Effect Of Overexpression Of CGRP On Proliferation And Osteogenesis In Rat Bone Mesenchymal Stem Cells

Background and Purposes:Periodontal disease is a common disease of the oral cavity with widely population and high incidence.It is a chronic inflammatory destructive disease and dental plaque is the initiating factor.With the development of the regeneration of tissue engineering,periodontal tissue regeneration has improved.The related cytokines and stem cell technology research has made significant achievements.Because of the complexity of the etiology and mechanism of periodontitis repair,there is still a large gap between the regeneration of periodontal tissue and the natural physiological periodontal tissue.Calcitonin gene related peptide(calcitonin gene-related peptide,CGRP)is a kind of nerve polypeptide and can adjust the growth and metabolism of bone tissue to realize the function of the bone tissue regeneration through various channels.Early experiments showed that:after the inferior alveolar nerve was cut off,the rats'regenerative bone was loose and bone trabecula was sparse.As one of the main substance neuropeptide,CGRP can regulate the growth and metabolism of bone tissue to realize the function of the bone tissue regeneration.Based on the above research background,we studied the effect of CGRP on proliferation and osteogenic differentiation of rat bone mesenchymal stem cells,providing theoretical support for further study of neural factors in the regeneration of periodontal tissue.Materials and methods:1.Primary culture and identification of rat bone mesenchymal stem cells.Rat bone mesenchymal stem cells were cultured in vitro.The characteristics of rBMSCs were identified by morphological observation,surface marker antigen detection and multiple differentiation potential.2.Construction of rat bone mesenchymal stem cells with overexpressing CGRP gene.Four plasmid lentivirus packaging system cotransfected 293T cells.After transfected for 48h and 72h,the supernatant with lentiviral particles was collected.Rat bone mesenchymal stem cells were infected with lentivirus and then were detected the mRNA and protein expressions of CGRP.The experiment was divided into three groups:CGRP group,empty virus group,control group.3.Effect of overexpression of CGRP on proliferation and osteogenesis in rat bone mesenchymal stem cells.1)The effect of CGRP on cell proliferation was detected by CCK-8 and flow cytometry.2)The mRNA and protein expressions of ALP,BSP,OPN and Runx2 were detected by Realtime PCR and Western Blot.3)The osteogenic differentiation of three groups was measured by Alizarin red staining.4)OPG/RANKL ratio was also detected by Western Blotting at 1 week after mineral-induction to explain related mechanisms of osteogenic differentiation.Results:1.Rat bone mesenchymal stem cells were cultured in vitro and identified the cell characteristics.In vitro isolation,rat bone mesenchymal stem cells(rBMSCs)were cultured and purificatied by whole bone marrow adherence method.rBMSCs were mainly spindle cells,and were arranged in a radial arrangement.Flow cytometry analysis was used to identify the surface makers of rBMSCs.It showed that the expression rates of CD34,CD44,CD45 and CD90 were respectively 0.3%,2.8%,92.3%and 97.6%.After adipogenic and osteogenic induction,oil red O and alizarin red staining of rBMSCs were positive.2.Construction of rat bone mesenchymal stem cells with overexpressing CGRP gene.After rBMSCs were infected with lentivirus,the protein and mRNA expression of CGRP obviously increased in the CGRP group(P<0.05).The protein and mRNA expression of CGRP had no significant difference between the empty virus group and control group(P>0.05).3.Effect of overexpression of CGRP on proliferation and osteogenesis in rat bone mesenchymal stem cells.The cell proliferation ability of CGRP group was higher than the empty virus group and control group through CCK-8 and flow cytometry.The expressions of ALP,BSP,OPN and Runx2 in the CGRP group at 1w and 2w were all higher compared with the empty virus group and control group(P<0.05).It had no significant difference between the empty virus group and control group(P>0.05).OPG was up-regulated,whereas RANKL was down-regulated in the CGRP groups,resulting in an increased OPG/RANKL ratio(P<0.05).No significant difference was found between Control group and Vector grou(P>0.05).CGRP promoted the osteogenic differentiation of rat bone mesenchymal stem cells by regulating OPG/RANKL.Conclusion:1.Overexpression of CGRP promotes the proliferation of rat bone mesenchymal stem cells.2.Overexpression of CGRP promotes the osteogenic differentiation of rat bone mesenchymal stem cells by regulating OPG/RANKL.