Expression Of Pcp4 Gene During Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells In Vitro

Bone marrow mesenchymal stem cells(BMSCs) have the potential to differentiate into a variety of cell lineages both in vitro and in vivo including osteogenic, chondrogenic, adipogenic, and myogenic cells in special conditions. However, many problems still exist in the bone tissue engineering research which use BMSCs as seeding cells. So far, the molecular mechanisms of the osteogenic differentiation of BMSCs are still unclear. The purpose of this study is to establish the separation, purification, and vitro osteogenic-inductive differentiation training system of rat BMSCs, apply gene chip technology to screen differentially expressed genes during osteoplastic differentiation of BMSCs, use real-time fluorescence quantitative PCR(RQ-PCR) to detect gene mRNA quantity and analyze the trend during the process of BMSCs' osteogenic differentiation, and lay the foundation in order to better understand the molecular mechanisms of BMSCs' osteogenic differentiation and study relative gene' function.We isolated and purified the rat BMSCs with density gradient centrifugation and adherent culture, carried out the original culture, passaged when adherent cells paved about 80% of the bottom of the bottle, and induced the third generation cells into osteoplastic differentiation with the conditioned medium. Detected the osteogenic characteristics through observing cells morphology, depicting cells growth curve, Calculating cells doubling time, the quantitative detection of alkaline phosphatase, immunohistochemistry detection for typeⅠCollegen, staining mineral nodules with alizarin red. On the basis of the same culture conditions, the osteogenic-inductive third generation BMSCs were set the experimental group and the third generation BMSCs, which were not induced, were set the control group. Then, we extracted cells RNA of the control group and the experimental group respectively and screened differentially expressed genes during osteoplastic differentiation of BMSCs through applying gene chip technology. Under the same conditions, we Set the osteogenic-inductive third generation BMSCs the experimental groups and the third generation BMSCs without osteogenic induction the control group, extracted cells RNA of the control group and the experimental groups at the 4th day, 7th day, 14th day and 21th day respectively, then, detected and analyzed the total RNA quality and the reverse transcription polymerase chain reaction(RT-PCR) product quality, and used RQ-PCR to detect the gene mRNA quantity at different time and analyze the trend during the process of BMSCs' osteogenic differentiation. Furthermore, the bioinformatics analysis was made to discuss osteogenic-related function of the gene primarily.The results showed that the high-purity BMSCs were acquired through the density gradient centrifugation with adherent culture and the third generation BMSCs were successfully induced into osteoplastic differentiation withα-minimal essential medium Containingβ-glycerophosphate, L-ascorbic acid, dexamethasone and vitamin D3. There were no significant difference for the growth of cells between the induction medium and the primary medium; after osteogenic induction, BMSCs displayed typical osteoblast changes; alkaline phosphatase activity increased significantly in the cells after induction while there was no significant changes in the cells without induction and alkaline phosphatase activity remained low; positive typeⅠcollagen immunohistochemical staining was observed after 7 days induction; mineral nodules were found after 2 weeks induction and stained positively with alizarin red. Differentially expressed Purkinje cell protein 4(Pcp4) gene was successfully screened during osteoplastic differentiation of BMSCs through applying gene chip technology. RQ-PCR results showed that Pcp4 gene expression had upward trend during osteoplastic differentiation of BMSCs, among which Pcp4 gene expression quantity respectively increased 7, 9 and 6 times in the experimental groups than the control group, and fully met the test results of gene chip technology. So, Pcp4 gene is upregulated expression gene during osteoplastic differentiation of BMSCs. Based on above results and bioinformatics analysis, we have made the assumption: mineral nodules are reliable indicators of bone formation ability and Pcp4 gene has the potency of regulating calcium binding kinetics, so, Pcp4 gene is Likely to be involved in promoting the deposition of calcium to form mineral nodules; during bone formation, Protein kinase(PKA) path activation is the key signal of BMPs activation, while Pcp4 relates with CaM-dependent PKA activation, involves in regulation of PKA and has synaptic transmission function, so, Pcp4 gene may have certain signal transduction relations with BMPs. In further research, we will continue to study relations between Pcp4 gene and bone formation.Based on these findings, much valuable information was obtained. Firstly, the high-purity BMSCs were acquired through the density gradient centrifugation with adherent culture and the third generation BMSCs were successfully induced into osteoplastic differentiation. Secondly, differentially expressed Pcp4 gene was screened during BMSCs' osteoplastic differentiation and bone formation through applying gene chip technology for the first time. Thirdly, RQ-PCR results showed that Pcp4 gene expression had upward trend during BMSCs' osteoplastic differentiation and bone formation and fully testified the test results of gene chip technology. In summary, Pcp4 gene is upregulated expression gene during BMSCs' osteoplastic differentiation and bone formation, involves in BMSCs' osteoplastic differentiation and bone formation. These methods and information will be very useful for studying the molecular mechanisms of BMSCs' osteogenic differentiation and bone formation.