Construction of Notch1(NICD) Eukaryotic Expression Vector and Its Influence on the Proliferation and Differentiation of Rat Bone Marrow Mesenchymal Stem Cells in vitro / 天津医药

ABSTRACT

Objective To investigate the effect of construct the Notch1 (NICD) eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Rat BMSCs were experimented as the object. NICD eukaryotic expression vector was constructed. pEGFP-N1-NICD expressing plasmids were used to transfect BMSCs. The study included control group (CON group), empty vector group (VEC group) and the trans-fection group (TRA group). After 48-hour transfection, BMSCs were observed for general morphology. The protein expres-sions of NSE, GFAP and Notch1 were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy-cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay. Results The DNA sequencing con-firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after 48-hour transfection. The relative expression levels of Notch1 and GFAP mRNA and pro-tein were significantly higher in TRA group than those in VEC group and CON group (P<0.05), and there was no significant difference between VEC group and CON group. After 48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group (P<0.05). The late apoptotic rate was significantly higher in VEC group than that of CON group. The proportion of G1/G0 cells was significantly higher in TRA group than that of CON group and VEC group, but S and G2/M cells were significantly lower (P<0.05). The value of growth curve was gradually de-creased in TRA group than that of CON group and VEC group (P<0.05). Conclusion The high expression of NICD gene might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentiation.