[In-vivo tracing of bone marrow stromal cell differentiating into chondrocytes by green fluorescent protein gene transfection].
AIM: To achieve highly efficient, stable and long-term expression of green fluorescent protein(GFP) reporter gene in bone marrow stromal cells(BMSCs) and to trace directly the distribution and differentiation of BMSCs in articular cartilage defects. METHODS: Recombinant RV-GFP expression vector was constructed and transfected into packaging cell PT67. After G418 screening and amplification, cell clones producing high level recombinant viruses were obtained and in-vitro expanded. The virus supernatant from infected PT67 cell culture was used to infect BMSCs directly. Autologous GFP-labeled BMSCs were seeded onto biodegradable polymer and implanted into porcine articular cartilage defects. 7 months later, the repaired tissue was evaluated by confocal microscope. RESULTS: The constructed recombinant expression vector RV-GFP was identified by restriction digestion. The transfection rates of PT67 cells reached 20%-50%, which reached 100% after G418 screening, bright green fluorescence could be seen under fluorescence microscope. BMSCs could be transfected successfully by the culture supernatant from infected PT67 cells and GFP was expressed efficiently in a long-term period. Confocal microscope revealed that GFP-labeled cells existed in many neocartilage lacunae after 7 months. CONCLUSIONS: The recombinant vector RV-GFP can provide a simple, sensitive and reliable tool to label BMSCs which can be used to study cytologic dynamic follow-up in-vivo. BMSCs can differentiate into chondrocytes and play an important role in repairing articular cartilage defects.