[In-vivo tracing of bone marrow stromal cell differentiating into chondrocytes by green fluorescent protein gene transfection].
OBJECTIVE
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.
Zhou
Guang-dong
GD
Department of Plastic and Reconstructive Surgery of the 9th People's Hospital, Shanghai Second Medical University, Shanghai Tissue Engineering Laboratory, Shanghai 200011, China. guangdongzhou@yahoo.com.cn
Wang
Xiao-yun
XY
Liu
De-li
DL
Cui
Lei
L
Liu
Wei
W
Cao
Yi-lin
YL
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D019902:Chondrocytes / Q000166:cytology*; D024043:Collagen Type II / Q000032:analysis; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D013552:Swine; D023822:Tissue Engineering; D014162:Transfection
D024043:Collagen Type II; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins