Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-l for repair of articular cartilage defects in rabbits  

作　　者：张绍昆 刘一 宋之明 付长峰 徐莘香 

机构地区：[1]Department of Orthopedics, First Affiliated Hospital, Jilin University, Changchun 130021, China

出　　处：《Chinese Journal of Traumatology》2007年第1期10-17,共8页中华创伤杂志（英文版）

基　　金：This study was supported by the National Natural Science Foundation of China （ No. 30400447 ）.

摘　　要：Objective： To label the primary articular chondrocytes overexpressing human insulin-like growth factor （ IdGF-1 ） with green fluorescent protein （GFP） for repair of articular cartilage defects in rabbits. Methods： GFP cDNA was inserted into PeDNA3.1- hlGF-1 to label the expression vector. The recombinnnt 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 hlGF-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 lI collagen. Results： The expression of hlGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hlGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfectton of IGF-1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type lI collagen. Conclusions： The hlGF-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 hlGF-1. The labeled articular chondrocytes overexpressing hlGF-1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

关 键 词：Humans Insulin-like growth factor I Green fluorescent proteins Cartilage  articular TRANSGENES 

分 类 号：R[医药卫生]