ox-LDL对VSMC表达SDF-1α的影响及其对VSMC和SPC趋化作用的研究
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摘要
目的
1、探讨氧化低密度脂蛋白(ox-LDL)对小鼠血管平滑肌细胞表达基质细胞衍生因子1α(SDF-1α)的影响;
2、比较VSMC的条件培养液及SDF-1α对血管平滑肌细胞(VSMC)和平滑肌祖细胞(SPC)趋化迁移的影响。
方法
1、组织块贴壁法培养小鼠主动脉平滑肌细胞,用免疫组织化学方法对培养的平滑肌细胞进行鉴定;
2、ox-LDL与VSMC共培养,用免疫组织化学方法、酶联免疫吸附试验(ELISA)测定VSMC表达SDF-1α蛋白的变化,用逆转录-聚合酶链式反应(RT-PCR)测定VSMC表达SDF-1αmRNA的变化,观察剂量-时间效应;
3、趋化迁移实验:将含有趋化因子SDF-1α的培养液置于Transwell小室下室,将VSMC或SPC悬液置于其上室,共孵育6h后,乙醇固定,高倍镜下计数滤膜背面(下室面)的细胞数,检测VSMC的条件培养液及SDF-1α对VSMC和SPC的趋化迁移的影响。
结果
1、培养的小鼠血管平滑肌细胞的纯度可以达到约90%。
2、小鼠血管平滑肌细胞有基础水平的SDF-1α的表达。ox-LDL的浓度在0～60ng/ml范围内,作用24h,SDF-1α蛋白的表达随ox-LDL浓度的增加而变化,SDF-1α蛋白表达的峰值在ox-LDL浓度为45ng/ml时,是ox-LDL浓度为0ng/ml时的1.4倍。在ox-LDL浓度为45ng/ml时,0～48h范围内,SDF-1α蛋白的表达变化随时间的延长而变化, SDF-1α蛋白表达的峰值在24h,是0h的1.5倍。在ox-LDL浓度为45ng/ml时,0～30h范围内,SDF-1αmRNA的表达变化随时间的延长而变化,SDF-1α基因mRNA的峰值在10h,是0h的3.2倍。
3、VSMC的条件培养液及SDF-1α对VSMC和SPC都具有明显趋化作用。而且VSMC的条件培养液及SDF-1α对SPC的趋化作用强于对VSMC的趋化作用。
结论
1.、ox-LDL可增强小鼠血管平滑肌细胞SDF-1α的表达。
2、VSMC的条件培养液及SDF-1α对SPC的趋化作用明显强于对VSMC的趋化作用。
Objective
1. To explore the effect of oxidized low density lipoprotein (ox-LDL) on expression of stromal cell derived factor-1α(SDF-1α) in mouse vascular smooth muscle cell (VSMC).
2. Compare migration of vascular smooth muscle cell and smooth muscle progenitor cell (SPC) by the effect of stromal cell derived factor-1αand conditioned medium of VSMC.
Methods
1. Culture VSMC of mouse by tissue—piece inoculation and identify VSMC by the method of immunochistochemistry (IHC).
2. VSMC was incubated with different concentration/time of ox-LDL, protein of SDF-1αwas revealed by the method of immunochistochemistry and immunosorbent assay (ELISA) and mRNA of SDF-1αwas revealed by transcription-polymerase chain reaction (RT-PCR), Observed the effect of concentration-time.
3. Experiment of migration: Put SDF-1αin the basic room of the Transwell chamber; Put VSMC and SPC in the upside room of the Transwell chamber; Put Transwell chamber in 37℃for 6 hours; Fixed by alcohol; Count the number of cell by Microscope; Reveal effect of migration in VSMC and SPC by SDF-1αand conditioned medium of VSMC.
