2023, Vol. 27 ›› Issue (19): 2999-3004

Effects of lentivirus mediated silencing of Smad2 gene on osteogenic differentiation of human dental pulp stem cells

Wang Chang1, Sun Shuai2, Chen Xiaotao3, Zhang Xiaoli3   

Abstract: BACKGROUND: The specific regulatory mechanism underlying osteogenic differentiation of human dental pulp stem cells is still unclear. Therefore, studying the specific mechanism is of great significance for the application of stem cells in tissue engineering.  
OBJECTIVE: To investigate the role of lentivirus mediated silencing of Smad2 gene in the osteogenic differentiation of human dental pulp stem cells.
METHODS: The third generation of human dental pulp stem cells were cultured and divided into blank control group, negative control group, Smad2-shRNA group, and Smad2-shRNA+transforming growth factor-β3 (TGF-β3) group. The mRNA expression of osteogenic related genes Runx2, osteocalcin, Smad2, Smad3, and Smad4 in human dental pulp stem cells was measured by qRT-PCR. The protein expression levels of Runx2, osteocalcin, Smad2/Smad3, p-Smad2/Smad3, and Smad4 in human dental pulp stem cells were measured by western blot assay. Alizarin red staining was performed in each group on the 7th and 14th days of culture.  
RESULTS AND CONCLUSION: (1) The mRNA expressions of Runx2, osteocalcin, Smad2, Smad3, and Smad4 in the Smad2-shRNA group were significantly lower than those in the negative control group and blank control group (P < 0.05). The mRNA expressions of osteocalcin, Smad2, Smad3, and Smad4 were significantly increased in the Smad2-shRNA+TGF-β3 group compared with the Smad2-shRNA group (P < 0.05). (2) The protein expressions of Runx2, osteocalcin, and Smad2/Smad3 in the Smad2-shRNA group were significantly lower than those in the negative control group and blank control group (P < 0.05). The protein expressions of RUNX2, osteocalcin, Smad2/Smad3, and Smad4 were significantly increased in the Smad2-shRNA+TGF-β3 group compared with the Smad2-shRNA group (P < 0.05). (3) Culture with conventional medium and transfection with lentiviral vector could not induce the osteogenic differentiation of human dental pulp stem cells, while TGF-β3 could positively regulate the osteogenic differentiation of human dental pulp stem cells. (4) All these findings indicate that theTGF-β/Smad2 signal transduction pathway plays an important role in the osteogenic differentiation of human pulp stem cells.

Key words: lentivirus, Smad2 gene, dental pulp stem cell, osteogenic differentiation, transforming growth factor-β