Effect Of Fluoride And Aluminum On Proliferation And Differentiation In MC3T3-E1 Cells

ObjectiveFluoride is an essential trace element for human. An appropriate intake of fluoride increase bone density and promote teeth growth, promote the growth and generation functions as well. However, chronic excessive intake of fluoride may cause fluorosis. Aluminum is the most abundant on earth, an appropriate intake of aluminum can stimulate bone cell proliferation and differentiation. However, excessive intake of aluminum could accumulate in the body, might cause senile dementia, osteomalacia, anemia and other disease. Studies have shown that, the capacity of Al+ binding with F-in neutral pH condition is better than other 60 kinds of metal ions. Fluoride in a complex with aluminum (fluoroaluminate, most likely AlF4-) binds to G proteins in vitro and activates G protein-mediated intracellular signaling pathways. In the fluorosis area, aluminum workers not only exposed to large number of aluminum, but also exposed to high fluoride. In water fluorosis areas, people use aluminum to remove fluoride, caused the coexistence of aluminum and fluoride in drinking water. A lot of instant foods are also detected aluminum fluoride content exceeded. Tea has a strong accumulation capacity of fluoride and aluminum, usually fluoride and aluminum are enriched in mature leaves and old leaves, which up to 1175mg/kg and 4381mg/kg, resulted in the tea drinking fluorosis. The epidemiological and animal experimental study found that fluoride can promote the accumulation of aluminum in bones, aluminum can promote the fluoride poisoning. Hence in the tea-drinking fluorosis ward, damage to the patients are often more serious. Therefore, the study of aluminum fluoride has an important practical significance and research value.The osteoblast cell was derived from mesenchymal stem cell. The development may experience several stages as follows:mesenchymal stem cell→progenitor cell→preosteoblast→mature osteoblast→osteocyte。We use MC3T3-E1 cells to explore the effect of different concentrations of fluoride, aluminum and aluminiunofluoride on proliferation and differentiation, investigate the mechanism of endemic fluorosis and provide experimental data.Methods1. Cell CultureMC3T3-E1 cells were cultured in aMEM conventional medium containing 10% fetal bovine serum,1×105U/L penicillin, streptomycin,5% CO2, cultured under the conditions of 37℃.2. MaterialsThe stock solutions of NaF and AlCl3 (100mM和10mM, respectively), as well as working solutions at 10 times higher concentration than final, were prepared in water and mixed only before the experiment.3. Cell proliferation dynamic detectedThe cell proliferation was measured by CCK method. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group. OD was measured with the microplate reader at 450 nm.4. Cell cycleThe change of cell cycle was measured by flow cytometry. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group.5. ALP activity assayThis experiment was determined by ALP activity test kit. Determination of cellular ALP activity based on the following principle:phosphorylation of p-nitrophenol in the presence of ALP can be transformed into p-nitrophenol and phosphate. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group.6. Extraction of total cellular RNAWe used Trizol to extract the MC3T3-E1 cell RNA. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group.7. Primer synthesisAccording to documents the cDNA sequences download from the GeneBank, and designed by software Primer 5. Designed and synthesized. The mouse OPQ RANKL, housekeeping gene (β-actin), Osterix, Runx2 cDNA sequence by the Shanghai Public Health Biology company designed and synthesized.8. RT-PCRTake total cellular RNA 3μl, detection of RNA concentration. Before using adjust the concentration to 0.5μg/μl. RT total reaction volume of 5μl. The total reaction volume of 20μl, concrete steps are as follows:cDNA5μl,5×PCR Buffer 5μl, upstream and downstream primers (10μM) of 0.25μl, Taq enzyme 0.125μl, replenishment 14.375μl, mixing in 0.5ml