The Study On Effect Of Fgf-2Gene On Proliferation And Chondrogenic Differentiation Of Mesenchymal Stem Cells C3H10

Objective: To construct an eukaryotic vector namedpIRES2-EGFP-FGF-2and investigate its effect on the proliferation,osteogenic and chondrogenic differentiation of mouse mesenchymal stemcells C3H10.Methods: FGF-2gene was amplified by PCR from plasmidpAd-Trace-FGF-2. After being digested with BglⅡ and Sal I, the PCRproduct was inserted into pIRES2-EGFP to construct pIRES2-EGFP-FGF-2.pIRES2-EGFP-FGF-2plasmid was transfected into C3H10by Liposomes.C3H10transfected with pIRES2-EGFP and untreated were set up as controls.The mRNA and protein expression levels of FGF-2were determined byRT-PCR and Western blot. The proliferation activity and cell cycle phase ofC3H10were examined by MTT and flow cytometry. The mRNAtranscription level of ColⅠ, OC, OPG, OPN, ColⅡ, ACAN and Wntpathway related members was detected by RT-PCR. The protein expressionof ColⅡ was detected by Western blot. Toluidine blue staining was used to test the secretion of cartilage oligomeric matrix protein by cells in culture.Results: Restriction analysis and sequencing proved that recombinantplasmid pIRES2-EGFP-FGF-2was constructed correctly. Both the mRNAand protein expression level of FGF-2increased significantly inpIRES2-EGFP-FGF-2transfected C3H10cells. Cell proliferation activity ofthe experimental group increased significantly (P＜0.05).The mRNAtranscription level of ColⅠ, ColⅡ，and ACAN in C3H10cells transfectedwith pIRES2-EGFP-FGF-2recombinant plasmid increased significantly(P<0.05), however there is no significant increase in the mRNA expression ofOC,OPG,OPN(P＞0.05). The transcription level of Wnt5a and Fzd8decreased significantly (P<0.05). Toluidine blue staining showed purplemetachromatic in FGF-2overexpressed C3H10cells，compared with fewpurple staining control cells.Conclusions: The recombinant eukaryotic expression vector for FGF-2gene is successfully constructed and overexpression of FGF-2can promotechondrogenic differentiation and proliferation activity of C3H10cells butnot affect osteogenic differentiation in short-term. The effect of FGF-2onchondrogenesis might be attributed to the downregulation of Wnt5a andFzd8.