Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits.
OBJECTIVE
To label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits.


METHODS
GFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen.


RESULTS
The expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen.


CONCLUSIONS
The hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.
Zhang
Shao-kun
SK
Department of Orthopedics, First Affiliated Hospital, Jilin University, Changchun 130021, China. szgnohuw@hotmail.com
Liu
Yi
Y
Song
Zhi-ming
ZM
Fu
Chang-feng
CF
Xu
Xin-xiang
XX
D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D005434:Flow Cytometry; D005819:Genetic Markers; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D049408:Luminescent Agents; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits
D005819:Genetic Markers; D049408:Luminescent Agents; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I