[The Mechanism of GREM1's Effect on Osteogenic/Odontogenic Differentiation of Stem Cells from Apical Papilla].

Objective: To study the effect of bone morphogenetic protein (BMP) antagonist Gremlin 1 (GREM1) on the function of stem cells from apical papilla (SCAPs) and explore its mechanism.
Methods: After isolation and culturing of stem cells from apical papilla in vitro , immunofluorescent staining was done to examine the subcellular localization of GREM1 in SCAPs. Transfection with lentiviral GREM 1 shRNA was done to knock-down the GREM 1. The SCAPs were subjected to osteogenic induction in both the GREM 1 knockdown group and the control group, and the knockdown effect of GREM 1 was examined using real time-PCR and Western blot. Two groups of cells were collected and the alkaline phosphatase (ALP) activity was measured 7 d after osteogenic induction. Alizarin red staining was done 3 weeks after osteogenic/odontogenic induction and real time-PCR was done after 0, 1, 2, 3 weeks of osteogenic induction to examine the expression of osteogenic/odontogenic marker genes, including osteocalcin ( OCN ), osteopontin ( OPN ), bone sialoprotein ( BSP ), dentin matrix protein 1 ( DMP 1), dentin sialophosphoprotein ( DSPP ) and and the critical transcription factor osterix ( OSX ), Runt-related transcription factor 2 ( RUNX 2), and distal-less homebox 2 ( DLX 2). Two groups of cells were collected, and CCK-8 and CFSE assay were used to evaluate changes in cell proliferation. In addition, real time-PCR was used to examine the expression of senescence-related genes p 53 and wide-type activated factor 1 ( Waf 1), a regulatory factor of the cell cycle, stemness associated gene krupple-like factor 4 ( KLF 4), and SRY related HMG box-2 ( SOX 2), and the expression of bone morphogenetic protein ( BMP ) 2, 4, 5, 6, 7, 9 after GREM 1 knockdown.
Results: Immunofluorescence staining showed that the expression of GREM1 in the nucleus was higher than that in the cytoplasm. Real time-PCR and Western blot affirmed that GREM 1 was knocked down steadily. The ALP activity of the GREM 1 knockdown group was higher than that of the control group ( P <0.05), and the alizarin red staining was lighter than that of the control group. The expression of OCN and DMP 1 increased in the first, second and third week, OPN was increased in the second week, BSP increased in the third week, DSPP increased in the first week, and the difference was statistically significant ( P <0.05). The key osteogenic transcription factors RUNX 2, OSX , and DLX 2 all increased at different stages, and the difference was statistically significant ( P <0.05). CCK-8 and CFSE assay showed that the proliferation ability of the GREM 1 knockdown group decreased ( P <0.05). In the GREM 1 knockdown group, the expression of BMP 2, 6, and 7 increased, the expression of SOX 2 and KLF 4 increased, while the expression of p 53 and Waf 1 decreased ( P <0.05).
Conclusions: The knockdown of GREM 1 enhanced the osteogenic/odontogenic differentiation and stemness of SCAPs and inhibited the proliferation and senescence of SCAPs. Effects of GREM1 on the function of SCAPs maybe achieved through regulating the gene expression of BMP 2, BMP 6, and BMP 7 at the mRNA level.
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