Yang Guo1, Michael Klüppel2, Hao Tang3, Sheng Tan1, Peidong Zhang4, Zhenzhou Chen5. 1. Department of Neurology, Zhujiang Hospital, Southern Medical University, Guangzhou, China. 2. Department of Pediatrics, Robert H. Lurie Comprehensive Cancer Center, Stanley Manne Children's Research Institute, Northwestern University, Chicago, IL, USA. 3. Department of Neurosurgery, National Key Clinic Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China. 4. Department of Cardiovascular Medicine, Zhujiang Hospital, Southern, Medical University, Guangzhou, China. 5. Department of Neurosurgery, National Key Clinic Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China. czz1020@163.com.
Abstract
OBJECTIVE: To test the feasibility of secretion of functional chondroitinase ABC (ChABC), a bacterial enzyme that promotes axonal regeneration after spinal cord injury, from human bone marrow stromal cells (hBMSCs). RESULTS: A lentiviral-expression vector, Lenti6.3-ChABC-3F, carrying the ChABC-3F gene without the bacterial leader sequence (aa 1-24) was constructed. Transfection of these Lenti6.3-ChABC-3F lentivirus led to stable expression in and secrection of ChABC proteins from hBMSCs for at least ten passages in culture in vitro, which was demonstrated by QRT-PCR, immunostaining, western blotting and ELISA. Moreover, the secreted ChABC proteins exhibited similar functional activity as the commercially-available ChABC. CONCLUSIONS: The lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence induced substantial long-term secretion of functional ChABC in hBMSCs.
OBJECTIVE: To test the feasibility of secretion of functional chondroitinase ABC (ChABC), a bacterial enzyme that promotes axonal regeneration after spinal cord injury, from human bone marrow stromal cells (hBMSCs). RESULTS: A lentiviral-expression vector, Lenti6.3-ChABC-3F, carrying the ChABC-3F gene without the bacterial leader sequence (aa 1-24) was constructed. Transfection of these Lenti6.3-ChABC-3F lentivirus led to stable expression in and secrection of ChABC proteins from hBMSCs for at least ten passages in culture in vitro, which was demonstrated by QRT-PCR, immunostaining, western blotting and ELISA. Moreover, the secreted ChABC proteins exhibited similar functional activity as the commercially-available ChABC. CONCLUSIONS: The lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence induced substantial long-term secretion of functional ChABC in hBMSCs.