| Background:Atherosclerosis and its main form coronary artery disease(CAD), leads to many fatalities all over the world. It has been proved that stem cells, especially mesenchymal stem cells(MSCs) may participate in the development of plaque:MSCs could migrate into the vessel wall under certain stimulates, and in situ MSCs could inhibit the proliferation and migration of smooth muscle cells(SMCs). MSCs co-cultured with mature endothelial cells(ECs) have the ability to undergo milieu-dependent differentiation toward ECs, thus depress the progress of atherosclerosis and regenerate the damaged endothelial layer. Howerver, MSCs can differentiate into SMCs that contribute to atherosclerotic lesions and SMCs from MSCs was able to differentiate into foam cells under the stimulate of ox-LDL. In mice with atherosclerosis, labeled donor marrow cells engraft plaque. Recipients have less atherosclerosis when donor marrow-derived progenitor cells are from younger mice. Notably, MSCs has been regarded as an ideal donor in cell transplantation. It will be very important to investigate the fate of MSCs under the risk factors of CAD.Hyperlipemia is one of the main risk factors in atherosclerosis. Low density lipoprotein, especially oxidized low density lipoprotein is critical for the instability of atherosclerotic plaque. Compared with endothelial progenitor cells and smooth muscle progenitor cells, the effects of ox-LDL on less differentiated MSCs are poorly understood.The membrane receptor (such as oxidized low density lipoprotein receptor1,LOX-1) mediated phagocytosis is considered as the main approach in ox-LDL induced toxic effects. Ox-LDL could inhibit the expression of Aktland subsequently the exression of endothelial nitric oxide synthase, thus increase the oxidative stress and inhibit cell migration and proliferation. However, there exist another Akt subtype-Akt2, which has totally different physiologic effects compared with Akt1. It could induce MSCs migration after being activated.However, the distinct role of ox-LDL on these two subtypes in MSCs is not clear. Let-7is the second discovered microRNA and let-7g could modulate the expression of LOX-1in SMCs. But its role in MSCs is not clear.Objective:To investigate the effects of ox-LDL on MSCs proliferation, migration and apoptosis and the associated mechanism.Methods:After primarily isolated and cultured the bone marrow MSCs:(1) Depict the cell growth curve;(2) Determine cell surface antigens:CD29. CD31、CD34、CD45with flow cytometry;(3) Test the adipogenic ability of MSCs;(4) Analyse MSCs apoptotosis after treated with ox-LDL with Annexin-V/PI;(5) Analyse MSCs apoptotosis after treated with ox-LDL with TUNEL;(6) Analyse the phagocytosis of MSCs on ox-LDL with ELISA;(7) Determine the expression of let-7g and LOX-1in MSCs pre and post ox-LDL treated;(8) determine the role of let-7g mimic and LOX-1monoclonal antibody on MSCs phagocytosis;(9) Detect the role of ox-LDL on MSCs migration and proliferation;(10) Investigate the expression of Akt2, MMP-2, Akt1, eNOS in MSCs pre and post ox-LDL treated;(11) Determine the role of let-7g mimic or LOX-1monoclonal antibody on ox-LDL induced MSCs migration and proliferation.Resultsl.The MSCs are CD29+CD31CD34CD45-cells and could differentiate into adipocytes2. Ox-LDL does not sinificantly induce MSCs apoptosis3. MSCs express LOX-1and let-7g. Ox-LDL could increase the expression of LOX-1and inhibit the expression of let-7g4. MSCs could phagocytose ox-LDL. Let-7g mimic and LOX-1monoclonal antibody was able to inhibit this process5. Ox-LDL could induce the proliferation and migration of MSCs, increase the expression of Akt2MMP-2and inhibit the expression of Aktl and eNOS;6.Let-7g mimic and LOX-1monoclonal antibody was able to inhibit the proliferation and migration induced by ox-LDL.Conclusion1. MSCs could phagocytose ox-LDL and induce MSCs migration and proliferation2. Mild to moderate concentration of ox-LDL has no significant effect on cell apoptosis3. The migration and proliferation of MSCs induced by ox-LDL may partly mediated by let-7g/LOX-1-Akt2signaling pathway. Part1The isolation and cultivation of mice bone marrow MSCs and the effects of ox-LDL on MSCs apoptosisObjective:In vitro isolate and culture mice bone marrow MSCs,and investigate the roles of ox-LDL on MSCs apotosisMethods:Combined with density gradient centrifugation and adherent culture method to amplify MSCs in vitro. Use MTT assay to gain growth curve of MSCs. Cellular surface