Effects Of MGF-Ct24E And Mechanical Stress On The Gene Expressing Of Osteoblasts By Microarray Analysis

Mechano growth factor (MGF) is a hotspot in the research fields of the biomechanics and growth factor recently. It is an alternative splicing variant of Igf-1 gene which is mainly expressed in stretched skeletal muscle, damaged muscle and neuron. Further studies found that MGF and its E peptide (MGF-Ct24E) played an important role in promote muscle hypertrophy, damage repaired and have a function of neuroprotective and myocardial protection. Our previous studies found that MGF mRNA had a high expressed in the cyclic stretched osteoblasts, which suggested that MGF might be a mechano-response molecule in bone and have a role in regulating the proliferation and differentiation of osteoblasts to benefit bone repair. Mechanical stress has an important role in regulating bone growth and reconstruction. Osteoporosis often appeared at the bone fracture site after healing because of the lack of mechanical stress. Therefore, in this study we compared the gene expression profiles of osteoblasts after treat with MGF-Ct24E and mechanical loading by microarray analysis. The aim of this study was to find out the relationship between the treatment of MGF-Ct24E and mechanical loading in bone metabolism, so that we can speculate whether MGF-Ct24E could replace the mechanical stress to benefit bone repair. The main work and conclusions were as follows:(1) The primary osteoblasts were cultured from the calvarias of new born SD rats by tissue piece culture method. The osteoblasts were purified by the difference of the adhesion time with fibroblasts. Osteoblasts were identified by morphology, ALP staining, matrix precursor staining and mineralization staining. The identified results showed that the cells had typical characteristics of osteoblasts. The third generation osteoblasts were used in the subsequent experiments.(2) Cells were suffered with cyclic-strain unit at 12% and 50ng/ml MGF-Ct24E treatment for 8h. Total RNA were extracted, microarray were used to analyze the differentially expressed genes. The results showed that 1866 genes had different expressions under mechanical loading group comparing with the untreated group, in which, 1113 genes were up-regulated while 753 genes were down-regulated. 1178 differentially expressed genes were found in the MGF-Ct24E treated group. Among them, 796 gene expressions were up-regulated and 382 gene expressions were down-regulated. Only 602 genes had different expressions between the MGF-Ct24E treated group and mechanical loading group, including 290 up-regulated genes and 312 down-regulated genes.(3) Differentially expressed genes were analyzed using gene ontology database. The GO terms have most of the same expressing both in mechanical loading group and MGF-Ct24E group. These common GO terms are mainly about the regulation of cell proliferation and differentiation, cell cycle, cell migration, bone development, regulation of ossification and response to mechanical stimulus. Besides the common expressing in the two groups, the GO terms of actin cytoskeleton organization, cell growth, protein domain specific binding and protein dimerization activity have differentially expressed in mechanical loading group, while cartilage development, skeletal system development, growth factor binding, protein kinase binding and receptor binding have differentially expressed in MGF-Ct24E group. Compared with mechanical loading group, the different expressing genes in the MGF-Ct24E group are mainly associated with GO terms of embryonic placenta development, positive regulation of transcription and gene expression and et al. The differentially expressed genes in mechanical loading group and MGF-Ct24E group have a high similarity in regulation of bone metabolism.(4) Cell proliferation assay, cell cycle stage distribution analysis and cell differentiation assay were used to study the influence of MGF-Ct24E on the biology activity of osteoblasts. The results suggested that the proliferation activity of MGF-Ct24E group was significantly higher while the ALP activity was inhibited at the first day compared with control. The significantly greater proportion of S and G2/M phase cells appeared in the MGF-Ct24E group than control. But when cells went into the late differentiation stage, MGF-Ct24E promoted the collagen expression and mineralization to facilitate osteoblast differentiation. This found revealed that MGF-Ct24E was not inhibited the osteoblast differentiation, but promoted the osteoblast proliferation and delayed the differentiation. Cell experiment results were consistent with the microarray analysis.To sum up, MGF-Ct24E and mechanical stress have an extremely similar effect in the regulation of the gene expression associated with bone metabolism. Cell experimental analysis again confirmed that MGF-Ct24E could promote osteoblast proliferation while delay the differentiation. This effect was finally promoted bone formation. Therefore, MGF-Ct24E is expected to replace mechanical stress to benefit bone repair.