Result
1. Purity of the cultured VSMC was about 90%.
2. SDF-1αwas constitutionally expressed in mouse VSMC. SDF-1αin mouse VSMC was changed by ox-LDL with 0～60ng/ml and the time of 24h,protein of SDF-1α's expression was peaked at 45ng/ml and was more 1.4times than basal expression. SDF-1αin mouse VSMC was changed by time of 0～48h and the ox-LDL of 45ng/ml, protein of SDF-1α's expression was peaked at 24h and was more 1.5 times than basal expression, mRNA of SDF-1α's expression was peaked at 10h and was more 3.2 times than basal expression.
3. Effect of migration in VSMC and SPC is obvious by the effect of SDF-1αand conditioned medium of VSMC and the effect of migration in VSMC is more obvious than the effect of migration in SPC.
Conclusion
1. SDF-1αin mouse VSMC was increased by ox-LDL.
2. The effect of migration in VSMC is more obvious than the effect of migration in SPC by the effect of SDF-1αand conditioned medium of VSMC.
1、探讨氧化低密度脂蛋白(ox-LDL)对小鼠血管平滑肌细胞表达基质细胞衍生因子1α(SDF-1α)的影响;
2、比较VSMC的条件培养液及SDF-1α对血管平滑肌细胞(VSMC)和平滑肌祖细胞(SPC)趋化迁移的影响。
方法
1、组织块贴壁法培养小鼠主动脉平滑肌细胞,用免疫组织化学方法对培养的平滑肌细胞进行鉴定;
2、ox-LDL与VSMC共培养,用免疫组织化学方法、酶联免疫吸附试验(ELISA)测定VSMC表达SDF-1α蛋白的变化,用逆转录-聚合酶链式反应(RT-PCR)测定VSMC表达SDF-1αmRNA的变化,观察剂量-时间效应;
3、趋化迁移实验:将含有趋化因子SDF-1α的培养液置于Transwell小室下室,将VSMC或SPC悬液置于其上室,共孵育6h后,乙醇固定,高倍镜下计数滤膜背面(下室面)的细胞数,检测VSMC的条件培养液及SDF-1α对VSMC和SPC的趋化迁移的影响。
结果
1、培养的小鼠血管平滑肌细胞的纯度可以达到约90%。
2、小鼠血管平滑肌细胞有基础水平的SDF-1α的表达。ox-LDL的浓度在0～60ng/ml范围内,作用24h,SDF-1α蛋白的表达随ox-LDL浓度的增加而变化,SDF-1α蛋白表达的峰值在ox-LDL浓度为45ng/ml时,是ox-LDL浓度为0ng/ml时的1.4倍。在ox-LDL浓度为45ng/ml时,0～48h范围内,SDF-1α蛋白的表达变化随时间的延长而变化, SDF-1α蛋白表达的峰值在24h,是0h的1.5倍。在ox-LDL浓度为45ng/ml时,0～30h范围内,SDF-1αmRNA的表达变化随时间的延长而变化,SDF-1α基因mRNA的峰值在10h,是0h的3.2倍。
3、VSMC的条件培养液及SDF-1α对VSMC和SPC都具有明显趋化作用。而且VSMC的条件培养液及SDF-1α对SPC的趋化作用强于对VSMC的趋化作用。
结论
1.、ox-LDL可增强小鼠血管平滑肌细胞SDF-1α的表达。
2、VSMC的条件培养液及SDF-1α对SPC的趋化作用明显强于对VSMC的趋化作用。
Objective
1. To explore the effect of oxidized low density lipoprotein (ox-LDL) on expression of stromal cell derived factor-1α(SDF-1α) in mouse vascular smooth muscle cell (VSMC).
2. Compare migration of vascular smooth muscle cell and smooth muscle progenitor cell (SPC) by the effect of stromal cell derived factor-1αand conditioned medium of VSMC.
Methods
1. Culture VSMC of mouse by tissue—piece inoculation and identify VSMC by the method of immunochistochemistry (IHC).
2. VSMC was incubated with different concentration/time of ox-LDL, protein of SDF-1αwas revealed by the method of immunochistochemistry and immunosorbent assay (ELISA) and mRNA of SDF-1αwas revealed by transcription-polymerase chain reaction (RT-PCR), Observed the effect of concentration-time.
