氧化低密度脂蛋白对小鼠骨髓源性平滑肌祖细胞CXCR4表达的影响
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摘要
背景基质细胞衍生因子(stromal cell derived-factor1,SDF-1)又名CXCL12或前B刺激因子,属于CXC族趋化因子成员,同基因编码两种蛋白:SDF-1α和SDF-1β。SDF-1主要在骨髓基质细胞及骨髓内皮细胞表达,在造血干细胞中也有相应的表达。SDF-1唯一的受体CXCR4是一个有七个跨膜结构域的G蛋白耦联的受体,在造血干/祖细胞表面及内皮祖细胞表面高表达,新近研究表明CXCR4也在骨髓间充质细胞(marrow stromal cells;MSC)上表达。文献报道,在心肌梗死后的心肌中立即出现了SDF-1的上调并导致过表达CXCR4的MSC向损伤心肌迁移。在Zhang等的研究中表明CXCR4+的MSC参与了新生血管的生成并分化为新的心肌细胞,动物实验表明SDF-1/CXCR4轴的相互作用在心肌修复时对MSC的募集起着重要作用,在SDF-1和/或CXCR4敲除老鼠的研究中表明该轴在细胞分化中起着重要作用。MSC是骨髓微环境中重要细胞成分,具有向各种细胞分化的潜能,且有文献报道MSC可向平滑肌祖细胞(Smooth muscle progenitor cell;SPC)分化。骨髓源性MSC或SPC是动脉粥样硬化(atherosclerosis,As)斑块中平滑肌细胞重要来源之一。那么在As的斑块形成及修复过程中SDF-1/CXCR4轴是否能募集MSC或骨髓源性的SPC的归巢致损伤动脉,参与As形成尚未见报道。
目的检测As危险因子氧化低密度脂蛋白(Oxidized Low Density Lipoprotein;ox-LDL)对小鼠骨髓源性SPC SDF-1受体CXCR4表达的影响,探讨SDF-1/CXCR4对骨髓源性SPC归巢至受损动脉的作用。
方法小鼠骨髓源性SPC与ox-LDL(50μg/mL)共同孵育,用逆转录聚合酶链反应法(Reverse transcription-polymerase chian reaction;RT-PCR)检测SPC CXCR4 mRNA的表达,免疫印迹法(immunoblotting)及免疫荧光染色和共聚焦激光扫描显微镜分析(immunofluorescence staining and confocal laser scanning microscope analysis)检测SPC CXCR4蛋白表达,观察ox-LDL对SPC表达CXCR4的时效关系。
结果未给予ox-LDL刺激的SPC有基础水平的CXCR4表达,50μg/mL的ox-LDL刺激SPC 0-72h,随刺激时间的延长,CXCR4表达逐渐增强,36h达峰值,其mRNA和蛋白水平分别为基础水平的5.73倍和5.02倍,差异均有统计学意义(p<0.05),随后逐渐下降,但仍高于基础水平。免疫荧光染色和共聚焦激光检测并观察到在0h时即未给予ox-LDL刺激时SPC的胞质和细胞膜表面有少量的CXCR4表达,随着作用时间的延长,CXCR4的表达量逐渐上调且在36h时其在SPC的胞质和细胞膜表面的表达最强,60h后CXCR4的表达量逐渐下降。
结论ox-LDL上调小鼠骨髓源性SPC CXCR4的表达,提示ox-LDL有可能通过上调SPC CXCR4的表达致SPC迁移和归巢至病变动脉参与As形成。
Background The chemokine stromal cell derived-factor1(SDF-1)also name CXCL12 or pro-B stimulating factor and belong to the intercrine CXC subfamily. The single SDF-1 gene encode two kinds protein:SDF-1αand SDF-1β.SDF-1 major express on hematopoietic stem cells.CXCR4 is the single chemokine receptor of SDF-1, which is a seven-transmembrane domain, GTP-binding protein-coupled receptor and is high expression in hematopoietic stem/progenitor cells and endothelial progenitor cells. Newly investigation indicate that CXCR4 also express on marrow stromal cells(MSC).Literature report that the myocardium after myocardial infarction(MI) will immediately up-regulate the SDF-1 and induce over-expression of CXCR4 to migration in ischemic myocardium. In the Zhang’s investigation manifest the CXR4+ MSC has participated in the form of the new vessels and differentiate to original myocardical cells; animal studies indicate the interaction of SDF-1/CXCR4 axis play a critical role in the repair myocardium through recruitment MSC. Study with the mouse which are knocked-out of SDF-1 and/or CXCR4 has indicate the SDF-1/CXCR4 axis also play a important role in the cell differentiation. MSC is a crucial cell component in the bone marrow microenvironment, which has potency to differentiate to various kinds of cells, and a literature report that MSC can differentiate into the smooth muscle progenitor cells(SPC).The bone marrow-derived MSC or SPC is the significant source of smooth muscle cell in the plaque of the atherosclerosis(As).Then ,we are still unknown that during the plaque to form and reparation of the As whether the SDF-1/CXCR4 recruitment MSC or SPC homing to damage arteries and take part in the shape of As or not.
