Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (27): 4964-4969.doi: 10.3969/j.issn.1673-8225.2010.27.006

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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*

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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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