Establishment of rat bone mesenchymal stem cell lines stably expressing Chondromodulin I.

ABSTRACT: To study the role of Chondromodulin I (ChM-I) in inducing bone mesenchymal stem cells (MSCs) into chondrocytes, the plasmid expressing pcDNA3.1 (+) / ChM-I was constructed, and transfected into MSCs. Stable expression verified that ChM-I is upregulated in MSCs, which make it a useful preparation for studying the further role of ChM-I in inducing bone MSCs toward a chondrogenic I phenotype and for tissue engineering constructs. MSCs were obtained from adult Sprague Dawley (SD) rats using density gradient centrifugation. Cell surface antigens were detected by flow cytometry. The experimental groups were MSCs transfected with the plasmid pcDNA3.1 (+)/ChM-I. The control one was with the plasmid pcDNA3.1 (+) and the negative control one was with no plasmid. Transcription and translation were detected in the level of mRNA and protein respectively in order to identify the stability of the expression of ChM-I in cell lines by RT-PCR and western blot analysis. The results of RT-PCR showed that the mRNA level of ChM-I in the experimental group was six times higher than the control. The quantitative optical density analysis showed that the expression of ChM-I in the experimental group was significantly higher than the control by western blot. We conclude that we have established MSCs lines stably expressing ChM-I. This work lays the foundation of studying the potential role of the ChM-I in inducing BMSCs into chondrocytes.

SUBMITTER: Xing S 

PROVIDER: S-EPMC3272684 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature