| Bone fracture healing is a complex biological process,and many cytokines involve in the regulations of healing process.Many researches show that the nervepeptides play anoticeable role in bone repair and reconstruction.Calcitonin gene-related peptide?CGRP?is a widespread peptide in vivo,secreted by nerve ending synapses.It has been confirmed to have the function of promoting bone reconstruction.CGRP receptor belongs to the G protein coupled receptors?GPCR?and is found on the cell membrane,including the calcitonin receptor-like receptor?CRLR?,receptor activity modifying protein?RAMP?and receptor component protein?RCP?.Clinical studies have found that bone fracture patients combined with central nervous system damage suffered a shorter healing time than those who suffered from bone fracture only.On the contrary,those patients merged peripheral nervous system injury went through a longer time to be healed.CGRP concentration increased in the plasma of bone fracture patients.The number of CGRP positiveneurons raised obviously in the injured location.Our previous studies have clarified that CGRP can accelerate the proliferation and osteogenic differentiation of osteoblasts,in the meantime,induce osteoclasts apoptosis.A great many signaling pathways,for example,c AMP/PKA and PANKL take part in this process.Nevertheless,the regulatory function of CGRP on bone mesenchymal stem cells?BMSCs?,the precursor cells of osteoblasts and osteoclasts,and relevant signal transductions are sitll ambiguous.BMSCs are a branch of adult stem cellsderived from the mesoderm.BMSCs can be induced to differentiate to osteoblasts,chondrocytes,neurons,adipocytes,and so on.Beyond that,BMSCs also have a lot of advantages such as rapid growth,high plasticity,and simple obtaining accesses.In a word,BMSCs have won a high attention in bone tissue engineering.In clinical practice,the liver is still able to continue developing after a two-thirds surgical resection.The body tissues and organs will not keep growing any longer when they achieve physiological needs.Such phenomena guide us to have a sight in signaling pathways that can modulate growth and development.There is no doubt that the Hippo signaling pathway is a pivotal one in size controling of tissues and organs.This pathway is highly conserved in both fruit flies and mammals.It functions as a size regulator mainly by lowering cell proliferation and strengthening apoptosis.There are three primary components in Hippo pathway:the upstream signaling molecule?Ex,Mer,Nf2,etc?,core kinase cascade chain?Mst1/2 ? Lats1/2 ? Sav,etc?and downstream effectors?Yap/Taz?.Molecules in the upstream receive intracellular and extracellular signals,then transmit into cells to cause the phosphorylation of core kinase cascades,inactivateion and nucleus/cytoplasm transfer of Yap/Taz,lead to thetranscription and expression of relatedtarget genes,ultimately.Studies of the Hippo pathway in mammals began in mouse liver.Overexpression of Yap gene in mouse liver cells led to an accelerated proliferation and the liver reached a 3-4 folds in size than normal.On the contrary,after the overexpressed effection removed,liver cells began to die and the liver shrank to a normal size.It is also reported that both Mst1/2 and Sav1 participate in liver development.Besides,it is worth noting that Hippo pathway plays an amportant role in embryonic development and adult stem cells differentiation fate.Objectives: By means of studying the cell proliferation and osteogenic differentiation mediated by CGRP in mouse BMSCs,we achieved the goal of further revealing the role CGRP plays in bone fracture healing.Besides,we discussed the Hippo pathway in this process.Methods:1.We used the adhesion method to isolate Balb/c mouse BMSCs in vitro and then observed the BMSCs morphology under a microscope.2.Osteogenesis ability of primary BMSCs was identified by Alizarin red staining and ALP staining.At the same time,flow cytometry was performed to identify the specific antigens?CD29 and CD45?on the surface of cell membrane.3.BMSCs were divided into 5 groups randomly,different concentrations of CGRP(0?10-?10??10-9?10-8?10-7 mol/L)were added into 5 groups above.Then we used CCK8 kit?flow cytometry and ALP kit to measure the proliferation and differentiation in every group.4.BMSCs were induced to osteogenic differentiation in vitro by different concentrations of CGRP(0?10-10?10-9?10-8?10-7 mol/L)for a screening of the optimal concentration.5.Added CGRP in BMSCs,then the activity of alkaline phosphatase?ALP?and the number of mineralized noduleswere examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days,respectively.6.The phosphorylation level of Hippo pathway's core component—Mst1/2 was measured by Western blot.7.We used Verteporfin to block the downstream Yap signaling,then detected the m RNA expression of collagen type??collagen I?and Runt-related transcription factor 2?Runx2?by RT-PCR.Results: 1.Wesuccessfully used the adhesion method and obtained rather purified Balb/c mouse BMSCs from the whole bone marrow.2.Compared to the blank group,different concentrations of CGRP(10-9to 10-7 mol·L-1),especially 10-8 mol·L-1,significantly increased the proliferation and differentiation of BMSCs?P<0.05?.3.Both Alizarin red staining and ALP staning showed a higher level of differentiation in the 10-8 mol·L-1 group,compared with the rest four groups.4.The protein expression of p-Mst1/2 rose in the CGRP group?P<0.05?.5.Verteporfin treatment effectively decreased the m RNA expression of collagen I and Runx2?P<0.05?.Conclusions:1.Using the adhesion method to isolate and purify BMSCs from the tibia and femur marrow of Balb/c mice is feasible and effective.2.CGRP can significantly up-regulate the proliferation and osteogenic differentiation of mouse BMSCs,especially at a concentration of 10-8mol/L.3.The Hippo signaling pathway plays a positive role in CGRP-induced osteogenic differentiation of mouse BMSCs. |
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