Effects Of Steroid On The MRNA Expression Of Neuropeptide Receptor In Human Bone Marrow Mesenchymal Stem Cells

BackgroundThere are many theories in the pathogenesis of steroid-induced osteonecrosis of femoral head(SONFH),and the effect of neuromodulation in SONFH has increased people’s attention.Nervous system of bone can regulate bone metabolism in which peptidergic nerve is very important. Peptidergic nerve paly biological effects through release neuropeptides,and calcitonin gene-related peptide(CGRP),substance P (SP) respectively act variety cells in the bone tissue,regulate bone repair and reconstruction, affect bone metabolism.Bone marrow mesenchymal stem cells(BMSCs) is important stromal cell in bone formation and metabolism,and is important cytological basis in the pathogenesis of SONFH.Study found that there are calcitonin gene-related peptide receptor(CGRPR),substance P receptor(SPR) in BMSCs surface,neuropeptide affect the expression of substance through by act CGRPR and SPR.ObjectiveIsolated and cultured human bone marrow mesenchymal stem cells(BMSCs) in vitro.Observed the effect of steroid on the mRNA expression of CGRPR, SPR, peroxisome proliferators-activated receptor y(PPARy),Runx2in BMSCs.Investigated the role and mechanism of neuropeptide in SONFH. MethodsBMSCs were isolated and purified by density gradient centrifugation combined plastic-adhering culture,Cell morphology were observed by inverted phase contrast microscope.Cell surface markers were identified with flow cytometry.Cell cycles of third,BMSCs were randomly divided into two groups,then were treated with or without10"7mol/L dexamethasone.Experiment observed changes in cell morphology and growth trend,drew the growth curve,oil red O stained adipocyte,analyzed the expression changes of mRNA with RT-qPCR.Results1. Adherent cells showed fibroblast-figure shape or spindle shaped morphology,swirl or radial growth,the positive rate of CD29, CD44was(91.4±0.7)%、(93.8±0.5)%,while CD34was(2.7±0.8)%.These accord with the biological characteristics of BMSCs, it showed that obtained adherent cells are BMSCs.2.Cells of experimental group grew slower than control group,while control group had more superiority in proliferation. Oil Red O staining certificated that lipid droplets appeared in BMSCs,the number of adipocytes in experimental group increased significantly,while no intracellular lipid droplets appeared in control group.3.CGRPR mRNA,SPR mRNA and Runx2mRNA in experimental group was significantlly lower than that in control group,decreased ratio by0.10±0.03,0.16±0.04,0.23±0.05(all P<0.01),the decrease of CGRPR mRNA was most obvious.while PPARy mRNA increased ratio was5.08±3.37(P<0.01),the differences was statically significant.Conclusion1.Steroid could induce BMSCs adipogenic differentiation,and in the process of adipogenic differentiation, the expression of PPARγ mRNA increased,while the expression of important osteogenic transcription factor Runx2mRNA descended.2.Steroid inhibit the expression of CGRPR mRNA,SPR mRNA,and then restrain cells growth, proliferativc ability and osteogenic differentiation of the BMSCs,result to bone repair capacity decline,which might be related to the pathogenesis of steroid-induced ONFH.