脂联素对内皮祖细胞增殖分化的影响及其机制探讨
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摘要
人们生活水平的提高以及一些不良生活和/或饮食方式的长期存在,导致肥胖症、糖耐量减低、胰岛素抵抗和2型糖尿病患病人群数量不断上升,这已经成为一个公共卫生问题,引起内分泌、心脑血管疾病等临床医生以及公共卫生研究学者的高度重视。临床研究和流行病学调查已经证实,这些代谢性疾病与心血管疾病的发生关系密切,代谢综合症和2型糖尿病已经被认为是冠心病的等危症。肥胖所导致的炎症因子分泌的增加是导致胰岛素抵抗的重要因素,也是导致内皮细胞损害和功能紊乱的原因之一。2型糖尿病患者过高的血糖状态同样对血管内皮尤其是微循环血管内皮会造成损害,这对增加动脉粥样硬化、冠心病等心血管疾病的发生和促进病变的进一步发展非常重要。
脂联素是脂肪组织分泌的特异性的脂肪细胞因子,与肥胖、糖耐量异常、胰岛素抵抗和2型糖尿病密切相关,患有这些疾病的人群血浆脂联素水平显著下降。最近研究表明,脂联素通过抑制泡沫细胞的形成、抑制细胞凋亡、减少炎症因子的产生和聚集从而发挥抗动脉粥样硬化病变的作用。血管内皮细胞的功能状态与各种心血管疾病的发生发展关系密切。在内皮损伤或高血糖等因素刺激下,血管内皮功能会发生紊乱。与正常人群相比,患有高血压、冠心病等心血管疾病的人群血浆脂联素水平显著降低。目前,低脂联素血症被认为是冠心病独立的危险因素和预测因子,同时与冠心病病变严重程度密切相关,因此也被作为预后预测的指标之一。
内皮祖细胞(endothelial progenitor cells,EPCs)是一类能增殖并分化为血管内皮细胞的前体细胞,不仅参与胚胎时期血管的形成,也参与出生后血管的新生和内皮损伤的修复,这对于预防和治疗各类与血管损伤有关的心血管疾病有着重要的病理和生理学意义。有关EPCs在各种缺血性疾病和创伤治疗中的应用以及如何有效促进机体EPCs的增殖分化是目前该领域的研究热点。已有研究发现在小鼠造血干细胞(hematopoietic stem cells,HSCs)表面存在脂联素受体,脂联素可促进HSCs在体内外的增殖并提高其功能,被认为是其不可缺少的生长因子。EPCs是血管内皮细胞的前体细胞,同时具有内皮细胞和祖细胞的一些生物学特性。EPCs是否也表达脂联素受体,脂联素对EPCs的增殖和分化功能有无影响目前尚不清楚。
基于上述的理论基础,我们提出假设:脂联素可能影响EPCs的数量和功能,从而影响血管内皮损伤的修复能力,维持内皮损伤和修复之间的动态平衡,促进内皮功能的恢复,进而延缓动脉粥样硬化病变的发生和发展。因此,本实验的目的是了解EPCs中是否存在脂联素受体的表达,以及脂联素是否影响EPCs的增殖和分化,同时探讨其可能的作用机制。以下分2部分对本研究的方法、结果和结论等作一简述。
第一部分内皮祖细胞的分离、培养、鉴定及脂联素受体的表达
目的:对脐血EPCs进行分离、培养和鉴定;探讨EPCs是否存在脂联素受体的表达。
方法:采用Ficoll密度梯度离心法从人脐静脉血中获得单个核细胞,接种于预包被有人纤维连接蛋白的培养板中,经贴壁筛选法纯化,用FITC-UEA-I和DiI-Ac-LDL双染色法和流式细胞技术检测EPCs表面标志(CD34、VEGFR-2和AC133)来验证分离、培养所得EPCs;并通过RT-PCR、Western blot和免疫细胞化学等方法检验EPCs中是否存在脂联素受体的表达。
结果:培养所得的细胞81.4±11.7%为FITC-UEA-I和DiI-Ac-LDL染色呈双阳性;用流式细胞技术检测其表达VEGFR-2(76.6±7.0%)、CD34(42.9±8.5%)、AC133(19.8±3.9%);脂联素受体AdipoR1和AdipoR2的mRNA和蛋白EPCs上均有表达。
结论:1.采用密度梯度离心后贴壁筛选培养的方法可以从人脐静脉血中成功分离培养EPCs。2.分离培养的EPCs中存在脂联素受体AdipoR1和AdipoR2的表达。
第二部分脂联素对内皮祖细胞增殖分化的影响及其机制探讨
目的:观察脂联素对分离培养的EPCs增殖分化的影响并初步探讨其可能的作用机制。
方法:分离的EPCs培养15-20天后,用MTT法观察脂联素对EPCs增殖的影响。用Real time PCR检测vWF、CD31、CD34、AC133相对表达量评价脂联素对EPCs分化的影响。用Western blot检测脂联素作用后不同时间p-JNK、JNK、p-p38、p38、ERK、p-ERK等蛋白的表达,并用信号转导抑制剂SB203580和SP600125干预加以验证脂联素对EPCs增殖分化影响的可能信号传导途径。
结果:脂联素作用后能使EPCs数量(及其MTT值)增加,30μg/ml和作用48h时最明显;脂联素(30μg/ml)干预48h后细胞表达vWF、CD31mRNA的相对量有所增加,而CD34、AC133 mRNA的表达水平则有下降;脂联素干预后5min,可检测到EPCs中p38和JNK磷酸化,30min达高峰,60min稍有下降,而ERK无磷酸化(p-ERK)表达。p38信号传导抑制剂SB203580和JNK信号传导抑制剂SP600125则可阻断脂联素的促进p38和JNK磷酸化作用;SB203580可减弱脂联素的促增殖效应;而SP600125可减弱其对EPCs的促分化作用。
结论:1.脂联素可促进EPCs的增殖和分化,该作用有一定的时间和剂量依赖性。2.MAPK途径参与脂联素对人EPCs增殖和分化的促进作用。
Improvement in people's living standards,long-term existence of some adverse living and dietary patterns have caused the number of patients with obesity,impaired glucose tolerance,insulin resistance,and type 2 diabetes increased greatly in the recently years in China.This has become a public health problem and drawn the attentions of endocrine,cardiovascular and cerebrovascular physicians,and researchers in public health.Clinical researches and epidemiological investigations have confirmed that cardiovascular diseases are closely related with metabolic disorders such as diabetes.Metabolic syndromes and type 2 diabetes have been considered as equivalent to the risk of coronary heart disease(CAD).The increasing of inflammatory cytokines caused by obesity leads to insulin resistance,which also leads to endothelial cell damage and functional disorders.High blood glucose in patients with type 2 diabetes causes vascular endothelial damage,especially microcirculation vascular endothelial cells,which is also an important factor for the development of atherosclerosis and CAD.
