| Objective: Arterial calcification is an active and strictly regulated process mainly driven by vascular smooth muscle cells(VSMCs).Periodic tension stimulation can affect the phenotypic transition of VSMCs and regulate arterial calcification.The mechanical stretch produced by the cardiovascular circulatory system on intravascular VSMCs is a kind of physical stimulation,which converts the physical stimulation into internal signals of the cell through ion channels of the cell membrane,thus affecting VSMCs.Mechanosensitive channel of large conductance(Msc L)is the most deeply studied mechanically sensitive ion channel at present.It only responds to the tension of cell membrane,and does not need any functional interaction with other cell elements.In this study,we intend to explore the effects of mechanical stretching on osteoblast-like differentiation of VSMCs by regulating Msc L and the mechanism.Methods: First of all,Msc L expression lentivirus and negative control lentivirus were constructed by Shanghai Taiting Biotechnology Co.,Ltd.according to the standard.Then the multiplicity of infection(MOI)of VSMCs was screened.VSMCs were infected with Msc L virus and negative control(NC)virus,and the VSMCs were purified with puromycin.The arterial calcification cell model was established with VSMCs expressed by Msc L induced by β-glycerophosphate disodium salt hydrate(β-GP)or control VSMCs,and the best induction days were selected.Alizarin red staining was used to further verify the establishment of arterial calcification cell model.The VSMCs induced by β-GP were divided into mechanical traction group and control group according to whether mechanical traction was given or not,and VSMCs without β-GP and without traction were established as controls respectively.The mechanical traction group was stimulated with 7% extension amplitude and frequency of 1Hz,while the control group was cultured in the same environment.After 6 days of culture,the expression of RUNX2 and BMP2 were detected by western blot to evaluate the degree of osteogenic differentiation of VSMCs,and the expression of ERK and p-ERK were further detected to explore whether ERK signaling pathway was involved in this process.Results:(1)Under fluorescence microscope,the transfection efficiency of lentivirus in VSMCs was satisfactory.There was significant difference in MOI(90,180,270,360)between each group and the control group(P<0.001),but there was no difference between MOI 270 and MOI360(P=0.982).(2)Flow cytometry showed that the survival of VSMCs screened by puromycin concentration(1.5,2,2.5,3,3.5ug/ml)was significantly different from that of the control group(P<0.001),and the purification efficiency was 95% when puromycin was 3.5ug/ml.(3)Western blot results showed that compared with the control group,the significant expression of calcification related factor RUNX2 could be detected on the 6th day after β-GP induction(P=0.024).Further alizarin red staining showed that the mineralized nodules formed by VSMCs induced by β-GP were significantly higher than those in the control group(P= 0.006).(4)The protein was extracted from the cells of each group after being cultured in the same environment for 6 days.Western blot showed that the RUNX2 and BMP2 of Msc+β-GP group were lower than those of the control group+β-GP group under mechanical stretch stimulation(P=0.016 、 P=0.024),while there was no significant difference in calcification-related protein RUNX2 and BMP2 between Msc+β-GP group and control group+β-GP group without stretch stimulation(P=0.160、P=0.097).(5)Western blot analysis showed that under mechanical stretch,the expression of phosphorylated ERK in Msc L+β-GP group was lower than that in control group+β-GP group(P < 0.001).Conclusion: Stimulation of Msc L by mechanical stretching may alleviate the osteogenic differentiation of VSMCs induced by β-GP through ERK signal pathway. |
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