The Experimental Study Of Effect Of The Peptide That Specifically Bind To Osteoblasts On Proliferation And Mineralization Of Human Calvarial Osteoblasts In Vitro

Human Calvarial osteoblasts were used as the target cells and human gingival fibroblst used as the absorber cells for whole-cell biopanning from a 12-mer peptide phage-display library.We obtained the peptide that specifically bind to osteoblasts whose amino acid sequence was: VLAVPWSEPGYL. Investigative studies the peptide that specifically bind to osteoblasts on proliferation and mineralization of human calvarial osteoblasts,and to explore the cells within the peptide concentration 10-4~10-8 mol/L range most suitable for the proliferation of concentration as well as the most appropriate concentration of osteogenic differentiation, into the peptide that specifically bind to osteoblasts to provide reliable theoretical basis for further laboratory studies and in vivo studies.The whole study consists of four parts as follows:Part I MTT examine the proliferation of human calvarial osteoblastsHuman calvarial osteoblasts were routinely cultured which were divided into 6 groups including experimental groups containing the peptide at various concentrations(10-4, 10-5, 10-6, 10-7, 10-8 mol/L) and a control group.The proliferative activity was examined by MTT on day 4 and day 7. Finally, experimental data were statistically analyzed.It turned out that the peptide that specifically binds to osteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the proliferation of human calvarial osteoblasts in vitro, and the best concentration is 10-5mol/L.Part II Examine the activity of alkaline phosphataseHuman calvarial osteoblasts were routinely cultured which were divided into 6 groups including experimental groups containing the peptide at various concentrations(10-4, 10-5, 10-6, 10-7, 10-8 mol/L)and a control group.The activity of alkaline phosphatase and the total protein of human calvarial osteoblasts was examined as the specification on day 4 and day 7. The ALP activity divided by the measured cell total protein can be unit relative ALP activity of protein content.Finally, experimental data were statistically analyzed.It turned out that the peptide that specifically binds to osteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the activity of alkaline of human calvarial osteoblasts in vitro, and the best concentration is 10-4mol/L.Part III Alizarin red staining and semi-quantitative analysis detect the calcification degree of human calvarial osteoblastsHuman calvarial osteoblasts were routinely cultured which were divided into 6 groups including experimental groups containing the peptide at various concentrations(10-4,10-5,10-6,10-7,10-8 mol/L)and a control group.The calcification degree of human calvarial osteoblasts was detectd by alizarin red staining and semi-quantitative analysis on day 14 ang day 21.Finally, experimental data were statistically analyzed.It turned out that Alizarin red staining cells cultured for 14 and 21 d, the deposition of the calcium in experimental group(10-5mol/L) were higher than the control group, but there was no statistically significant difference.Part IV RT-QPCR detect human calvarial osteoblasts osteocalcin(OCN) m RNA expressionHuman calvarial osteoblasts were routinely cultured which were divided into 6 groups including experimental groups containing the peptide at various concentrations(10-4,10-5,10-6,10-7,10-8mol/L)and a control group.Human calvarial osteoblasts osteocalcin(OCN) m RNA expression was detected by RT-QPCR on day 14 and day 21.Finally, experimental data were statistically analyzed.It turned out that the peptide that specifically binds to osteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the expression of human calvarial osteoblasts osteocalcin(OCN) m RNA in vitro, and the best concentration is 10-6mol/L.Conclusion The peptide that specifically binds to osteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the proliferation and mineralization of human calvarial osteoblasts in vitro, and the best concentration is 10-5mol/L.