3. Experiment of migration: Put SDF-1αin the basic room of the Transwell chamber; Put VSMC and SPC in the upside room of the Transwell chamber; Put Transwell chamber in 37℃for 6 hours; Fixed by alcohol; Count the number of cell by Microscope; Reveal effect of migration in VSMC and SPC by SDF-1αand conditioned medium of VSMC.
Result
1. Purity of the cultured VSMC was about 90%.
2. SDF-1αwas constitutionally expressed in mouse VSMC. SDF-1αin mouse VSMC was changed by ox-LDL with 0～60ng/ml and the time of 24h,protein of SDF-1α's expression was peaked at 45ng/ml and was more 1.4times than basal expression. SDF-1αin mouse VSMC was changed by time of 0～48h and the ox-LDL of 45ng/ml, protein of SDF-1α's expression was peaked at 24h and was more 1.5 times than basal expression, mRNA of SDF-1α's expression was peaked at 10h and was more 3.2 times than basal expression.
3. Effect of migration in VSMC and SPC is obvious by the effect of SDF-1αand conditioned medium of VSMC and the effect of migration in VSMC is more obvious than the effect of migration in SPC.
Conclusion
1. SDF-1αin mouse VSMC was increased by ox-LDL.
2. The effect of migration in VSMC is more obvious than the effect of migration in SPC by the effect of SDF-1αand conditioned medium of VSMC.
引文
[1]戴恩来,薛国,武俊斌,杨应兄大鼠血管平滑肌细胞的贴块法培养中医儿科杂志2007 .3 1673- 4297 ( 2007) 02- 0047- 03
[2]任立群,张秀云,孙波,王金风大鼠胸主动脉平滑肌细胞的组织贴块法培养及鉴定吉林大学学报(医学版) 2002 Q813 1 R-332
[3] Brunner S, Winogradow J, Huber BC, Zaruba MM, Fischer R, David R, Assmann G, Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4/SDF-1 axis. FASEB J. 2009 Feb;23(2):351-61. Epub 2008 Sep 30.
[4] Jarczowski F, Jahreis G, Erdmann F, Schierhorn A, Fischer G, Edlich F. FKBP36 is an inherent multifunctional glyceraldehyde-3-phosphate dehydrogenase inhibitor J Biol Chem. 2009 Jan 9;284(2):766-73. Epub 2008 Nov 10.
[5]余俊阮秋蓉利用Psm22α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1007-3949( 2007) 15-08-0579-01
[6] Younes S , Mach F , Sauty A The stromal cell derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques 1999 86 1312138
[7] Hideyasu Sakihama Taro Masunaga Kenichiro The stromal Cell Derived Factor-1 and CXCR4 Interaction Is Critical for Development of Transplant Arteriosclerosis Circulation 2004 110 2 9242930.
[8] Bjokeurd B M,Bjorkerud S.Contrary effects of lightlyand strongly oxidized LDL with potent promotion ofgrowth apoptosis in arterial smooth muscle cells,nlaerophagesand fibroblasts[J].After Thro Vasc Biol,1996,16(3):416—424.
[9] Sacre.Selective Use of TRAM in Us and LTA InducedNF-κB Activation and Cytokine Production in PrimaryHuman Cells:TRAM Is an Adaptor for LPSand mSignaling[J].Immunol,2007,178(2):148—154.
[10] Kanters E,Gijbels M J,Vergouwe M N,et.HematopoieticNF-kappaBldeficiency Resnlts in small atheroselerotielesions with an inflammatory phenotype[J].Blood,2004,103(3):934—940.
[11] Kucia M,Jankowski K,Reca R,et a1.CXCR4一SDF-1 signalling,locomotion,chemotaxis and adhesion.J Mol Histol,2004,89}265-272.