Object Oxidized Low Density Lipoprotein (ox-ldl) is a risk factor of As. Now to detect the effect of ox-ldl on CXCR4, which is the receptor of SDF-1, expression in mouse bone marrow-derived SPC .To explore the contribution of SDF-1/CXCR4 to homing SPC to arterial damage.
Methods SPC was incubated with the same concentration (50μg/mL), CXCR4 mRNA and protein was revealed by RT-PCR, Western-Blot ,immunofluorescence staining and confocal laser scanning microscope analysis respectively. To observe the time-effect relationship between ox-ldl and the SPC expression of CXCR4.
Results Ox-ldl did not give to the SPC but it has the foundational level of CXCR4 expression,50μg/mL of ox-ldl stimulate SPC 0-72h,during the stimulation time ,CXCR4 expression gradually,36h reached peak, and its mRNA and protein levels were 5.73 folds and 5.02 folds of the basis level ,the differences were statistically significant(p<0.05),then decreased gradually, but still higher than the basis level. Immunofluorescence staining and confocal laser scanning microscope analysis detection in 0h observed when ox-ldl did not give SPC stimulated the cytoplasm and cell membrane surface of a small number of CXCR4 expression, with the extension of time ,CXCR4 expression gradually increases the volume when in 36h in the SPC and the cycoplasmic membrane surface expression of the strongest,60h the expression of CXCR4 decreased gradually.
Conclusions Ox-ldl can increase in mouse bone marrow-derived SPC CXCR4 expression ,suggesting the possibility of ox-ldl by upregulate the expression of SPC CXCR4 to induced migration and homing of SPC to the artery lesions to participate in the formation of As.
目的检测As危险因子氧化低密度脂蛋白(Oxidized Low Density Lipoprotein;ox-LDL)对小鼠骨髓源性SPC SDF-1受体CXCR4表达的影响,探讨SDF-1/CXCR4对骨髓源性SPC归巢至受损动脉的作用。
方法小鼠骨髓源性SPC与ox-LDL(50μg/mL)共同孵育,用逆转录聚合酶链反应法(Reverse transcription-polymerase chian reaction;RT-PCR)检测SPC CXCR4 mRNA的表达,免疫印迹法(immunoblotting)及免疫荧光染色和共聚焦激光扫描显微镜分析(immunofluorescence staining and confocal laser scanning microscope analysis)检测SPC CXCR4蛋白表达,观察ox-LDL对SPC表达CXCR4的时效关系。
结果未给予ox-LDL刺激的SPC有基础水平的CXCR4表达,50μg/mL的ox-LDL刺激SPC 0-72h,随刺激时间的延长,CXCR4表达逐渐增强,36h达峰值,其mRNA和蛋白水平分别为基础水平的5.73倍和5.02倍,差异均有统计学意义(p<0.05),随后逐渐下降,但仍高于基础水平。免疫荧光染色和共聚焦激光检测并观察到在0h时即未给予ox-LDL刺激时SPC的胞质和细胞膜表面有少量的CXCR4表达,随着作用时间的延长,CXCR4的表达量逐渐上调且在36h时其在SPC的胞质和细胞膜表面的表达最强,60h后CXCR4的表达量逐渐下降。
结论ox-LDL上调小鼠骨髓源性SPC CXCR4的表达,提示ox-LDL有可能通过上调SPC CXCR4的表达致SPC迁移和归巢至病变动脉参与As形成。
Background The chemokine stromal cell derived-factor1(SDF-1)also name CXCL12 or pro-B stimulating factor and belong to the intercrine CXC subfamily. The single SDF-1 gene encode two kinds protein:SDF-1αand SDF-1β.SDF-1 major express on hematopoietic stem cells.CXCR4 is the single chemokine receptor of SDF-1, which is a seven-transmembrane domain, GTP-binding protein-coupled receptor and is high expression in hematopoietic stem/progenitor cells and endothelial progenitor cells. Newly investigation indicate that CXCR4 also express on marrow stromal cells(MSC).Literature report that the myocardium after myocardial infarction(MI) will immediately up-regulate the SDF-1 and induce over-expression of CXCR4 to migration in ischemic myocardium. In the Zhang’s investigation manifest the CXR4+ MSC has participated in the form of the new vessels and differentiate to original myocardical