| Bone reconstruction, has been the focus of oral medical research, because of thealveolar bone is one of the most active part of the metabolism of bone tissue in thebody, reconstruction of bone tissue can be a lifetime. Many oral diseases orphysiological bone reconstruction are closely related with the absorption, defects,repair or metabolism of alveolar bone. The biological basis of orthodontics isthat tooth movement is under the action of the appropriate force. Osteogenesis andbone resorption occurred in the reconstruction process of alveolar bone, and thebalance of these two major processes is the key to maintain the normal bonemetabolism process. In physiological conditions, bone reconstruction process iscontrolled by a complex network of osteoblasts,osteoclasts,local cytokines andsystemic factors. A variety of factors were used to affect osteoblasts and intervene inthe bone formation process. All these studies ultimately need to go to the level ofsignaling pathways to explore the further mechanisms. Due to the stimuli aredifferent, the signal transduction pathways are different. It says that the ways toregulate bone formation is not the single and there is overlapping between each path.The ERK1/2，p38MAPK are researched more advanced. ERK1/2MAPK has beenfound to play an important role in regulating cell proliferation and differentiation,while p38MAPK foten involve in the regulation of cell differentiation and apoptosisprocess. Runx2plays an important role in osteoblast regulation,and it is the mostimportant transcription factor. A number of signaling pathways affect the function ofosteoblast by influencing the activity of Runx2.ODN (oligodeoxynucleotides, ODN) refers to the multi-nucleotide chainscontaining20-50nucleotide monomers. As ODN also has the advantages ofhigh-efficiency, easy synthesis and properties of stable, so in recent years it becomesthe hot spot in the field of immunology and basic medical researches. ODN isabsorbed into cells through the receptor-mediated form, and then perform important function in cells. Thio-modified phosphate could enhance the capability of ingestingand resisting the digestion of nuclease, which is good for keeping stable in vivo andincreasing the intracellular uptake rate. Our Previous studies have shown that aspecific sequence of ODN can promote the osteogenetic differentiation from bonemarrow mesenchymal stem cells, and it can promote osteoblast proliferation anddifferentiation. The specific sequences ODN promote bone reconstruction process asthe research provides a theoretical basis and experimental basis. Because theprevious research mainly focused on immune cells to absorb ODN, this topic willfocus on whether ODN MT01could be taken into the osteoblasts, as well as its timedependencies into osteoblast. Reviewed previous literatures, we found thatosteoblasts are regulated by MAPK signal pathway,especially by ERK1/2and p38MAPK, as the main transcription factor Runx2is involved, therefore hypothesis isproposed: ODN MT01could promote osteoblast differentiation mainly throughERK1/2and p38MAPK pathway, Runx2would participate in the regulationfunction. The laser confocal microscope will be used to observate the fluorescentlabeling of ODN MT01. Using western blot technology, realtime PCR technologyand alkaline phosphatase (ALP) staining techniques to detect the activation ofMAPK pathways. Protein kinase inhibitors of ERK1/2MAPK and p38MAPKsignaling pathway were used. Then we observed the changes of the ALP, OC andColla I.We also detected the phosphorylation level of Runx2. Our findings willfurther clarify the mechanism that ODN MT01promoting the osteoblastdifferentiation, and to lay the theoretical basis for study of ODN MT01regulationthe process of bone reconstruction.The content of this research is divided into five parts:Part i: InternalizationAnalysis of ODNsTo investigate whether the effects of ODN required internalization into cells,MG63cells were incubated with ODN MT01which was labeled with Cy5accordingto the test of time, confocal laser microscope was used to detect the results.Part ii: Effects of ODN MT01on MAPK Signaling Proteins ERK1/2and p38 MAPKTo investigate the effect of ODN MT01on MAPKs, both total andphosphorylated levels of MAPKs were investigated by Western blotting after0,30,60min,24h and48h of treatment with1μg/mL ODN MT01. To better demonstratethe effects of MT01in MG63cells were the specific sequence we tested anotherODN sequence, ODN FC003, a24-merODN(5’-TCTCTCTCTCTCTCTCTCTCTCTC-3’). The design of ODN FC003wasalso based on human mitochondrial DNA, and contained successivethymidine-cytosine nucleotides,we