[12] Gazitt Y . Homing and mobilization of hematopoietic stem cellsand hematopoletic cancer cells are mirror image processes,uti—lizing similar signaling pathways and occurring concurrently icirculating cancer cells constitute an ideal target for concur—rent treatment with chemotherapy and antilineage—specific an—tibodies.Leukemia,2004,18t1.
[13] Kucia M,Ratajezak J bone marrow cells plasticor heterogenous 2005,33(6):613-623.
[14] Kueia M,Wojakowski W The migration of bone derived non-hematopoietic tissue-committed stem cells is regulated in an LIF, HGF and SDF-1 dependent manner 2006,54(2):121—135.
[15]张林李玉泉张炎杨向群张传森骨髓间质细胞中组织定向干细胞的鉴定解剖学杂志2008年第31卷第l期
[16]陈飞兰,张华蓉,徐承平,卞修武SDF-1/CXCR4轴活化诱导人内皮祖细胞增殖、迁移及管型形成2008年5月1001—6325(2008)05—0428·04
[17]王佐吕运成危当恒姜志胜等.基质细胞衍生因子1α对大鼠血管平滑肌细胞与单核细胞黏附的影响2006 , 14 (9) : 7592762中国动脉硬化杂志,
[18] Ymaguchi J,Kusano KF The stromal cell derived factor-1 effects on erive expanded endothelial progenitor cell re-cruitment for is chemicneovas 2003,107(9):1322-1328
[19] Majka M,Janueska.ieezorrk A The stromal derived factor-1 regulate distinct aspecets of human 2000,96(13):4142—4151 Blood
[20] Peled A,Kollet O The chemokine SDF-1a vates the integrins VLA-5,VLA-4 and LFA-l in immature human CD34+cells role in transendothelial stromal migration and engraftment of nod-scid-mice 2000,95(11):3289-3296. Blood
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[2] Zengin E, Gehling UM, Chalajour F. Vascular wall resient progenitor cells source for postnatal vasculogenesis 2006, 133 (8): 1543-1551. Development
[3] Rafii S, Benezra R, Lyden D, novel targets for anti-angiogenesis therapy? Vascular and hematopoieticstem cells 2002, 2 (11):8262835 NatRev Cancer.
[4] PeichevM Expression ofAC133and VEGFR-2 by circulating human CD34 cells identifies a population of functional endothelial precursors Blood
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[2]任立群,张秀云,孙波,王金风大鼠胸主动脉平滑肌细胞的组织贴块法培养及鉴定吉林大学学报(医学版) 2002 Q813 1 R-332
[3] Brunner S, Winogradow J, Huber BC, Zaruba MM, Fischer R, David R, Assmann G, Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4/SDF-1 axis. FASEB J. 2009 Feb;23(2):351-61. Epub 2008 Sep 30.
[4] Jarczowski F, Jahreis G, Erdmann F, Schierhorn A, Fischer G, Edlich F. FKBP36 is an inherent multifunctional glyceraldehyde-3-phosphate dehydrogenase inhibitor J Biol Chem. 2009 Jan 9;284(2):766-73. Epub 2008 Nov 10.
[5]余俊阮秋蓉利用Psm22α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1007-3949( 2007) 15-08-0579-01
[6] Younes S , Mach F , Sauty A The stromal cell derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques 1999 86 1312138
[7] Hideyasu Sakihama Taro Masunaga Kenichiro The stromal Cell Derived Factor-1 and CXCR4 Interaction Is Critical for Development of Transplant Arteriosclerosis Circulation 2004 110 2 9242930.
[8] Bjokeurd B M,Bjorkerud S.Contrary effects of lightlyand strongly oxidized LDL with potent promotion ofgrowth apoptosis in arterial smooth muscle cells,nlaerophagesand fibroblasts[J].After Thro Vasc Biol,1996,16(3):416—424.