cells; animal studies indicate the interaction of SDF-1/CXCR4 axis play a critical role in the repair myocardium through recruitment MSC. Study with the mouse which are knocked-out of SDF-1 and/or CXCR4 has indicate the SDF-1/CXCR4 axis also play a important role in the cell differentiation. MSC is a crucial cell component in the bone marrow microenvironment, which has potency to differentiate to various kinds of cells, and a literature report that MSC can differentiate into the smooth muscle progenitor cells(SPC).The bone marrow-derived MSC or SPC is the significant source of smooth muscle cell in the plaque of the atherosclerosis(As).Then ,we are still unknown that during the plaque to form and reparation of the As whether the SDF-1/CXCR4 recruitment MSC or SPC homing to damage arteries and take part in the shape of As or not.
Object Oxidized Low Density Lipoprotein (ox-ldl) is a risk factor of As. Now to detect the effect of ox-ldl on CXCR4, which is the receptor of SDF-1, expression in mouse bone marrow-derived SPC .To explore the contribution of SDF-1/CXCR4 to homing SPC to arterial damage.
Methods SPC was incubated with the same concentration (50μg/mL), CXCR4 mRNA and protein was revealed by RT-PCR, Western-Blot ,immunofluorescence staining and confocal laser scanning microscope analysis respectively. To observe the time-effect relationship between ox-ldl and the SPC expression of CXCR4.
Results Ox-ldl did not give to the SPC but it has the foundational level of CXCR4 expression,50μg/mL of ox-ldl stimulate SPC 0-72h,during the stimulation time ,CXCR4 expression gradually,36h reached peak, and its mRNA and protein levels were 5.73 folds and 5.02 folds of the basis level ,the differences were statistically significant(p<0.05),then decreased gradually, but still higher than the basis level. Immunofluorescence staining and confocal laser scanning microscope analysis detection in 0h observed when ox-ldl did not give SPC stimulated the cytoplasm and cell membrane surface of a small number of CXCR4 expression, with the extension of time ,CXCR4 expression gradually increases the volume when in 36h in the SPC and the cycoplasmic membrane surface expression of the strongest,60h the expression of CXCR4 decreased gradually.
Conclusions Ox-ldl can increase in mouse bone marrow-derived SPC CXCR4 expression ,suggesting the possibility of ox-ldl by upregulate the expression of SPC CXCR4 to induced migration and homing of SPC to the artery lesions to participate in the formation of As.
引文
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2. Sata M.Circulating vascular progenitor cells contribute to vascular repair,remodeling,and lesion formation [J].Trends Cardiovase Med, 2003, 13 (6):249-253.
3. Yeh ET,Zhang S,Wu HD,et al.Transdifferentiation of human peripheral blood CD34+-enriched cell population into cardiomyocytes,endothelial cells,and smooth muscle cells in vivo [J].circulation,2003,108(17):2070-2073.
4. Saiura A,Sata M,Hurata Y.et al.Circulating smooth muscle progenitor cells contribute to atherosolerosis[J].Nat Med,2001,7(4):382-383.
5. S.Abi-Younes,A.Sauty,F.Mach,et,al. The stromal cell derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaque.J Circ. Res.2000;86:131-138.
6.王新均,唐俊明,孔霞等HSDF-1/CXCR4介导骨髓源间充质干细胞迁移的信号转导。J Fourth Mil Med Univ 2007;28(19):1753-1756.