Adiponectin is an adipocyte-specific protein and hypoadiponectinemia is often observed in patients with obesity,impaired glucose tolerance,insulin resistance,and type 2 diabetes.Recent studies have showed that adiponectin's overexpression could decrease atherosclerosis by attenuating the endothelial inflammatory response and macrophage-to-foam cell transformation in vivo.Therefore,adiponectin has anti-atherogenic properties through anti-inflammatory effects.Vascular endothelial functions are closely related to cardiovascular diseases.Endothelial function's disorders happen in the situation of high blood glucose and endothelial injury.Low plasma adiponectin level was also observed in patients with cardiovascular diseases such as hypertension and CAD.In a recent study,adiponectin affected plaque components,and low plasma adiponectin,a well-known independent risk factor for CAD,might enhance the vulnerability of atherosclerotic plaques and vessels,which could lead to acute coronary syndrome(ACS).Therefore,plasma adiponectin level is now considered as one of the indicators of its prognosis prediction.
Endothelial progenitor cells(EPCs) are a cell population that have the capacity to circulate,proliferate,and differentiate into mature endothelial cells,but have neither acquired characteristic mature endothelial markers nor formed a lumen.EPCs have been shown to be incorporated into sites of physiological and pathological neovascularization in vivo.More recently,it has been suggested that circulating EPCs may also be a marker of endothelial function and cardiovascular risk.Endothelial dysfunction is usually one of the earlier markers of atherosclerosis,and predisposes the vasoconstriction and thrombosis.EPCs may exert an important function as an endogenous repair mechanism to maintain the intergrity of the endothelial monolayer by replacing denuded parts of the artery.These beneficial propertities of EPCs are attractive for cell therapy that targets endothelial regeneration for treatment of ischemic diseases and wound healing,Therefore,how to promote expansion,proliferation,and differentiation of EPCs effectively is a current research hotspot.Recent research has shown that adiponectin receptors expressed in mouse hematopoietic stem cells(HSCs) and adiponectin could promote HSCs proliferation in vitro /vivo and improve their functions.EPCs are also precursor cells and have some biological characteristics of both endothelial cells and progenitors cells.Whether adiponectin receptors are expressed in EPCs or adiponectin affects proliferation and differentiation of EPCs and its mechanism is unclear.
