中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (27): 4964-4969.doi: 10.3969/j.issn.1673-8225.2010.27.006

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

骨髓间充质干细胞-胞嘧啶脱氨酶/5-氟胞嘧啶自杀基因治疗系统的构建及效应

曹始波1，孟庆海1，金  澎1，王洪伟1，栗世方1，窦以河1，孟承东2，阎晶杰3   

  1. 青岛大学医学院附属医院，1神经外科，2放射科，山东省青岛市  266003；3临邑县德平医院，山东省临邑县  251500
  • 出版日期:2010-07-02 发布日期:2010-07-02
  • 通讯作者: 孟庆海，博士生导师，教授，青岛大学医学院附属医院神经外科，山东省青岛市 266003 qhmeng301@ sohu.com
  • 作者简介:曹始波★，男，1972年生，山东省临邑县人，汉族，青岛大学医学院神经外科在读硕士，主治医师，主要从事胶质瘤的基因治疗研究 QYC020542@ 126.com
  • 基金资助:

    山东省自然科学基金重点项目(Z2006C02)，课题名称：基因工程化神经干细胞治疗胶质瘤的实验研究。

Construction and effects of bone marrow mesenchymal stem cells-cytosine deaminase/5-fluorocytosine suicide gene therapy system

Cao Shi-bo1, Meng Qing-hai1, Jin Peng1, Wang Hong-wei1, Li Shi-fang1, Dou Yi-he1, Meng Cheng-dong2, Yan Jing-jie3   

  1. 1 Department of Neurosurgery, 2 Department of Radiology, Affiliated Hospital of Qingdao University, Qingdao  266003, Shandong Province, China; 3 Linyi County Deping Hospital, Linyi  251500, Shandong Province, China
  • Online:2010-07-02 Published:2010-07-02
  • Contact: Meng Qing-hai, Doctoral supervisor, Professor, Department of Neurosurgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China qhmeng301@sohu. com
  • About author:Cao Shi-bo★, Studying for master’s degree, Attending physician, Department of Neurosurgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China QYC020542@126. com
  • Supported by:

    the Key Program of Natural Science Foundation of Shandong Province, No. Z2006C02*

PDF

340

被引次数

7

摘要：

背景：体内研究已经证实骨髓间充质干细胞能够像神经干细胞一样在脑内迁移和整合，如果骨髓间充质干细胞用于系统途径移植成为可能，将会显著简化移植的步骤。
目的：构建含胞嘧啶脱氨酶(CD)基因的重组表达质粒pEGFP-N3-CD，用脂质体Lipofectamine2000转染大鼠骨髓间充质干细胞，体外观察CD基因的表达及BMSCs-CD/5-氟胞嘧啶自杀基因治疗系统对C6胶质瘤细胞的杀伤作用。
方法：构建pEGFP-N3-CD质粒，酶切、DNA测序鉴定。全骨髓贴壁法分离培养骨髓间充质干细胞，脂质体Lipofectamine2000介导重组表达质粒pEGFP-N3-CD转染大鼠骨髓间充质干细胞。G418筛选培养获取阳性克隆(BMSCs-CD细胞)，免疫细胞化学染色检测BMSCs-CD细胞的CD基因蛋白表达。Transwell小室共培养BMSCs-CD细胞和C6胶质瘤细胞，24 h后加入前体药物5-氟胞嘧啶，72 h后TUNEL、MTT、流式细胞术检测C6胶质瘤细胞的凋亡情况。
结果与结论：构建的重组表达质粒pEGFP-N3-CD含完整的CD基因序列，CD基因转染至骨髓间充质干细胞并在基因及蛋白水平有完整表达。BMSCs-CD和C6胶质瘤细胞体外共培养条件下，C6胶质瘤的凋亡率呈剂量依赖性，与对照组相比差异有显著性意义(P < 0.01)。结果提示体外共培养条件下，BMSCs-CD/5-氟胞嘧啶自杀基因治疗系统对C6胶质瘤细胞生长有显著的抑制作用。

关键词: 胞嘧啶脱氨酶, 转染, 骨髓间充质干细胞, transwell共培养, 胶质瘤, 基因疗法

Abstract:

BACKGROUND: In vivo study has verified that bone marrow mesenchymal stem cells (BMSCs) can migrate and integrate in the brain as neural stem cells (NSCs). If BMSCs can be used in system pathway transplantation, it will simplify the procedures of transplantation.
OBJECTIVE: To construct recombinant expression plasmid pEGFP-N3-CD containing cytosine deaminase (CD). Liposome Lipofectamine2000 was used to transfect rat BMSCs. To observe CD gene expression and suicide effects of BMSC-CD/5-fluorocytosine (FC) suicide gene therapy system on C6 glioma cells in vitro. 
METHODS: The pEGFP-N3-CD plasmid was constructed, and determined by enzyme digestion and DNA sequence. BMSCs were harvested and cultured using the whole bone marrow method. The pEGFP-N3-CD plasmid was used to transfect rat BMSCs in vitro using Lipofectamine2000. CD gene protein expression of G418-resistant clones (named BMSCs-CD cells) was detected by immunocytochemistry. Apoptosis of C6 cells cultured with BMSCs-CD cells in Transwell culture system induced by 5-FC (24 hours later) in BMSCs-CD cells genetically modified to express CD was investigated by applying TUNEL, MTT and flow cytometry analysis techniques (72 hours later).
RESULTS AND CONCLUSION: The pEGFP-N3-CD plasmid containing complete CD gene sequence was constructed. CD gene was transfected to BMSCs, and fully expressed in gene and protein levels. Under in vitro co-culture conditions of BMSCs-CD and C6 glioma cells, apoptotic rate of C6 glioma cells showed dose-dependent effects, and significant difference was detected as compared with control group (P < 0.01). Results have indicated that BMSCs-CD/5-FC suicide gene therapy system exhibits significant inhibitory effects on growth of C6 glioma cells.

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