antigens were tested with flow cytometry and the adipogenic ability was also tested. Use Annexin-V/PI and TUNEL to investigate the cell apoptosis.Results:we successfully obtained the mice bone marrow MSCs, which illustrated both CD29positive, but CD31, CD34and CD45negative. Under certain condition, MSCs could differentiate into adipocytes. Variate concentration of ox-LDL (0,10,20,40μg/mL) did not significantly induce cell apoptosis:the apoptosis rate were2.0±0.9%,3.2±1.2%,3.9±1.8%,4.3±2.2%respectively with Annexin-V/PI assay (P>0.05) and2.1±1.0%,2.9±1.2%,4.0±0.9%,3.7±1.3%respectively with TUNEL assay (P>0.05).Neither, Ox-LDL did not induce cell apoptosis in a time dependent manner:the apotosis rates in0,6,12,24h were2.3±0.8%,3.4±1.2%,4.2±1.6%,3.9±1.4%respectively with Annexin-V/PI assay (P>0.05) and2.5±0.7%,3.7±1.3%,4.2±1.2%,4.0±1.0%respectively with TUNEL assay (P>0.05) Conclusion:Mice bone marrow MSCs could be successfully obtained after density gradient centrifugation and adherent culture. Mild to moderate concentration of ox-LDL in less than24hours did not significantly induce MSCs apoptosis. Part II The expression of let-7g and LOX-1in MSCs and their roles during phagocytosis of MSCs on ox-LDLObjective:To investigate the phagocytosis of mice bone marrow MSCs on ox-LDL and the roles of microRNA let-7g or membrane receptor LOX-1in this processMethods:We detected the content of ox-LDL inside cells with ELISA and detected the expression of let-7g with Quantitative real-time PCR or LOX-1with both Quantitative real-time PCR and western blot. We also detected the roles of LOX-1Monoclonal antibody or let-7g mimic on the process of phagocytosis.Results:The concentration of ox-LDL inside MSCs was significantly increased after incubated with20μg/mL ox-LDL for24hours,6.0±0.8vs control, P<0.05. Ox-LDL could dose dependent induce the expression of LOX-1in both mRNA and protein levels:After MSCs were treated with0,10,20or40μg/mL ox-LDL for24hours, the mRNA and protein levels of LOX-1were1.0±0.2,2.2±0.15,3.3±0.3,3.5±0.2and1.0±0.3,2.0±0.2,3.2±0.3,3.1±0.4respectively, both P<0.05. Ox-LDL could also time dependent induce the expression of LOX-1in both mRNA and protein levels:After MSCs were treated with20μg/mL ox-LDL for0,6,12or24hours, the mRNA and protein levels of LOX-1were1.0±0.15,1.9±0.2,2.7±0.2,3.3±0.3and1.0±0.2,1.7±0.3,2.7±0.3, 3.2±0.2respectively, both P<0.05. The expression of LOX-1induced by ox-LDL could be significantly inhibited by50nM Let-7g:the mRNA and protein levels of LOX-1were1.8±0.15vs3.3±0.15and2.0±0.25vs3.2±0.2respectively, both P<0.05. Conversely, the expression of microRNA let-7g was greatly inhibited after cells were treated with20μg/mL ox-LDL(0.4±0.06vs1.0±0.08, P<0.05); and this inhibitory effects could be partly reversed by10μg/mL LOX-1monoclonal antibody(0.8±0.04for LOX-1vs0.4±0.06for control, P<0.05). Either LOX-1monoclonal antibody or let-7g mimic could antagonize the phagocytosis of MSCs on ox-LDL(2.0±0.2vs6.0±0.8, and1.7±0.3vs6.0±0.8respectively, both P<0.05).Conclusion:Mice bone marrow MSCs could phagocyte ox-LDL and this role was related to microRNA let-7g and membrane receptor LOX-1. Part Ⅲ Let-7g/LOX-1-Akt2pathway mediate the proliferation and migration of MSCs induced by ox-LDLObjective:To investigate the role of ox-LDL on mice bone marrow MSCs proliferation and migration and to investigate whether these effects were mediated by Let-7g/LOX-1-Akt2pathwayMethods:Use MTT assay to detect the role of ox-LDL on MSCs proliferation and transwell assay to detect the role of ox-LDL on MSCs migration. Both Quantitative real-time PCR and western blot were chosen to detect the associated gene expression in Let-7g/LOX-1-Akt2pathway. We also detected the roles of LOX-1monoclonal antibody or let-7g mimic on the process of MSCs migration and proliferation.Results:The migration and proliferation of MSCs were significantly improved after ox-LDL treated. Either LOX-1monoclonal antibody or let-7g mimic could inhibit these effects. Ox-LDL could time and dose dependently enhance the expression of Akt2, MMP-2, and could time and dose dependently downregulate the expression of Aktl, eNOS. Either LOX-1monoclonal antibody or let-7g mimic could inhibit these effects.Conclusion:Ox-LDL could promote proliferation and migration of MSCs. This effects were mediated by Let-7g/LOX-1-Akt2pathway, followed by increased expression of MMP-2and decreased expression of Aktl and eNOS. |
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