bone or marrow cavity was injured or contaminated. Compared with before culture, the number of BMCs after 72 hours increased significantly [(8.1±2.0)×107 vs. (4.7±0.7)×107, P < 0.01], while the cell survival rate did not change remarkably [(97.5±1.1)% vs. (95.4±1.0)%, P > 0.05]. No visible microbe was found in the culture media. To harvest mouse BMCs using the retrograde isolation of the femur and tibia is a practical, feasible and rapid method.

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