Expression of connexin 43 in cartilage and chondrocyte of osteoarthritis and construction of shrna lentiviral vector targeting connexin 43 / 中国组织工程研究

ABSTRACT

BACKGROUND: Connexin 43 (Cx43) plays an important role in occurrence and development of osteoarthritis. However, the specific mechanisms involved remain unclear. OBJECTIVE: To verify the possibility of the dominant position of Cx43 in connexin family in osteoarthritis by detecting the expression of Cx43 in articular cartilage and chondrocyte cell line, and to construct shRNA lentivirus vector of Cx43 gene and establish a stable transfer cell line of chondrocyte (SW1353). METHODS: Animal models of osteoarthritis were established in six C57BL/6 mice by anterior cruciate ligament transection. The differences of Cx43 expression between osteoarthritic and normal knees were investigated by immunohistochemistry. Expression of Cx43 mRNA in chondrocyte (SW1353) was detected by RT-PCR, and the expression levels of Cx37, Cx40, Cx45 and Cx46 in SW1353 cells were detected as control. Cx43 were connected to the lentiviral vector carrying the EGFP gene, to reconstruct the lentiviral vector plasmid. The viral particles were generated by co-transfection of 293T cells with Cx43-shRNA. After transfection of Cx43-shRNA lentiviral vector into chondrocytes (SW1353), the expression level of Cx43 was detected by western blot assay and RT-PCR. The study protocol was approved by the Ethics Committee of State Key Laboratory of Oral Diseases, approved No. SKLODLL2013A172. RESULTS AND CONCLUSION: The expression level of Cx43 was significantly increased in the articular cartilage of osteoarthritic knees. The expression level of Cx43 mRNA was significantly higher than that of Cx37, Cx40, Cx45 and Cx46 in chondrocytes (SW1353). In SW1353 cells, Cx43 occupied the dominant position in connexin family. Cx43 shRNA lentiviral vector could inhibit the expression of Cx43 mRNA in SW1353 cells. The stably transfected SW1353 cell line was screened, laying a foundation for verifying the role of Cx43 in osteoarthritis.