The Role and Mechanism of Exogenous Calcitonin Gene-Related Peptide on Mesenchymal Stem Cell Proliferation and Osteogenetic Formation

Abstract

The aims of this study were to expose the function of calcitonin gene-related peptide (CGRP) in the proliferation and intracellular communication of mesenchymal stem cells (MSCs), to observe the change in IGF-1, BMP-2 and their receptor in the MSCs cell with exogenous CGRP, and to explore whether exogenous CGRP will induce MSCs to express the osteoinduced factor and it's receptor. MSCs were separated from bone marrow and collected by gradient centrifugation and adherent culture. Real-time polymerase chain reaction (RT-PCR) was used to detect CGRP receptor in MSCs in logarithmic growth phase [1]. Hybridoma technique was used to produce rabbit-anti-human CGRP receptor, which was used in the Western blot test to detect CGRP receptor protein produced in human MSCs. Then, MSCs were parted into 3 groups decided by the concentration of CGRP. Cell proliferation was detected through methylthiazol tetrazolium (MTT) test. Cell form in each group was detected through optical microscope, in the same time point. Cell cycle was detected with flow cytometric to analyze the ratio of cell in the mitotic time. MSCs collected from healthy volunteer were parted into 3 groups: the control group, the anagen group, and the experimental group. Intracellular communication medium molecule was detected through radioimmunoassay; intracellular communication and signal conduction were detected through carboxyfluorescein diacetate fluorescent dye. The expression of Cx43mRNA was detected through real-time PCR. The mRNA expressions of proliferation-related biological factor of MSCs were detected through real-time PCR. MSCs collected by gradient centrifugation and cultured by adherent culture have high purity and proliferation effect. It was proved through RT-PCR that MSCs express CGRP receptor mRNA, and it was also be proved through Western blot that MSCs express CGRP receptor protein. The MTT test showed similar result, the 10-8 mol/L CGRP group had the highest proliferation speed, and the control group had the lowest. There is statistical difference between experimental group and control group. There also had static difference between the 10-8 mol/L CGRP group and the other two experimental group. Expression of Cx43mRNA in experimental group was higher than the other two groups, but, without static difference. It was proved that the mRNA expressions of IGF-1, IGF-1 receptor, and BMP-2 receptor in experimental group were higher than that in control group with static difference. The mRNA expressions of BMP-2 in all the groups had no static difference. And Ct index in all the groups were higher than 35. It was proved that MSCs express CGRP receptor mRNA and protein. With MTT test, it had been proved that exogenous CGRP can accelerate the proliferation speed in the logarithmic growth phase. With flow cytometric, it had been proved that exogenous CGRP can raise the ratio of the cell in the DNA synthesis period and mitosis prophase. CGRP can promote not only intracellular communication of MSCs but also the expression of Cx43mRNA. The exogenous CGRP can increase the mRNA expression of IGF-1, IGF-1 receptor, and BMP-2 receptor of MSCs. In all the groups, the Ct indexes of BMP-2mRNA were higher than 35, which could be considered as negative expression.