CGRP On Vascular Endothelial Cells And Osteoblasts Training System Into The Impact Of Bone Cells

Objective: To establish osteoblasts and vascular endothelial cellco-culture system by the intervention from vitro neuropeptide CGRP（Calcitonin Gene Related Protein） to co-cultured system in the cells, wewish to study the role of CGRP on the regulation of cell interactions, and toprove of the effect on the bonecallus formation and osteogenic whileCGRP in the wound healing process of the jaw bone.Methods: The first part of the experiment, the MG63and the EC in a2:1ratio, according to the concentration of5.0*10⁴cells/well seeded in tinplates, each plate hole1to hole4in the culture medium containing10^-710^-8,10^-9,10^-10mol/L CGRP, hole5MG63on EC is simply a totalculture medium without adding CGRP, holes6separate culture MG63anddo not add of CGRP. Co-culture week and two weeks later under amicroscope to observe the co-culture system growth and Alizarin redstaining observed observe calcified nodules The second part of the experiment, the EC DiI staining （DiIconcentration of10-10mol/L, dyeing time of10minutes）, the MG63andthe EC in a2:1ratio, according to a cell concentration of2.0x104cells/mlseeded in cover glasschip, were placed in6-well plates, culture, divided intosix groups, the culture medium, respectively, containing10-7,10-8,10-9,10-10mol/LCGRP and without CGRP in the control groupMG63cultured alonethe control group, the third day of culture collagenΙimmunofluorescence staining using DAPI lining into the nucleus,fluorescence microscopy record results.The third part of the experiment, the MG63were seeded in96-wellplates in2.0x104cells/well, cultured with the culture medium of EC whichwas cultured with corresponding concentrations of CGRP for one day,divided into6groups, divided into6groups, culture medium of group1-4contained10^-7,10^-8,10^-9,10^-10mol/L CGRP respectively, group5did notcontain CGRP, the last sixth group was cell-free control group. thenumber of osteoblasts was measured by MTT on the third day of cultivationand the experiment was repeated twice, the data obtained was statisticalanalysed using Spss16.0software and cell proliferation curve of differentconcentrations of CGRProle was mapped.In the fourth part of these experiment, MG63were vaccinated at6hole plateto1.0x105/holes, with the culture medium of EC which was cultured withcorresponding concentrations of CGRP for one day, divided into6groups,culture medium of group1-4contained10^-7,10^-8,10^-9,10^-10mol/L CGRPrespectively,the fifth group was CGRP free, the last sixth group was culturewith common culture solution, the protein concentration measurement andAKP activity determination were conducted after one week’s co-cultivation; the experiment was repeated two times,and the experimental data wereanalyzed by Spss16.0software and the ALP activity curve of differentconcentrations of CGRP osteoblasts were mapped.The fifth part of experiment,we use transwell method to isolationand culture of EC and MG63,EC（2.0x104/hole） vaccination in transwellcells, MG63（4.0x104/hole） vaccination chamber that beneath the cultureboard,the experiment divide into6groups,and the culture mediumrespectively contain10-7,10-8,10-9,10-10mol/LCGRP and EC andMG63and MG63pure culture.After a week of culture together,we uesHuman collagen type I enzyme immunoassay kit to determine thecollagenΙ.Result:CGRP plays a role in promoting the proliferation ofosteoblasts, and the10^-7and the10-10groups were significantly different （P<0.05）,10-10group and control group were significantly different （P <0.05）.The10-10l/LCGRP stimulate the proliferation of osteoblasts more obviouslywhile using various concentrations, so it is visible that CGRP isdose-related with osteoblast cell proliferation in a co-culture system.Co-culture system is more earlier than separated bone cells in calcification,to promote asosteocalcin role, accordingly it shows that the vascularendothelial cells play a catalytic role in osteoblast differentiation andmaturation. The formation of calcium nodules and the levels of the CGRPadding showed the opposite trend. The activity of ALP that adds10^-7,10^-8,10^-9,10^-10mol/L CGRP relative to normal cultured alone MG63’s inthe control group showed obvious inhibition, of which inhibition of 10^-7mol/L CGRP group is the most obvious, and the use of10^-7mol/L CGRPculture group and other groups were statistically significant （P <0.05）. It isvisible that the AKP enzyme activity of osteoblasts cultured alone is evenhigher while comparing co-cultured group and individually culturedosteoblasts group. The10^-9,10^-10mol/L CGRP group relative to the controlgroup showed a more significant role in promoting the formation of collagenΙ,precisely speaking other groups is not obvious.Conclusion: The high concentrations of CGRP forcalcification inhibitionmay be present in the co-culture system, and high concentrations of CGRPshowed inhibition to the AKP activity. CGRP at low concentrationspromoted formation of organic collagen Ι in the matrix of osteoblasts, andalso play a stimulated role in the proliferation of the osteoblasts ofco-culture system.