Stably upregulating expression of chondromodulin-Ⅰ in bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Chondromodulin-Ⅰ is expressed mainly in the cartilage, but it is little expressed in mesenchymal stem cells. Combined with the previous study of our group, we concluded that chondromodulin-Ⅰmaybe play an important role in inducing mesenchymal stem cells into chondrocytes accurately. OBJECTIVE:To construct an expression plasmid stably carrying chondromodulin-Ⅰ to up-regulate the expression of chondromodulin-Ⅰ in bone marrow mesenchymal stem cells. METHODS:Specific primers were designed in rat cartilage for chondromodulin-Ⅰ gene, then the pcDNA3.1 (+) plasmid expression vector was digested by enzyme and directional connected gene to construct pcDNA3.1(+)/ChM-Ⅰ expression vector. Bone marrow mesenchymal stem cells were obtained from rats using the method of density gradient centrifugation combined with adherent culture. Recombinant plasmid pcDNA3.1(+)/ChM-Ⅰ was transfected into rat bone marrow mesenchymal stem cells with liposome method, and G418 selection was used for stable screen of transfected cells. Reverse transcription-PCR and western blot were used to detect chondromodulin-Ⅰ expression in celllines. RESULTS AND CONCLUSION:The positive clones were digested by enzyme and were identified and sequenced. The results showed that the reality length and sequence of chondromodulin-Ⅰ gene were consistent with the theoretical values, and reading frame was correct. Reverse transcription-PCR and western blot results showed that the expressions of chondromodulin-ⅠmRNA and protein were markedly up-regulated in bone marrow mesenchymal stem cells. Recombinant plasmid pcDNA3.1(+)/ChM-I was successful y constructed, and transfected into rat bone marrow mesenchymal stem cells. After G418 selection, expression of chondromodulin-Ⅰ was up-regulated stably in rat bone marrow mesenchymal stem cells.