7. Mayumi Inoue,Hiroshi Itoh,Tokuji Tanaka,et,al.Oxidized LDL Regulates Vascular Endothelial Growth Factor Expression in Human Macrophages and Endothelial Cells Through Activation of Peroxisome Proliferator-Activated Receptor-r [J]. Arterioscler Thromb Vasc Biol.2001;21:560-566.
8. Qiao C,Zhang K,Xia J.Influence of oxidized low density lipoprotein on the proliferation of human artery smooth muscle cells in vitro.J HuaZhong Univ Sci Technolog Med Sci.2007,27(1):20-3.
9. Zhao GF,Seng JJ,Zhang H,She Mp.Effects of oxidized low density lipoprotein on the growth of human artery smooth muscle cells[J].Chin Med J(Engl) . 2005, 118(23):1973-8.
10. Leah MC,Ali J,Kathleen D,et,al.Carboxypeptidase M expressed by human bone marrow cells cleaves the C-terminal lysine of SDF-1:Another player in hematopoietic stem/progenitor cell mobilization[J]. Stem Cell,2008;print issn:1066-5099.
11. Crump MP,Gong JH,Loetscler p,et,al.Solution structure and basis for functional activity of stromal cell-derived factor-1;dissociation of CXCR4 activation from binding and inhibition of HIV-1[J]. Embo 1997;16:6996-7007.
12. Sierra MD,Yang FQ,Narazaki M,et,al.Differential processing of stromal-derived factor-1 alpha and stromal-derived factor-1 beta explains functional diversity [J].Blood,2004;103:2452-2459.
13. Dar A,Goichberg P,Shinder V,et,al.Chemokine receptor CXCR4-depedent internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and stromal cells[J].Nat Immunol.2005;6:1038-1046.
14. Feng Y,Broder CC,Kennedy PE,et,al.HIV-1 entry cofactor:functionalcDNA cloning of a seven-transmembrane,G protein-coupled receptor[J]. Science, 1996; 272 (5263)872-877。
15. Kucia M,Jankowskik,Reca R,etal. CXCR4-SDF-1 Singnalling, locomotion, chemotaxis and adhension[J].Mol Histol,2004,35(3):233-245.
16. Entschladen F,Bastian P,Niggemann B.et al.Ihibition of cell migration via G protein-coupled receptoes to opioid peptides and angiotansin[J].Ann N Y Acad Sci,2004,1028:320-328.
17. Mohle R,Bautz F,Denzlinger C,et al.Transendothelial migration of hematopoietic progenitor cells role of chemotactic factors[J].Ann N Y Acid Sci,2001, 938:26-34;discussion 34-35.
18. Pittenger MF,Martin BJ.Mesenchymal stem cells and their potential as cardiac therapeutics[J].Circ Res 2004;95(1):9–20.
19. Dongsheng Zhang,Guo-chang Fan,Xiaoyang Zhou et al.Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infracted myocardium[J].Mol Cell Cardiol.2008;44(2):281-92.
20. Abi-Younes S, Sauty A, Mach F, et al.The stromal cell-derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques[J].Circ Res ,1999;86:131-138。
21.余俊,阮秋蓉,利用PSM22a增强型绿色荧光蛋白1质粒重组体从小鼠间充质干细胞中分选平滑肌祖细胞。中国动脉硬化杂志[J].2007,15(8):579。
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2. Sata M.Circulating vascular progenitor cells contribute to vascular repair,remodeling,and lesion formation [J].Trends Cardiovase Med, 2003, 13 (6):249-253.
3. Yeh ET,Zhang S,Wu HD,et al.Transdifferentiation of human peripheral blood CD34+-enriched cell population into cardiomyocytes,endothelial cells,and smooth muscle cells in vivo [J].circulation,2003,108(17):2070-2073.
4. Saiura A,Sata M,Hurata Y.et al.Circulating smooth muscle progenitor cells contribute to atherosolerosis[J].Nat Med,2001,7(4):382-383.
5. S.Abi-Younes,A.Sauty,F.Mach,et,al. The stromal cell derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaque.J Circ. Res.2000;86:131-138.
