| Objective: In the whole growth process, the mechanical force plays an important role in stimulating the body all the time, especially in bone developing, bone fracture healing, bone remodeling and so on. Osteoblasts are sensitive to external stimulation, and they play an important role in the bone formation process. Past scholars believe that pressure can promote bone absorption, and it is not conducive to the bone formation. However in recent years, more and more studies show that suitable force may play an important role in the proliferation and differentiation of bone cells, as well as bone formation, but current research on the signal transduction mechanism of biomechanics in cells is unknown. Cultivating mouse embryos osteoblast MC3T3-E1 in vitro to form 3D cell membrane,we observe the cells which are under pressure after 1, 4, 8 hours to find out the changes of proliferation activities by using FX-5000 C loading system with 10% deformation rate, the effect of compress on the proliferation of mouse osteoblast cells was detected by MTT method. Affymetrix gene expression profile microarray technology was also used to analyze the effect of stress on the differential gene expression of osteoblasts in mice, and to explore the mechanism of the effect of stress on osteoblasts. It provides theoretical basis and experimental reference for the clinical study of the mechanism of bone remodeling as well as the treatment and recovery of related diseases.Methods:1 Mouse osteoblast MC3T3-E1 cultured in vitroThe primary mouse embryo osteoblast MC3T3-E1 cells which were recovered from cryopreserved were observed growth under microscope. When the cells grown well and covered the bottom of the bottle, they would be passaged. MC3T3-E1 cells with good growth condition and that were in logarithmic phase of growth would be prepared into single cell suspension, cultured in 12 well plates as 1×105 cells/ hole for 28 days to form a layer of about 1~2 mm cell membrane.2 Establishment of mouse osteoblast loading model in vitroHarvested the cell membranes and put 6 drops into 6 wells pressure plate randomly, FX-5000 C cell pressure loading system is applied to the frequency 1 Hz(sine wave), the compression ratio of 10% and constant pressure for 1 hour(group A1), 4 hours(group B1), 8 hours(group C1), while control groups for 1 hour(group A2), 4 hours(Group B2), 8 hours(group C2) without pressure.3 Detection of mouse osteoblast proliferation activityAfter pressurized, the proliferation activity of the cells was determined by MTT assay, and detected the absorbance of each pore at the wavelength of 490 nm by enzyme-linked immunosorbent assay method(ELISA); the data were analyzed by computer with the SPSS statistic software.4 Extraction of RNA from mouse osteoblastsCells from both experimental group and control group that have the best proliferation activity were selected to extract the content of RNA, and determined concentration, purity and integrity.5 Preparation and detection of mouse osteoblast by gene chipThe extracted RNA is prepared by whole genome expression profile chip; scan the RNA after wash and stain, analysis the data with Transcriptome Analysis Console 3.0 software, to screen different genes.Results:1 Cell proliferation was measured by MTT,the osteoblast cells were divided into three groups were pressurized 1, 4 and 8 hours separately. Experimental data was expressed with the mean value of absorbance( x ± s), A1: 0.323±0.047, A2: 0.307±0.034; B1: 0.850±0.060, B2: 0.313±0.027; C1: 0.530±0.081, C2: 0.307±0.025. Compared group A1 with A2 there was no significant difference(P>0.05), while it was statistically different between group B1 and B2(P<0.001), as well as group C1 and C2(P<0.001), all groups were analyzed by t-text. The data of three control groups showed no significant change(P>0.05), while the data of three experimental groups was statistically significant(P<0.05). Single factor analysis of variance was adopted to analyze the data of group A1, B1 and C1, significant difference was between each two groups. It may indicate that there was no significant difference between the experimental groups and the control groups in cell proliferation activity by 1 hour; after 4 hours, the proliferation of osteoblast activity reached to the peak, but cell proliferation activity went down gradually with the increasing loading time.2 The concentration, purity and integrity of RNA detectionBy using the Nanodrop 2000/2000 C spectrophotometer, the result shows that the RNA OD260/OD280 ratios of group B1(the study group which was pressured by 4 hours) and group B2(4 hours in control group) were located between 1.8 and 2.0. The gel electrophoresis results shows that B1 group(4 hours in study group) and group B2(4 hours in control group) were isolated three RNA strips using automated gel imaging system(GBOXF3), the brightness of 28 S bands is about twice of 18 S band and 5 S strip is the faintest. These show that RNA extracted from each group completely and could satisfy experimental request.3 Mouse embryo osteoblasts MC3T3-E1 whole genome expression profiles in m RNAIn the analysis of the 34472 genes of mice, we screen out the genes which up-regulated or down-regulated differences over 2 times and between B1 group(4 hours in study group) and group B2(4 hours in control group), there are a total of 3609(which up-regulated genes have 1657, down regulated genes have 1952) genes between embryo osteoblasts with significant different expression of genes, of whom the products involves multiple aspects of cell signaling, proliferation, differentiation and apoptosis.Conclusions:1 A suitable compressure on osteoblasts in a fit time can enhance the proliferation activity of osteoblasts in a certain time range.2 The osteoblast gene changes under pressure, Myc, Jun, Fos, Runx2, Wnt10 b, Lrp5 and other genes was significantly raised.3 Myc, Jun, Fos, Runx2, Wnt10 b, Lrp5 and other osteogenic related genes may participate in promoting the proliferation and differentiation of osteoblasts through the TGF- beta signaling pathway and Wnt signaling pathway. |
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