Expression of neuropilin 1 in osteogenic differentiation of preosteoblastic cells and in mouse tibial tissue
Objective To investigate the expression of neuropilin 1(NRP1)mRNA and protein during the osteogenic dif-ferentiation of MC3T3-E1(a preosteoblastic cell line),as well as the expression of NRP1 in the mouse tibial tissue,in or-der to provide an experimental foundation for the clinical application of Semaphorin 3A/NRP1 signaling pathway in the treatment of bone resorption in chronic periapical inflammation.Methods This study was conducted from April to June 2023 in the Central Laboratory of the Stomatological Hospital of China Medical University.MC3T3-E1 cells were cul-tured for osteogenic differentiation induction and grouped based on the duration of osteogenic differentiation.Western blot analysis was used to detect the expression of NRP1 protein in the control group(0 days)and in the differentiated groups at 1,3,5,7,10,14,and 20 days.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of NRP1 mRNA in the control group and in the differentiation groups at 1,3,5,7,10,and 14 days.Immunofluorescence staining was performed on cells in the control group and in the differentiation groups at 3 and 5 days,and subcellular lo-calization of NRP 1 during the early stage of osteogenic dif-ferentiation was observed using confocal laser scanning microscopy.Six tibiae from three 4-day-old mice were equally divided into NRP1 and negative control IgG groups,immunofluorescence staining was performed,and an inverted fluorescence microscope was used to demonstrate NRP1 expression in mouse tibial tissues.Results The relative expression levels of NRP1 protein and mRNA were com-pared among the groups,and significant differences were found(F values were 48600.000 and 60.030,respectively,both P＜0.001).Specifically,the relative expression levels of NRP1 protein in the 7-day and 10-day differentiation groups were higher than those in other time groups,and the relative expression level of NRP1 mRNA in the 10-day differentia-tion group was the highest,with statistically significant differences observed(all P＜0.05).In the Western blot analysis,a secondary band corresponding to the secreted form of NRP1(soluble NRP1,sNRPl)was observed below the main NRP1 band.In the control group and the 3 and 5-day differentiation groups,NRP1 was expressed in the cytoplasm,with its subcellular localization surrounding the nucleus.NRP1 was expressed in the metaphyseal region of the mouse tibia,with widespread expression in osteoblasts within the bone-forming areas.Conclusion NRP1 participates in the osteo-genic differentiation process of MC3T3-E1,primarily expressed in the cytoplasm during the early stage of osteogenic dif-ferentiation,and contributes to the formation of the mouse tibia.
万方数据
维普
