| Part1Effect of MSCs on the formation of atherosclerosis plaque in apoliprotein E knock-out miceObjective To explore the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the formation of atherosclerosis plaque in hypercholesterolemic apoliprotein knock-out (ApoE-/-) mice.Methods BM-MSCs isolated from 4 week-old C57BL/6J background ApoE-/- mice were evaluated by flow cytometry for expression of MSC-specific markers. Thirty 8 week-old ApoE-KO mice were randomly divided into three experimental groups (n=10 per group):1. MSC group- received BM-MSCs intravenously; 2. Vehicle group-received DMEM only; 3. Control group- did not receive any treatment. The plaque area of all subjects were compared, the concentration of cytokines in blood were determined by ELISA.Results Compared with controls, BM-MSCs resulted in a significant decrease of the atherosclerotic plaques size in aortic root (15292.78 ± 3309.10 um2 versus 20605.52 ± 5920.42 um2, P<0.05; 15292.78 ± 3309.10 um2 versus 25044.32 ± 4920.42 um2, P<0.05). While, the atherosclerotic plaque size in Vehicle has no obvious change when campared to Control (20605.52 ± 5920.42 um2 versus 25044.32 ± 4920.42 um2, P>0.05). The ratio of plaque area and vessel caliber in MSC was decreased significantly when compared with Control and Vehicle group. The blood levels of TGF-β and IL-10 in MSC group were increased while hs-CRP, IFN-y and MMP-1 decreased significantly (P<0.05).Conclusion BM-MSCs play an important role in regulating the immune inflammatory response and may significantly inhibit the formation and progression of the atherosclerotic plaque in ApoE-/- mice.Part 2Effect of msenchymal stem cells on CD4+CD25+ regulatory T cells in apoliprotein E knock-out miceObjective To explore the effect of bone marrow-derived msenchymal stem cells (BM-MSCs) on CD4+CD25+ regulatory T cells in hypercholesterolemic apoliprotein E knock-out (ApoE-/-) mice.Methods At 8 weeks of age,30 male C57BL/6J background ApoE-/- mice were randomly divided into three groups. Labeled:negative control (Control) group-did not receive any treatment, positive control (Vehicle) group-received DMEM only, msenchymal stem cells (MSC) group-received BM-MSCs intravenously (n=10 per group). To culture and identify msenchymal stem cells isolated from bone marrow of ApoE-/- mice. Analysis of plaque stability was performed, percentage of CD4+CD25+ regulatory T cells in splenocytes were analyzed by FACS, proliferation response of splenocytes to msenchymal stem cells was detected and cytokines in the superanant were determined by ELISA.Results Compared with control animals, BM-MSCs resulted in a significant increase in CD4+CD25+ regulatory T cells in spleen (P<0.05). Specifical proliferation response of CD4+CD25+ regulatory T cells in splenocytes to BM-MSCs was significantly suppressed and the level of TGF-β and IL-10 in the superanant increased while IFN-y, MMP-1 and hs-CRP decreased significantly (P<0.05). The expression of Foxp3 at both mRNA and protein levels was significantly increased in the MSC group (P<0.05).Conclusion BM-MSCs plays an important role in anti-inflammatory response within the atherosclerotic lesion. It may make the atherosclerotic plaque stablization in mice.Part 3Msenchymal stem cells inhibit macrophage-derived foam cells formation by down-regulating scavenger receptor expressionObjective To investigate whether and how bone marrow-derived msenchymal stem cells(BM-MSCs) affect macrophage foam cells formation, and thereby investigate the potential mechanism of BM-MSCs in the development of atherosclerosis.Methods BM-MSCs isolated from four week-old C57BL/6J background mice, were evaluated by flow cytometry for expression of MSCs-specific markers by flow cytometry. Macrophages were cultured alone, with DMEM, or MSCs in the presence of oxLDL for 72 h to transform macrophages into foam cells. Oil red O staining and cellular lipid meagurement were used to identify macrophage foam cells formation. Semi-quantitative PCR, quantitative real-Time PCR and Western blot analysis were carried out to explore the potential mechanism of MSCs-mediated suppression on macrophage foam cells formation.Results Foam cells formation as identified by oil red O staining was readily apparent in cells treated with DMEM and without any treatment cells. After treatment with MSCs. A marked decrease (12.8 ± 5.3%) in foam cells count was observed when compared with untreated cells (12.8 ± 5.3% versus 48.9 ± 8.9%, P<0.001) or Vehicle (DMEM)-treated cells (12.8 ± 5.3% versus 46.7 ± 15.2%, P<0.001). Similar effect was observed when the level of Oil Red O extracted from MSCs-treated and control macrophages cultures was measured on a spectrophotometer. The accumulation of cholesteryl ester was also used to quantify foam cells formation. Consistent with above results, the accumulation of lipids in macrophages was significantly lower than untreated control and DMEM-treated cells. Total cellular cholesterol (TC) and cellular cholesteryl ester (CE) was significantly reduced in MSCs cultures relative to with untreated cells (TC:52.2 ± 7.5 μg/mg versus 132.48 ± 15.46 μg/mg, p<0.01; CE: 22.36 ± 6.75 μg/mg versus 92.76 ± 14.25 μg/mg, p<0.01) or DMEM-treated cells (TC:52.2 ± 7.5 μg/mg versus 120.34 ± 22.09 μg/mg, p<0.01; CE:22.36 ± 6.75 μg/mg versus 86.20 ± 9.3 μg/mg, p<0.01). Moreover, PCR and Western blot analysis showed that the expression of Scavenger receptor both CD36 and SRA in MSCs-treated macrophage foam cells was significantly down-regulated (p<0.01).Conclusion Results collectively suggest that BM-MSCs may inhibit the conversion of macrophages into foam cells in vitro, which is largely attributed to a down-regulated expression in scavenger receptor in BM-MSCs-treated macrophage foam cells. |
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