Construction of bone marrow mesenchymal stem cells modified by ciliary neurotrophic factor gene in SD rat / 中华实验眼科杂志

ABSTRACT

Background The application of mesenchymal stem cells to transfer specific genes is under investigation in various diseases.Using this strategy may provide a more effective method to supply exogenous neurotrophic factors to the cental nervous system,including retina.Objective This study was to construct ciliary neurotrophic factor(CNTF)-overexpressing bone marrow mesenchymal stem cells (BMSCs) using lentiviral vectors.Methods Rat secreted-CNTF gene cDNA was synthesized and subcloned into a lentiviral vector plasmid pHⅣ-dTomato to construct recombinant vector CNTF-dTomato.CNTF-dTomato/pH Ⅳ-dTomato plasmid were co-transfected into 293T packaging cell line with packaging plasmid psPAX2 and enveloped plasmid pMD.2G to produce recombinant lentivirus CNTF-lenti and control-lenti.Rat BMSCs were infected with CNTF-lenti/control-lenti to produce CNTF-BMSCs and control-BMSCs.Expression of dTomato and efficiency of infection was evaluated under the fluorescence microscope.Uninfected BMSCs(pure BMSCs) served as the blank control.CNTF protein level in the supernate was detected by enzyme-linked immunosorbent assay (ELISA) and compared among the blank-BMSCs group,control-BMSCs group and CNTF-BMSCs group.Adipogenic and osteogenic differentiation of CNTF-BMSCs were induced using adipogenic-inducible medium and osteogenic-inducible medium and identified using oil-red O staining and alizarin red S (ARS) staining.Results After CNTF-dTomato plasmid was transfected into Stbl3 competent cells,the colony PCR product was 1 033 bp.The inserted sequence in the pHⅣ-dTomato plasmid was coincident with the expected one.The results of DNA sequencing showed that CNTF-dTomato plasmid was successfully constructed.The infection rate of CNTF-lenti was approximately 95％.ELISA showed that on the post-infected day 2,3,7,the CNTF protein levels in the supernate were significantly higher in the CNTF-BMSCs group than those in the blankBMSCs group and control-BMSCs group (all at P=0.000).In the CNTF-BMSCs group,the CNTF protein levels in the supernate were significantly increased on post-infected day 2,3,7 compared with day 1 (P =0.013,0.004,0.042).The adipogenic-induced cells showed the red staining to oil red O,and osteogenic-induced cells presented the orange staining to ARS.Conclusions BMSC line with stable expression of CNTF is successfully established by lentiviral vectors.CNTF-BMSCs have the potential to differentiate towards adipocytes and osteoblasts.