[9] Sacre.Selective Use of TRAM in Us and LTA InducedNF-κB Activation and Cytokine Production in PrimaryHuman Cells:TRAM Is an Adaptor for LPSand mSignaling[J].Immunol,2007,178(2):148—154.
[10] Kanters E,Gijbels M J,Vergouwe M N,et.HematopoieticNF-kappaBldeficiency Resnlts in small atheroselerotielesions with an inflammatory phenotype[J].Blood,2004,103(3):934—940.
[11] Kucia M,Jankowski K,Reca R,et a1.CXCR4一SDF-1 signalling,locomotion,chemotaxis and adhesion.J Mol Histol,2004,89}265-272.
[12] Gazitt Y . Homing and mobilization of hematopoietic stem cellsand hematopoletic cancer cells are mirror image processes,uti—lizing similar signaling pathways and occurring concurrently icirculating cancer cells constitute an ideal target for concur—rent treatment with chemotherapy and antilineage—specific an—tibodies.Leukemia,2004,18t1.
[13] Kucia M,Ratajezak J bone marrow cells plasticor heterogenous 2005,33(6):613-623.
[14] Kueia M,Wojakowski W The migration of bone derived non-hematopoietic tissue-committed stem cells is regulated in an LIF, HGF and SDF-1 dependent manner 2006,54(2):121—135.
[15]张林李玉泉张炎杨向群张传森骨髓间质细胞中组织定向干细胞的鉴定解剖学杂志2008年第31卷第l期
[16]陈飞兰,张华蓉,徐承平,卞修武SDF-1/CXCR4轴活化诱导人内皮祖细胞增殖、迁移及管型形成2008年5月1001—6325(2008)05—0428·04
[17]王佐吕运成危当恒姜志胜等.基质细胞衍生因子1α对大鼠血管平滑肌细胞与单核细胞黏附的影响2006 , 14 (9) : 7592762中国动脉硬化杂志,
[18] Ymaguchi J,Kusano KF The stromal cell derived factor-1 effects on erive expanded endothelial progenitor cell re-cruitment for is chemicneovas 2003,107(9):1322-1328
[19] Majka M,Janueska.ieezorrk A The stromal derived factor-1 regulate distinct aspecets of human 2000,96(13):4142—4151 Blood
[20] Peled A,Kollet O The chemokine SDF-1a vates the integrins VLA-5,VLA-4 and LFA-l in immature human CD34+cells role in transendothelial stromal migration and engraftment of nod-scid-mice 2000,95(11):3289-3296. Blood
[1] Peichev M , Percira D, Naiyer AJ Expression of VEGFR-2 and AC-33 by circulating human CD34 + cells identifies a population of functional endothelial precursors 2000, 95(3)952-958 Blood
[2] Zengin E, Gehling UM, Chalajour F. Vascular wall resient progenitor cells source for postnatal vasculogenesis 2006, 133 (8): 1543-1551. Development
[3] Rafii S, Benezra R, Lyden D, novel targets for anti-angiogenesis therapy? Vascular and hematopoieticstem cells 2002, 2 (11):8262835 NatRev Cancer.
[4] PeichevM Expression ofAC133and VEGFR-2 by circulating human CD34 cells identifies a population of functional endothelial precursors Blood
[5] Jin H, Hyo-Soo K,Chang-Hwan Y, Characterization of two types of Endothelial progenitor cells and their different contribution to neovasculogenesis 2004, 24 (2) : 2882293,Arterioscler Thromb Vasc Biol.
[6] Asahara T,Sullivan A, Marohara T, Isolation of putative progenitor endothelial cells for angiogenesis , 1977,275 (5302): 9642967, Science.
[7] Werner N, Junk S , Laufs U, Link A, Walenta K, Bohm M, etal .Intravenous transfusion of endothelial progenitor cells reduces neointimaformation after vascu -larinjury[J]. Circ Res,2003 , 93 (2) :17 224.
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