In vitro study of micro-dystrophin gene-modified mesenchymal stem cells

BACKGROUND: Studies have shown that the transplantation of mesenchymal stem cells (MSCs) improves dystrophin expression in muscle cell membrane of mdx mice and plays a role in ameliorating sport injuries to the myocyte. In addition, dystrophin gene plasmid injection exhibits therapeutic effect in mdx mice. However, these two methods exhibit shortcomings, such as low rate of post-transplantation expression. Therefore, the present study determined the combinatorial effects of these two methods. OBJECTIVE: To transfect and observe effects of pSL139 plasmid carrying the micro-dystrophin gene into MSCs, as well as in vitro micro-dystrophin gene expression in transfected MSCs. DESIGN, TIME AND SETTING: A comparative, molecular biology study was performed at the Laboratory of Tissue Engineering, West China Medical Center, Sichuan University from March 2007 to February 2008. MATERIALS: The pSL139 plasmid was cloned and provided by the Department of Neurology, Washington University, USA. Lipofectamine 2000 was purchased from Invitrogen, USA. Mouse anti-human dystrophin N-based terminal monoclonal antibody was purchased from Chemicon, USA. METHODS: Differential velocity adherent technique and density gradient centrifugation were combined to separate and culture MSCs from C57/BL10 mice. The cells were induced to trans-differentiate into osteoblasts. Subsequently, the Lipofectamine 2000 method was used to mediate transfection of plasmid pSL139 into third generation MSCs. MAIN OUTCOME MEASURES: Semi-quantitative reverse transcription polymerase-chain reaction and immunofluorescence were respectively employed to detect micro-dystrophin mRNA and protein expressions in MSCs. RESULTS: At 48 hours after MSC transfection with plasmid pSL139, a 379-kb target band was observed by agarose gel electrophoresis. Immunofluorescence revealed micro-dystrophin expression up to 45%-55%. CONCLUSION: Micro-dystrophin mRNA and protein were highly expressed in pSL139-transfected MSCs, which provided a method for efficient expression of dystrophin.