| Bone tissue is the most easily affected tissue by mechanical surroundings change，soas to change in morphology and bone mass. Appropriate mechanical stimulation caninhibit bone resorption and increase bone formation. Studies have shown that there aremany ion channels associated with the osteogenetic differentiation of cells, such as Ca2+,K+, etc..But as the main anion channel-chloride channel in the body, the role of it is stillpoorly understood. ClC-3is one of the voltage-gated chloride channel,it was defined asvolume sensitive chloride channel earlier. In recent years, studies have shown that the roleof ClC-3is not only confined to the cell volume regulation but also with the relevant ofcell proliferation, apoptosis, intracellular acidification regulation of osteoclast, etc..Osteoblast cell volume changes when under the mechanical stimulation,it’s not clear thatwhether this change is mediated by ClC-3. This study will explore the possibility of ClC-3is a mechanical stimulation sensitive chloride channel through the technologies such likecells cultured in vitro, afterburner experiment in vitro, siRNA etc.. ClC chloride channel is a gene family, and is a voltage-gated chloride channel.Among them, the ClC-3and ClC-4, ClC-5become a major branch due to its geneticsequence similarity,are the chloride channel of endosomal system. In recent years, manystudies have found ClC-3chloride channel may be associated with the osteogeneticdifferentiation of cells. In this study, we used the augmentor designed of ourself to givepersistent static pressure stimulation to cultured mouse osteoblast-like cell line MC3T3-E1in vitro, established a pressurized model in vitro successfully. Morphological changeswere observed by inverted microscope，and detected the relative mRNA expressionchanges of related bone factors and intracellular signaling pathways molecules whichregulating osteogenic differentiation. We found that osteoblasts cell morphologychanged after persistent static pressure. The relative mRNA expression of osteogenicfactor (Alp of Ocn, Bsp), Runx2and Wnt pathway signaling molecules (β-catenin, Lrp6)have significantly increased, the relative mRNA expression of Tgf-β is decreased whilePvrl has no significant change after the mechanical stimulation.This indicates that themechanical stimulation can influence the differentiation and proliferation ofosteoblasts.Tgf-β, Runx2and Wnt signaling pathway are interactional, and to participate inthe cell development and differentiation jointly.The relative mRNA expression of Chloride ion channel ClC-3in osteoblasts wassignificantly increased after persistent static pressure. Through the method of genetransfection to inhibit and overexpress the CLC-3chloride channel in osteoblastsrespectively, we found that after inhibition of CLC-3chloride channel, in the relatedmolecules we detected, the relative mRNA expression of Alp, Ocn, Bsp, Runx2, Lrp6and β-catenin was also decreased, but the relative mRNA expression of Tgf-β and Pvrlwas elevated. But over-expression of ClC-3chloride channel, the results were opposite toinhibition results. This was confirmed from both positive and negative that ClC-3chloridechannel has a regulating effect on the expression of osteogenesis related gene and therelated signal pathways. To give continuous static pressure stimulation to cells after genetransfection, inhibit and overexpress the CLC-3chloride channel in osteoblastsrespectively, the tendency of relative mRNA expression level of each gene’s response to stress between transfection cells and the normal cells is the same. This shows that theCLC-3chloride channel in osteoblasts can be directly activated or indirectly activate bythe volume change when the cell in a certain mechanical stimulus environment.And thenthe CLC-3chloride channel can balance the relationship between osteoblast activity andosteoclast activity, which is also involved in osteoblast differentiation.Through this study, we established a pressure model in vitro of mouse osteoblast-likecell line MC3T3-E1,observed that pressure stimulation can affect osteoblast differentiation,and ClC-3chloride channel is sensitive to pressure stimulation. And by the method ofgene transfection, confirmed that the ClC-3chloride channel plays an important role onthe adjust expression of bone-related genes and signaling pathways. And the pressurestimulating effect on osteoblasts is mediated by CLC-3chloride channel. The presentstudy view the osteoblast stress problem from the perspective of cell physiology, andthere’s some new interpretation and supplements to the signal transmission mechanism ofosteoblast mechanics which have been obtained. |
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