中国组织工程研究

中国组织工程研究

• 干细胞培养与分化 • 上一篇    下一篇

人脐带间充质干细胞在3种不同培养体系中的生长状况及腺病毒感染效率

洪敬欣1，张茜真2，3，韩俊领1，4，刘 辉2，刘 剑1，邱录贵1，4   

  1. 1协和干细胞基因工程有限公司，天津市脐带血造血干细胞库，天津市 300384；
    2解放军第二军医大学东方肝胆外科医院病毒基因治疗实验室，上海市 200438；
    3浙江理工大学生命科学学院新元医学与生物技术研究所，浙江省杭州市 310018；
    4中国医学科学院，北京协和医学院，血液学研究所，血液病医院，天津市 300020
  • 出版日期:2010-01-04 发布日期:2010-01-04
  • 通讯作者: 邱录贵，主任，主任医师，教授，博士生导师，协和干细胞基因工程有限公司，天津市脐带血造血干细胞库，天津市 300384；中国医学科学院，北京协和医学院，血液学研究所，血液病医院，天津市 300020 drqiu99@medmail.com.cn
  • 作者简介:洪敬欣☆，女，1978年生，天津市人，汉族，2007年天津医科大学毕业，博士，助理研究员，主要从事血液学及干细胞方面的研究。 hjx78@126.com
  • 基金资助:

    天津市科技创新专项资金（08 FDZDSH03000）。

Growth state and adenovirus infection efficiency of human umbilical cord-derived mesenchymal stem cells in 3 different culture systems

Hong Jing-xin1, Zhang Qian-zhen2,3, Han Jun-ling1,4, Liu Hui2, Liu Jian1, Qiu Lu-gui1,4   

  1. 1Union Stem Cell & Gene Engineering Co.,Ltd., Tianjin Cord Blood Bank, Tianjin   300384, China;
    2Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University of Chinese PLA, Shanghai   200438, China;
    3Xinyuan Institute of Medicine and Biotechnology, College of Life Science, Zhejiang Sci-Tech University, Hangzhou   310018, Zhejiang Province, China;
    4Institute of Hematology, Blood Disease Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin 300020, China
  • Online:2010-01-04 Published:2010-01-04
  • Contact: Qiu Lu-gui, Chief physician, Professor, Doctoral supervisor, Union Stem Cell & Gene Engineering Co.,Ltd., Tianjin Cord Blood Bank, Tianjin 300384, China; Institute of Hematology, Blood Disease Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin 300020, China drqiu99@medmail.com.cn
  • About author:Hong Jing-xin☆, Ph.D., Assistant researcher, Union Stem Cell & Gene Engineering Co.,Ltd., Tianjin Cord Blood Bank, Tianjin 300384, China hjx78@126.com
  • Supported by:

    Special Fund for Scientific and Technological Innovation of Tianjin City, No. 08 FDZDSH03000*

PDF

488

被引次数

13

摘要：

背景：目前所报道的脐带间充质干细胞体外培养条件及培养效率不尽相同，尚缺乏统一标准。而且由于不同来源的间充质干细胞生物学特征尚有一定差异，因此建立脐带间充质干细胞简便、高效的培养体系十分必要。

目的：观察人脐带来源的间充质干细胞在体外不同培养体系中的生长状态，以及不同腺病毒感染的效率。

方法：采用胶原酶消化法从正常足月新生儿脐带中分离出间充质干细胞，贴壁法纯化培养，细胞贴壁后利用低糖DMEM，MesenPRO RS™ Medium和STEMPRO® MSC SFM这3种培养体系进行体外扩增。取对数生长期的第3~5脐带间充质干细胞，应用腺病毒Ad5-EGFP，Ad5/11-EGFP，Ad5/35-EGFP分别以感染复数=1，10，100进行感染，分别于感染后24，56，72 h倒置荧光显微镜观察病毒感染及绿色荧光表达情况。

结果与结论：使用低糖DMEM培养的细胞初期融合时间长，STEMPRO® MSC SFM培养的细胞虽然连接紧密，但消化传代后不易贴壁，而MesenPRO RS™ Medium培养的细胞在相同时间内能达到较高的细胞密度，更适于脐带间充质干细胞的体外扩增。Ad5/35-EGFP感染脐带间充质干细胞的效率明显高于其他两种腺病毒，但可导致细胞凋亡；腺病毒Ad5/11-EGFP对脐带间充质干细胞的感染效率较佳，随着感染复数的升高，所表达的荧光强度也逐渐增大。

关键词: 培养体系, 腺病毒, 感染效率, 脐带间充质干细胞, 干细胞

Abstract:

BACKGROUND: In vitro culture condition and culture efficiency are different in reported umbilical cord-derived mesenchymal stem cells, and lacked of unified standards. Different derived mesenchymal stem cells have different biological properties. Therefore, it is very necessary to establish a simple and high-performance culture system for umbilical cord-derived mesenchymal stem cells.
OBJECTIVE: To observe the growth state of human umbilical cord-derived mesenchymal stem cells in different culture systems in vitro and adenovirus infection efficiency.
METHODS: Mesenchymal stem cells were separated from healthy full-termed delivery fetus using collagenase digestion method and purified by adherent culture. These cells were cultured and amplified in DMEM (low glucose), MesenPRO RS™ Medium and STEMPRO® MSC SFM in vitro. The 3-5 passage mesenchymal stem cells were infected by the Ad5-EGFP, Ad5/11-EGFP, Ad5/35-EGFP as multiplicity of infection (MOI)=1, 10, 100. Viral infection and green fluorescence expression were observed at post-infection 24, 56 and 72 hours using inverted fluorescence microscope.
RESULTS AND CONCLUSION: The cell morphology in STEMPR® MSC SFM was different from other two culture system and these cells were not easy to adherent after trypsin digestion. Cell doubling time in the MesenPRO RS™ Medium was shorter than other two groups. Mesenchymal stem cells were infected by Ad5/35-EGFP with higher efficiency than other two kinds of adenovirus, but part of cells appeared apoptosis. The infection efficiency of Ad5/11-EGFP was highest. The fluorescence intensity was gradually increased with increased MOI.

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