On the basis of these considerations,we hypothesized that adiponectin affected EPCs number and functional activity,thus influenced endothelial repair process and maintained the balance between the magnitide of injury and the capacity for repair, which prevented atherosclerotic lesion developing.Therefore,the aim of this study was to investigate the expression of adiponectin receptors in EPCs,and whether adiponectin affect the proliferation and differentiation of EPCs and the possible mechanism.The following 2 Parts would describe the methods,results and conclusions of the study briefly.
Part 1 Isolation,Culture and Identification of EPCs from Human Umbilical Blood and Expression of Adiponectin Receptors in EPCs.
Objective:To isolate,culture and identify EPCs from human umbilical blood,and investigate the expression of adiponectin receptors in EPCs.
Methods:Total mononuclear cells(MNCs) were isolated from human umbilical blood by Ficoll density gradient centrifugation,and plated on fibronection-coated culture dishes.EPCs were characterized as adherent cells double positive for DiI-Ac-LDL-uptake and FITC-UEA-I lectin binding by direct fluorescent staining under a fluorescent microscope.EPCs were further documented by demonstrating the expression of VEGFR2,CD34,and AC133 with flow cytometry.The expression of adiponectin receptors were measured using RT-PCR,Western blot and immunocytochenistry.
Results:Adherent cells isolated and cultured from human umbilical blood DiI-Ac-LDL uptake and FITC-UEA-I lectin binding were assessed under direct fluorescent microscope,and the percentage of double positive cells were 81.4±11.7%. Analysis of VEGFR2,CD34 and AC133-expression by flow cytometry.Ex vivo EPCs were positive by 76.6±7.0%for VEGFR2,42.9±8.5%for CD34,and 19.8±3.9%for AC133.Adoponectin receptors' mRNA and protein were expressed in EPCs.
Conclusions:EPCs can be isolated from human umbilical blood and cultured by Ficoll density gradient centrifugation and adherence to fibronectin-coated culture dishes. Both AdopR1 and AdopR2 are expressed in EPCs.
Part 2 Effects of Adiponectin on Proliferation and Differentiation of Endothelial Progenitor Cells and the Possible Mechanism
Objective:To investigate the effects of adiponectin on proliferation and differentiation of EPCs and the possible signaling pathway.
Methods:After isolated EPCs were cultured 15-20 days,EPCs' proliferation was assayed with MTT assay.EPCs' differentiation was determined by Real-time PCR by measureing relative expression of surface markers including vWF,CD31,CD34,and AC133.The expression of p-JNK,JNK,p-p38,p38,ERK,p-ERK were measured by Western blot.MAPK's inhibitor SB203580 and SP600125 were also used for testing the signaling pathway.
Results:Adiponectin increased the number of EPCs with dose and time dependence, with maximum at 30μg/ml,48h.Adiponectin significantly increased vWF and CD31 expression of EPCs with maximum at 30μg/ml,48h,but decreased CD34 and AC133 expression.Western blot analysis showed that adiponectin induced activation of p38 and JNK,but not activation of ERK1/2 in EPCs.These effects were blocked by JNK inhibitor(SB600125) and p38 inhibitor(SB203580).Pretreatment of EPCs with p38 inhibitor(SB203580) and JNK inhibitor(SB600125) partly diminished adiponectin-induced cell proliferation and the expression of vWF,CD31,CD34,and AC133.
Conclusions:Adiponectin can promote EPCs proliferation and differentiation by dose-and time-dependent manner.Adiponectin enhances EPCs proliferation and differentiation through MAPK signaling pathway.