6.王新均,唐俊明,孔霞等HSDF-1/CXCR4介导骨髓源间充质干细胞迁移的信号转导。J Fourth Mil Med Univ 2007;28(19):1753-1756.
7. Mayumi Inoue,Hiroshi Itoh,Tokuji Tanaka,et,al.Oxidized LDL Regulates Vascular Endothelial Growth Factor Expression in Human Macrophages and Endothelial Cells Through Activation of Peroxisome Proliferator-Activated Receptor-r [J]. Arterioscler Thromb Vasc Biol.2001;21:560-566.
8. Qiao C,Zhang K,Xia J.Influence of oxidized low density lipoprotein on the proliferation of human artery smooth muscle cells in vitro.J HuaZhong Univ Sci Technolog Med Sci.2007,27(1):20-3.
9. Zhao GF,Seng JJ,Zhang H,She Mp.Effects of oxidized low density lipoprotein on the growth of human artery smooth muscle cells[J].Chin Med J(Engl) . 2005, 118(23):1973-8.
10. Leah MC,Ali J,Kathleen D,et,al.Carboxypeptidase M expressed by human bone marrow cells cleaves the C-terminal lysine of SDF-1:Another player in hematopoietic stem/progenitor cell mobilization[J]. Stem Cell,2008;print issn:1066-5099.
11. Crump MP,Gong JH,Loetscler p,et,al.Solution structure and basis for functional activity of stromal cell-derived factor-1;dissociation of CXCR4 activation from binding and inhibition of HIV-1[J]. Embo 1997;16:6996-7007.
12. Sierra MD,Yang FQ,Narazaki M,et,al.Differential processing of stromal-derived factor-1 alpha and stromal-derived factor-1 beta explains functional diversity [J].Blood,2004;103:2452-2459.
13. Dar A,Goichberg P,Shinder V,et,al.Chemokine receptor CXCR4-depedent internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and stromal cells[J].Nat Immunol.2005;6:1038-1046.
14. Feng Y,Broder CC,Kennedy PE,et,al.HIV-1 entry cofactor:functionalcDNA cloning of a seven-transmembrane,G protein-coupled receptor[J]. Science, 1996; 272 (5263)872-877。
15. Kucia M,Jankowskik,Reca R,etal. CXCR4-SDF-1 Singnalling, locomotion, chemotaxis and adhension[J].Mol Histol,2004,35(3):233-245.
16. Entschladen F,Bastian P,Niggemann B.et al.Ihibition of cell migration via G protein-coupled receptoes to opioid peptides and angiotansin[J].Ann N Y Acad Sci,2004,1028:320-328.
17. Mohle R,Bautz F,Denzlinger C,et al.Transendothelial migration of hematopoietic progenitor cells role of chemotactic factors[J].Ann N Y Acid Sci,2001, 938:26-34;discussion 34-35.
18. Pittenger MF,Martin BJ.Mesenchymal stem cells and their potential as cardiac therapeutics[J].Circ Res 2004;95(1):9–20.
19. Dongsheng Zhang,Guo-chang Fan,Xiaoyang Zhou et al.Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infracted myocardium[J].Mol Cell Cardiol.2008;44(2):281-92.
20. Abi-Younes S, Sauty A, Mach F, et al.The stromal cell-derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques[J].Circ Res ,1999;86:131-138。
21.余俊,阮秋蓉,利用PSM22a增强型绿色荧光蛋白1质粒重组体从小鼠间充质干细胞中分选平滑肌祖细胞。中国动脉硬化杂志[J].2007,15(8):579。
1. Luster AD.Chemokines:chemotactic cytokines that mediate inflammation.N Engl J Med.1998;338:436-445。
2. Rollins BJ. Chemokines.Blood.1997;90:909-928.
3. Premack BA,Schall TJ.Chemokine receptors:gateways to inflammation and infection.Nat Med.1996;2:1174-1178.
4. Nelken NA,Coughlin SR,Gordon D,et al.Monocyte chemoattractant protein -1 in human atheromatous plaques.J Clin INVEST.1991;88:1121-1127.
5. Takeya M,Yoshimura T,Leonard EJ,et al.Detection of monmcyte chemoattractant protein-1 in human atherosclerotic lesions by an anti-monocyte chemoattractant protein-1 monoclonal antibody. Hum Pathol.1993;24:534-539.
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