Message: file_get_contents(https://...@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests
Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Research Institute of Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China.
Published: May 2021
AI Article Synopsis
Article Abstract
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 , immunofluorescent staining was done to examine the subcellular localization of GREM1 in SCAPs. Transfection with lentiviral 1 shRNA was done to knock-down the 1. The SCAPs were subjected to osteogenic induction in both the 1 knockdown group and the control group, and the knockdown effect of 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 ( ), osteopontin ( ), bone sialoprotein ( ), dentin matrix protein 1 ( 1), dentin sialophosphoprotein ( ) and and the critical transcription factor osterix ( ), Runt-related transcription factor 2 ( 2), and distal-less homebox 2 ( 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 53 and wide-type activated factor 1 ( 1), a regulatory factor of the cell cycle, stemness associated gene krupple-like factor 4 ( 4), and SRY related HMG box-2 ( 2), and the expression of bone morphogenetic protein ( ) 2, 4, 5, 6, 7, 9 after 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 1 was knocked down steadily. The ALP activity of the 1 knockdown group was higher than that of the control group ( <0.05), and the alizarin red staining was lighter than that of the control group. The expression of and 1 increased in the first, second and third week, was increased in the second week, increased in the third week, increased in the first week, and the difference was statistically significant ( <0.05). The key osteogenic transcription factors 2, , and 2 all increased at different stages, and the difference was statistically significant ( <0.05). CCK-8 and CFSE assay showed that the proliferation ability of the 1 knockdown group decreased ( <0.05). In the 1 knockdown group, the expression of 2, 6, and 7 increased, the expression of 2 and 4 increased, while the expression of 53 and 1 decreased ( <0.05).
Conclusions: The knockdown of 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 2, 6, and 7 at the mRNA level.
