Study Of The Role And Mechanism Of Notch Signal Pathway And Key Factor Aph1in Osteoblastic Stress Effects

Backgrounds:Mechanical stress plays an important role in the remodeling of bone, and the effect of fluid shear stress on osteoblasts has always been an hot spot in this field. Notch pathway is regarded as a crucial signal involved in the differentiation of osteocytes, however, the signal transduction associated with bone remolding under stress is less studied. In the previous experiment of our project,appropriate magnitude of fluid shear stress was applied on MC3T3-E1cells and the results of microarray showed that APHl in the Notch pathway was elevated significantly, demonstrating an important role that Notch pathway plays in the response of osteoblasts to stress. In-depth study in the mechanism of Notch signal pathway makes it clear that mechanosensing characteristics of Notch receptor participates in the activation of this pathway. This project may provide theoretical foundation for tissue engineering, development in wound healing quality, treatment of bone diseases and exogenous gene therapy.Objective:On the above foundation, this study is to explore the role and mechanism of Notch signal pathway in the FSS-induced biological response in MC3T3-E1cells, in order to make clear further the molecular mechanism of the biological reaction of osteoblasts under stress.Methods:1. MC3T3-E1cells were cultured in vitro and model of mechanical stimulus was established through building parallel plate flow chamber system.2. Cells were divided into four groups randomly, which were control group, FSS group(0.12mN·cm-2for1h), inhibitor group (10μmol/ml DAPT) and FSS combined with inhibitor group.3. Methyl thiazol tetrazolium (MTT) and alkaline phosphatase (ALP) were adopted to test the proliferation and differentiation of osteoblasts.4. OPN Western blot and NO assay were conducted to explicit the role of Notch signal in the biological process.5. Statistically analysis were conducted through SPSS17.0software.Results:1. MC3T3-E1cells were in good condition and responded well on mechanical stimulus generated through parallel plate flow chamber system.2. Fluid shear stress can significantly increase the ALP activity compared with control groups(P<0.05), while MTT were not significantly changed(P>0.05); the ALP activity and MTT decreased with the application of inhibitor both in FSS and static group(P<0.05).3. Fluid shear stress can significantly increase the expression of OPN and NO production compared with the control group(P<0.05); treatment of Notch signal inhibitor leaded to the marked decline of OPN and NO both in FSS and static group(P<0.05).Conclusions:1. Parallel plate flow chamber system can be applied to study the biological responses to FSS of MC3T3-E1cells adheres to the walls, it can simulate the growth environment and realize the mechanical loading, thus establishing the foundation of further study on the mechanotransduction in osteoblasts.2. Notch signals exist in the normal physical environment and participate in proliferation and differientation of MC3T3-E1cells, FSS of0.12mN·cm-2for1h regulates the biological processes partially through Notch signal pathway.3. Notch signals exist in the normal physical environment and participate in OPN protein and NO production of MC3T3-E1cells, FSS of0.12mN·cm-2for4h regulates the production of OPN and NO partially through Notch signal pathways.