中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (40): 7441-7445.doi: 10.3969/j.issn.1673-8225.2011.40.008

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

慢病毒介导增强型绿色荧光蛋白基因修饰人骨髓基质细胞

汤  浩，项永生，秦玲莎，姜晓丹，徐如祥，陈镇洲   

  1. 南方医科大学珠江医院神经外科，广东省广州市510282
  • 收稿日期:2011-07-28 修回日期:2011-09-07 出版日期:2011-10-01 发布日期:2011-10-01
  • 通讯作者: 陈镇洲，博士，副主任医师，南方医科大学珠江医院神经外科，广东省广州市 510282 czz1020@163.com
  • 作者简介:汤浩☆，男，1982年生，广东省深圳市人，汉族，南方医科大学在读博士，主要从事骨髓基质细胞及其应用研究。 tanghao6251@gmail.com
  • 基金资助:

    国家自然科学基金(30801184)“强化表达VEGF 的骨髓基质细胞系的构建及其在脑缺血的应用”；广东省医学科研基金(B2009159)“慢病毒介导的hTERT和VEGF双基因修饰人骨髓基质细胞的研究”；广东省科技计划重点专项(2011A030400007)“自体骨髓基质细胞及血管内皮祖细胞移植治疗脑缺血：双盲、随机、对照的前瞻性临床研究”。

Enhanced green fluorescent protein gene-modified human bone marrow stromal cells induced by lentivirus

Tang Hao, Xiang Yong-sheng, Qin Ling-sha, Jiang Xiao-dan, Xu Ru-xiang, Chen Zhen-zhou   

  1. Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou  510282, Guangdong Province, China
  • Received:2011-07-28 Revised:2011-09-07 Online:2011-10-01 Published:2011-10-01
  • Contact: Chen Zhen-zhou, Doctor, Associate chief physician, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China czz1020@163.com
  • About author:Tang Hao☆, Studying for doctorate, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 　510282, Guangdong Province, China tanghao6251@gmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30801184*; the Medical Science Research Foundation of Guangdong Province, No. B2009159*; the Special Foundation for Science and Technology Key Plan of Guangdong Province, No. 2011A030400007*

PDF

412

被引次数

3

摘要：

背景：原代细胞的转染效率低下一直是制约其发展的主要因素之一，慢病毒转染以其独特的优势成为各种干细胞基因修饰良好的转染方法。
目的：验证慢病毒载体介导增强型绿色荧光蛋白基因修饰人骨髓基质细胞的可行性。
方法：将携带增强型绿色荧光蛋白基因的慢病毒在含血清或无血清条件下按不同的MOI值分别转染人骨髓基质细胞，长期观察荧光蛋白表达情况。将经修饰的细胞通过立体定向移植入大鼠纹状体内，观察移植细胞的存活及目的基因的表达情况。
结果与结论：基因转染48 h后，可见人骨髓基质细胞成功表达绿色荧光蛋白，无血清条件下的转染效率高于含血清条件下的转染效率，转染成功的细胞在体外持续培养2个月以上未见明显的荧光消减。转染后的细胞可在大鼠纹状体存活并持续表达绿色荧光蛋白2个月以上。提示慢病毒是一种高效的转染人骨髓基质细胞的载体，慢病毒载体介导增强型绿色荧光蛋白基因转染可以作为人骨髓基质细胞的示踪标志用于体内移植研究。

关键词: 增强型绿色荧光蛋白, 人骨髓基质细胞, 慢病毒, 转染, 体内移植

Abstract:

BACKGROUND: Transfection of primary culture cells is an important investigation field on cell biology, but the low transfection rate is one of the main obstacles which have limited its development and utility for a long time. Lentivirus transfection has become a good method because of its unique advantages in transfection of primary culture cells.
OBJECTIVE: To investigate the feasibility of transfection of human bone marrow stromal cells (hBMSCs) with lentivirus mediated enhanced green fluorescent protein (EGFP) gene.
METHODS: The hBMSCs were transfected by lentivirus carrying EGFP gene (LV-EGFP) at different MOI values under fetal bovine serum medium or serum-free condition respectively. The expressions of EGFP in these cells were observed for a long-term. These gene-modified cells were transplanted into the striatum rats of stereotaxicly. Survive of the grafted cells and EGFP expressions were detected by fluorescent microscope.
RESULTS AND CONCLUSION: After 48 hours of transfection, green fluorescent protein was successfully detected in hBMSCs. Transfection efficiency of cells in serum-free medium was higher than that in serum medium. The transfected hBMSCs could persistently express EGFP and had no obvious decline of fluorescence during 2-month culture in vitro. The EGFP positive cells could survive in the rat corpus striatum for more than 2 months and express EGFP persistently. Lentivirus is an efficient vector for gene modification of hBMSCs. Lentivirus mediated EGFP gene transfection can be used as efficient trance marker for hBMSCs transplantation study in vivo.

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