used ODN FC003as the negative control.Part iii: Effects of ERK and p38Inhibitors on Up-Regulation of ALP ActivityInduced by ODN MT01To investigate the involvement of ERK1/2and p38MAPK in modulation ofALP activity induced by ODN MT01, specific chemical inhibitors of ERK and p38(U0126and SB203580, respectively) were used. Cells were pre-treated with orwithout10μM U0126or SB203580for1h, and then treated with or without MT011μg/mL.Part iv: Effects of ERK and p38Inhibitors on the Expression of Osteocalcin andColla ITo further confirm our results, we analyzed the expression of subsets ofosteogenic marker genes including osteocalcin and Colla I. We used ERK and p38inhibitors to pre-treat cells for1h followed by treatment with1μg/mL ODN MT01or ODN FC003for15days. Then, the mRNA and protein expression ofosteocalcin and Colla I were analyzed by real-time PCR and Western blot,respectively.Part v: Effect of ERK and p38Inhibitors on Runx2Expression in Response to ODNMT01To investigate the role of Runx2, which is one of the most important factors forosteoblast differentiation, we used the ERK inhibitor U0126and p38inhibitorSB203580to pre-treat cells for1h followed by treatment with1μg/mL ODN MT01 for72h. The total and phosphorylated protein levels of Runx2were detected usingWestern blotting.Through the above five experiments, the results were as follows:Part iODN MT01entered cells by endocytosis after incubation with MG63cells for15min. The fluorescence intensity was enhanced over time, and the intensity at6hwas significantly higher than that at3h, and the intensity at12h reached the peak. Itwas apparent that ODN MT01stimulation of osteoblasts required internalization.Part iiPhosphorylation of ERK1/2was observed after30min of treatment with ODNMT01, which increased after60min compared with that at0min （P<0.05）.Furthermore, the active effect lasted up to48h（P<0.05）, while the total level ofERK1/2protein remained unchanged. For p38, there was no obvious change in thephosphorylation level before30min. However, p38phosphorylation increased after60min of treatment with MT01,and showed an increase at24h（P<0.05）, whichlasted up to48h. The FC003sequence had no active effect on MG63cells viaERK1/2or p38MAPKs.Part iiiCompared with the control, ALP activity was significantly up-regulated afterODN MT01treatment at48and72h（P<0.05）. Notably, U0126and SB203580significantly inhibited the up-regulation induced by ODN MT01（P<0.05）.Part ivCompared with the control, the protein expression of osteocalcin was increasedby ODN MT01treatment（P<0.05）. Compared with ODN MT01treatment, theprotein level of osteocalcin was significantly decreased when cells were pretreatedwith the ERK and p38inhibitors（P<0.05）. The expression level of Colla I exhibiteda similar tendency with osteocalcin. The mRNA levels were concurrent with theprotein expression. Part vThe total and phosphorylated protein levels of Runx2were detected usingWestern blotting. There were obvious differences in the phosphorylation levels ofERK and p38between the MT01-treatment and control groups; p-ERK and p-p38were significantly inhibited in the presence of U0126and SB203580, which wereinduced by MT01. For Runx2, there was no obvious change in protein levels inthese groups（P>0.05）, but MT01induced notable phosphorylation of Runx2（P<0.05）. However, the up-regulated phosphorylation of Runx2induced byMT01was inhibited in the presence of U0126and SB203580.Through the above results we confirmed:ODN MT01enter into the cytoplasm, but not the nucleus and the process wasin time dependence. ODN MT01launched a series of signal transduction pathways,transduction to the nucleus. ODN FC003did not show the activation effect ofERK1/2or p38MAPK. ODN MT01(1mg/L) can activate ERK1/2and p38MAPKsignal pathway, the activation is sequence specificity. ODN MT01activated theERK1/2earlier than p38MAPK protein kinase. The ERK1/2and p38MAPKsignaling pathways involved in the process that ODN MT01upregulating the ALPexpression.The ERK1/2and p38MAPK signaling pathways involved in the processthat ODN MT01promoting the osteoblasts to express OC and Colla I; ODN MT01promoted the activation of Runx2, inhibitors inhibit the promoting effect respectively,ODN MT01regulating the osteoblast differentiation via the ERK1/2and p38MAPK signal pathway, and Runx2also involved in the process.The above results of the study are applied in areas other than the immunology,and further expound the mechanism of specific sequence ODN MT01inosteogenesis adjustment, and lay the theoretical basis of the application of ODNMT01to regulate bone reconstruction. |
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