Construction of S100A10 Overexpression Vector and Its Effect on Osteogenesis of Antler Stem Cells
JIA Ning1, HU Pengfei2, CHEN Weijia1, LIU Hongyuan1, JIANG Tiantian1, HE Zhongmei1 *, LI Chunyi1,2*
The purpose of this study was to construct and identify the lentiviral vector overexpressing sika deer S100A10 gene, and to study the effect of S100A10 on osteogenic differentiation of antler RMCs (reserve mesenchyme cells). The coding region of S100A10 was amplified by PCR and ligated with the lentiviral expression vector plasmid PCDH after double digestion to construct a recombinant plasmid overexpressing S100A10. The virus was packaged and infected with antler RMCs. The infection efficiency of the cells was detected by qRT-PCR (real-time fluorescence quantitative polymerase chain reaction) and Western blot. ALP (alkaline phosphatase) staining and activity quantification were used to detect the early osteogenic differentiation level of cells. Alizarin red staining was used to detect the late osteogenic differentiation level of cells. The mRNA expression levels of BMP-2 (bone morphogenetic protein-2), Runx2 (Runt-related transcription factor 2) and OCN (osteocalcin) were detected by qRT-PCR. The results showed that the S100A10 overexpression lentiviral vector was successfully constructed, and the mRNA and protein levels of S100A10 in the overexpression group were significantly higher than those in the control group (P<0.001). Compared with the control group, the ALP activity, the formation of calcified nodules (P<0.01) and the mRNA levels of osteogenic differentiation marker genes BMP-2, Runx2 and OCN in the overexpression group were significantly increased. In summary, S100A10 can promote the osteogenic differentiation of antler RMCs, which lays a foundation for further study on the mechanism of S100A10 promoting antler osteogenesis.
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