EP tubes.Statistical analysisUsing SPSS 16.0 statistical software for statistical analysis, group and between groups using two independent samples t-test; groups compared using single factor analysis of variance (ANOVA),22 compared the use of LSD-t test.Results1. MC3T3-E1 cell morphologyThe suspensioned MC3T3-E1 cells were round. After cultured 24 h, in an inverted microscope could see the cell had been adherent, in the form of triangular, spindle-shaped, and a variety of polygonal forms. Piles of morphologically similar cells appeared cytoplasmic extension, with 2 to 4 and protruding nuclei obvious nucleolus definition (see as Figurel). After 3-4 d, cells in exponential growth, cell volume increased, rich in cytoplasm. After 6-7 d, cell fusion was stone pavement-like arrangement (see as Figure 2).2. Fluoride on the MC3T3-E1 cell proliferationTable 1 shows that after 24-72 h, compared with the control group, fluoride alone did not promote MC3T3-E1 cells proliferation.3. Fluoride, aluminum and aluminiunofluoride on cell proliferationTable 2 shows that after 72 h, compared with the control group, fluoride (50μM) or aluminum (5μM), did not promote MC3T3-E1 cells proliferation; Aluminiunofluoride stimulated proliferation in MC3T3-E1 cells (p<0.01). Compared with the control group, aluminiunofluoride group the proliferation rate had increased by 9.2%.4. cell cycleAs seen from Table 3, compared with control group, fluoride (50μM), aluminum (5μM) had no obvious effect on cell cycle. Aluminiunofluoride significantly induced increase of G2/M phase, PI (proliferation index) and DNA relative content.5. Fluoride, aluminum and aluminiunofluoride on the ALP activity.As can be seen from table 3 after 5 d mineralization fluid exposure, compared with the control group, F (50μM) or aluminum (5μM) group, ALP expression had no significant change; aluminiunofluoride significantly promoted ALP expression (p<0.05), stimulated MC3T3-E1 cell differentiation.6. RT-PCR results6.1. Fluoride, aluminum and aluminiunofluoride on the OPG, RANKL mRNA expression.As can be seen from graph7,8 after 72 h exposure, compared with the control group, aluminiunofluoride group (F 50μM+Al 5μM) significantly promoted the OPG mRNA expression (p<0.05). The RANKL mRNA did not have difference in expression. Compared with the control group, the aluminiunofluoride group (F 50μM+Al 5μmol /L), RANKL/OPG ratio decreased, there is significant difference (p<0.05).6.2. Fluoride, aluminum and aluminiunofluoride on the Runx2, Osterix mRNA expression.As can be seen from graph 10,11,12, after 72 h exposure, compared with the control group, simple fluoride group or pure aluminum group had no significant change in the expression of Runx2, Osterix mRNA; aluminiunofluoride group significantly enhanced Runx2 mRNA expression (p<0.05); in the aluminiunofluoride group Osterix mRNA expression was significantly increased (p<0.01).Conclusions1. Fluoride alone did not elicit any proliferation in osteoblast, while the aluminiunofluoride could significantly improve MC3T3-E1 cells proliferation and the content of M-phase cell, modulate MC3T3-E1 cells from S phase to the M phase transformation.2. Aluminiunofluoride could increase the activity of ALP. Aluminiunofluoride could significantly increase MC3T3-E1 cells differentiation capacity.3. Aluminiunofluoride can significantly improve the expression of Runx2 and osterix mRNA, stimulate MC3T3-E1 cells differentiation and bone formation. Aluminiunofluoride could significantly enhance the expression of OPG mRNA and reduce the RANKL/OPG ratio, balance the RANKL/OPG in reconstruction.Innovative self-evaluationWe study the initiation links of fluorosis in the perspective of aluminiunofluoride, study fluoride, aluminum and aluminiunofluoride on the MC3T3-E1 cells proliferation, differentiation, and mineralization capability. At present, bone reconstruction is a hot issue. According to OPG/RANKL ratio change and Runx2,Osterix mRNA expression we explore how fluoride modulates bone formation and absorption relationship in the process of signal transduction.