The Peptide-gel Scaffold Induce The BMSCs Gene-modified By Nt-3 Differentiate Into Nerve Cells

Objective:Construct the tissue engineering module in vitro to investigate the differentiation of the BMSCs gene-modified by NT-3 under the culture of Peptide-gel scaffold containing IKVAV.Methods:1.By getting BMSCs from rat by whole marrow adherence method,then via detecting surface antigens through flow cytometry and induce adipogenic differentiation to identify this stem cells,draw the growth curve of cells according to CCK-8.2.The BMSCs were observed under fluorescent microscope after gene-modified by Ad-NT-3-RFP 48 h later for the expression of the Labeled protein RFP.To delect the content of NT-3 by ELISA.3.The petide solution was triggered to from the 3D culture module by the addition of cell culture fluid containing the BMSCs.The experiments were divided into three groups the Gel-NT3 group,the Gel-RFP group and the NT-3 group.The expression of MAP-2 and GFAP at mRNA and protein levels in different groups were detected by qRT-PCR and Western blot.Results:1.The cells we obtained which likely spindle and highly expressed CD29,did not express CD34.generated lipid droplets after induced to adipogenic differentiation for two weeks,and oil red O staining positively.2.The expression of RFP were observed under fluorescent microscope in the cells transfected by Ad-NT-3-RFP 48 h later,and The transfection efficiency was more than 90%.ELISA was used to detect the NT-3 content in the cell culture solution,the cell fluid begins to appear NT-3 protein on the first day after transfection and significantly increased on the third day.5 days later,there was no significant difference in the concentration every day.3.The results of qRT-PCR and Western blot showed that the expression ofMAP-2 and GFAP at protein and mRNA levels was significantly increased in the Gel-NT3 group compared with the Gel-RFP group and the NT-3 group,the difference was statistically significant(P < 0.05).Conclusions:The Peptide-gel scaffold containing IKVAV had a significant role in promoting the differentiation of BMSCs gene-modified by NT-3 into nerve cells.