| Objective:One of the bone defect repair has been the focus of medical research. Clinical caused by trauma, infection and tumors on the repair of large bone defect is not found the ideal solution yet. Currently most widely used methods including autologous bone and Allogenic bone and artificial bones. But these tools have their own shortcomings. Over the past more than10years, stem-cell research are becoming hot spots. Bone marrow-derived mesenchymal stem cell (bone marrow derived mesenchymal stem cells,BMSCs) to its biological characteristics in attracting the attention of many scholars. Bone marrow-derived mesenchymal stem cells exist in many adults in the matrix, which is a kind of undifferentiated cells, or the original cell, BMSCs are self-replicating ability of pluripotent cells. Which can differentiate into osteoblasts, adipocytes and chondrocytes in vitro, until the formation of several types of tissues and organs. since1970in mouse embryonic stem cells cultured in vitro after success, stem cell research have become more and more important attracting the attention of scholars at home and abroad. Because MSCs for bone cell precursors provides resources, so they have an important role in bone remodeling. In addition, in various animal models of the MSCs derived from bone marrow to repair the bone deficiency of a certain size. Stem cell proliferation and differentiation has been the focus of the study.Oxygen which is an indispensable condition as one of a necessary life activities and environmental factors, play an important regulating role on the proliferation and differentiation of mesenchymal stem cells. but oxygen is approximately1%-7%pO2in the bone marrow, this is much lower than those in the general environment. Visible in the repair of bone defect in the process,MSCs have to undergo such low oxygen environment to finish its proper role. Study the effects of hypoxia on osteogenic differentiation of MSCs and its relevant mechanisms, it is meaningful.This study consists of three parts, namely:(1) discussing on the effect of hypoxia on osteogenic differentiation of MSCs and (2) researing the hypoxia in osteogenesis differentiation of MSCs and the relationship between Notch Signaling;(3) the Notch signal under low oxygen conditions on preliminary study on the mechanism of osteogenic differentiation of MSCs. As described below.Methods:In the first part of the work, observing the effects of hypoxia on osteogenic differentiation of MSCs. We used density gradient centrifugation method, uniform morphology of rat bone marrow derived MSCs were obtained and confirmed its bone marrow derived MSCs. After14days of the original culture, the cells with insulin/EDTA end, the night after adsorption, replace medium with osteogenic medium in order to study the effects of low oxygen, and normal oxygen,1%pO2(low-oxygen) or21%pO2(normal oxygen) under oxygen training, on purpose to ALP activity of cells, respectively; analysis of real time PCR gene expression is specified; ARS coloring of mine detection.. In MSCs differentiation by PCR in real time during the7-day,14-day,21-day test when Notch1expression level. Compared with normal oxygen, Notchl expression significantly higher in low oxygen conditions. In addition, under low-oxygen conditions, Notchl expression of osteogenic differentiation criteria groups than in control much in differentiated groups, suggesting that Notchl expression of low oxygen and Osteoblast differentiation of MSCs is negative. And consistent levels of mRNA, Notchl under low oxygen conditions of MSCs7days,14days,21day marked increase in protein expression levels in various points in time Control shRNA Notchl specificity chronic virus is injected into MSCs to inhibit Notch signaling activity. Notchl expression of shRNA obviously inhibit. Control shRNA Notchl shRNA and MSCs in the transformation of culture under condition of osteogenic induction after7days, exam ALP activity, Runx2, mRNA expression of OPN and OCN level.Results:ALP activity in cultured cells under low-oxygen conditions is much lower than under normal oxygen conditions (p<0.01); three genes expression in hypoxia group levels are much lower than under normal oxygen conditions (p<0.01), differentiation of cultured MSCs in low oxygen conditions compared to the level of mineralization under normal oxygen conditions significantly reduced (p<0.01). These results suggest that low oxygen inhibits osteoblastic differentiation of MSCs., it also validated the results of the mRNA levels. The above experiments, we can conclude that, hypoxia can enhance and Notchl expression leading to activation of Notch Signaling.Control shRNA Notchl specificity chronic virus is injected into MSCs to inhibit Notch signaling activity. Notch1expression of shRNA obviously inhibit. Control shRNA Notch1shRNA and MSCs in the transformation of culture under condition of osteogenic induction after7days, in control cells, ALP activity under low oxygen conditions than under normal oxygen conditions is much lower; in the inhibition of Notchl cells, ALP activity under two conditions do not obvious. In addition, Runx2, mRNA expression of OPN and OCN level control shRNA transformation group under low oxygen, and normal oxygen varied widely, but is not evident in the Notchl shRNA into groups. The same, mine is not apparent in the differences in levels of shRNA transformed cells, but is quite clear in the control group. Therefore, Notchl signal for differentiation of MSCs into osteoblasts hypoxic injury is crucial. Clarifies the Notch signal for differentiation of MSCs into osteoblasts after hypoxic injury of necessity, we further study of hypoxia by Notch Signaling inhibition mechanism of differentiation of MSCs into osteoblasts. Runx2Osteoblast differentiation is a key transcription factor, we also studied the effect of low oxygen and Notchl are activity of Runx2, this initial full account under low-oxygen conditions of Notch signaling mechanism on osteogenic differentiation of MSCs. First, in conditions of low oxygen, and normal oxygen measuring Runx2against Notchl-shRNA MSCs transfected neutralized Notchl-shRNA protein expression levels in MSCs transfected. Whether in low or normal oxygen conditions, Notchl inhibition can cause Runx2hike. In addition, immuno-precipitation experiments have show the Notchl physiological binding on Runx2. To further investigate the functional consequence of the the binding between Notch1and Runx2,the transcriptional activity of Runx2by using fibronectin or osteocalcin promoter through adenoviruses.The transcriptional activity of Runx2was upregulated by the Notchl knockdown. Therefore, Notchl inhibits Runx2activity by physical binding.Conclusion:(1) low oxygen inhibition of osteogenic differentiation of MSCs.(2) in osteogenic differentiation of MSCs during hypoxia activates the Notch signal.(3) the Notch signal for hypoxic injury of osteogenic differentiation of MSCs is a must.(4) the Notch on the Runx2by binding directly inhibits the transcriptional activity of Runx2. |
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