Construction of eukaryotic vector expressing FGF-2 gene and its effect on proliferation and cartilage differentiation of mesenchymal stem cells C3H10

Abstract:

Objective：To construct an eukaryotic vector of pIRES2-EGFP-FGF-2 and to investigate its effect on the proliferation，os－teogenesis and chondrogenesis of mouse mesenchymal stem cells C3H10. Methods：FGF-2 gene was amplified by PCR from plasmid pAd-Trace-FGF-2. After being digested with BglⅡ and SalⅠ，the PCR product was inserted into pIRES2-EGFP to construct pIRES2-EGFP-FGF-2. pIRES2-EGFP-FGF-2 plasmid was transfected into C3H10 by Liposomes，and C3H10 transfected with pIRES2-EGFP and untreated were set up as controls. The mRNA and protein expression levels of FGF-2 were determined by RT-PCR and Western blot respectively. The proliferation activity and cell cycle phase of C3H10 were examined by MTT and flow cytometry. The mRNA transcription levels of collagenⅠ（ColⅠ），osteocalcin（OC），osteoprotegerin（OPG），osteopontin（OPN），collagenⅡ（ColⅡ），aggrecan（ACAN） and Wnt pathway molecules were detected by RT-PCR. The protein expression of ColⅡ was detected by Western blot. Toluidine blue staining was carried out to measure the secretion of cartilage oligomeric matrix protein by cells in culture. Results：Re－striction analysis and sequencing proved that recombinant plas－mid pIRES2-EGFP-FGF-2 was constructed correctly. Both the mRNA and protein expression level of FGF-2 increased signifi－cantly in pIRES2-EGFP-FGF-2 transfected C3H10 cells. Cell proliferation activity of the experimental group increased sig－nificantly（P＜0.05）. The mRNA transcription levels of ColⅠ，ColⅡ and ACAN in C3H10 cells transfected with pIRES2-EGFP-FGF-2 recombinant plasmid increased significantly（P<0.05），however there was no significant increase in the mRNA expression of OC，OPG，OPN（P ＞0.05）. The transcription levels of Wnt5a and Fzd8 decreased significantly（P<0.05）. Toluidine blue staining showed purple metachromatic in the cells FGF-2 overexpressed C3H10 cells. Conclusion：The recombinant eukaryotic expression vector for FGF-2 gene is successfully constructed and overexpression of FGF-2 can promote chondrogenesis and proliferation activity of C3H10 cells but there is no significant effect on osteogenic differentiation in short-term. The effect of FGF-2 on chondrogen－esis maybe involve in the downregulation of Wnt5a and Fzd8.