脂联素是脂肪组织分泌的特异性的脂肪细胞因子,与肥胖、糖耐量异常、胰岛素抵抗和2型糖尿病密切相关,患有这些疾病的人群血浆脂联素水平显著下降。最近研究表明,脂联素通过抑制泡沫细胞的形成、抑制细胞凋亡、减少炎症因子的产生和聚集从而发挥抗动脉粥样硬化病变的作用。血管内皮细胞的功能状态与各种心血管疾病的发生发展关系密切。在内皮损伤或高血糖等因素刺激下,血管内皮功能会发生紊乱。与正常人群相比,患有高血压、冠心病等心血管疾病的人群血浆脂联素水平显著降低。目前,低脂联素血症被认为是冠心病独立的危险因素和预测因子,同时与冠心病病变严重程度密切相关,因此也被作为预后预测的指标之一。
内皮祖细胞(endothelial progenitor cells,EPCs)是一类能增殖并分化为血管内皮细胞的前体细胞,不仅参与胚胎时期血管的形成,也参与出生后血管的新生和内皮损伤的修复,这对于预防和治疗各类与血管损伤有关的心血管疾病有着重要的病理和生理学意义。有关EPCs在各种缺血性疾病和创伤治疗中的应用以及如何有效促进机体EPCs的增殖分化是目前该领域的研究热点。已有研究发现在小鼠造血干细胞(hematopoietic stem cells,HSCs)表面存在脂联素受体,脂联素可促进HSCs在体内外的增殖并提高其功能,被认为是其不可缺少的生长因子。EPCs是血管内皮细胞的前体细胞,同时具有内皮细胞和祖细胞的一些生物学特性。EPCs是否也表达脂联素受体,脂联素对EPCs的增殖和分化功能有无影响目前尚不清楚。
基于上述的理论基础,我们提出假设:脂联素可能影响EPCs的数量和功能,从而影响血管内皮损伤的修复能力,维持内皮损伤和修复之间的动态平衡,促进内皮功能的恢复,进而延缓动脉粥样硬化病变的发生和发展。因此,本实验的目的是了解EPCs中是否存在脂联素受体的表达,以及脂联素是否影响EPCs的增殖和分化,同时探讨其可能的作用机制。以下分2部分对本研究的方法、结果和结论等作一简述。
第一部分内皮祖细胞的分离、培养、鉴定及脂联素受体的表达
目的:对脐血EPCs进行分离、培养和鉴定;探讨EPCs是否存在脂联素受体的表达。
方法:采用Ficoll密度梯度离心法从人脐静脉血中获得单个核细胞,接种于预包被有人纤维连接蛋白的培养板中,经贴壁筛选法纯化,用FITC-UEA-I和DiI-Ac-LDL双染色法和流式细胞技术检测EPCs表面标志(CD34、VEGFR-2和AC133)来验证分离、培养所得EPCs;并通过RT-PCR、Western blot和免疫细胞化学等方法检验EPCs中是否存在脂联素受体的表达。
结果:培养所得的细胞81.4±11.7%为FITC-UEA-I和DiI-Ac-LDL染色呈双阳性;用流式细胞技术检测其表达VEGFR-2(76.6±7.0%)、CD34(42.9±8.5%)、AC133(19.8±3.9%);脂联素受体AdipoR1和AdipoR2的mRNA和蛋白EPCs上均有表达。
结论:1.采用密度梯度离心后贴壁筛选培养的方法可以从人脐静脉血中成功分离培养EPCs。2.分离培养的EPCs中存在脂联素受体AdipoR1和AdipoR2的表达。
第二部分脂联素对内皮祖细胞增殖分化的影响及其机制探讨
目的:观察脂联素对分离培养的EPCs增殖分化的影响并初步探讨其可能的作用机制。
方法:分离的EPCs培养15-20天后,用MTT法观察脂联素对EPCs增殖的影响。用Real time PCR检测vWF、CD31、CD34、AC133相对表达量评价脂联素对EPCs分化的影响。用Western blot检测脂联素作用后不同时间p-JNK、JNK、p-p38、p38、ERK、p-ERK等蛋白的表达,并用信号转导抑制剂SB203580和SP600125干预加以验证脂联素对EPCs增殖分化影响的可能信号传导途径。
结果:脂联素作用后能使EPCs数量(及其MTT值)增加,30μg/ml和作用48h时最明显;脂联素(30μg/ml)干预48h后细胞表达vWF、CD31mRNA的相对量有所增加,而CD34、AC133 mRNA的表达水平则有下降;脂联素干预后5min,可检测到EPCs中p38和JNK磷酸化,30min达高峰,60min稍有下降,而ERK无磷酸化(p-ERK)表达。p38信号传导抑制剂SB203580和JNK信号传导抑制剂SP600125则可阻断脂联素的促进p38和JNK磷酸化作用;SB203580可减弱脂联素的促增殖效应;而SP600125可减弱其对EPCs的促分化作用。
结论:1.脂联素可促进EPCs的增殖和分化,该作用有一定的时间和剂量依赖性。2.MAPK途径参与脂联素对人EPCs增殖和分化的促进作用。
Improvement in people's living standards,long-term existence of some adverse living and dietary patterns have caused the number of patients with obesity,impaired glucose tolerance,insulin resistance,and type 2 diabetes increased greatly in the recently years in China.This has become a public health problem and drawn the attentions of endocrine,cardiovascular and cerebrovascular physicians,and researchers in public health.Clinical researches and epidemiological investigations have confirmed that cardiovascular diseases are closely related with metabolic disorders such as diabetes.Metabolic syndromes and type 2 diabetes have been considered as equivalent to the risk of coronary heart disease(CAD).The increasing of inflammatory cytokines caused by obesity leads to insulin resistance,which also leads to endothelial cell damage and functional disorders.High blood glucose in patients with type 2 diabetes causes vascular endothelial damage,especially microcirculation vascular endothelial cells,which is also an important factor for the development of atherosclerosis and CAD.
Adiponectin is an adipocyte-specific protein and hypoadiponectinemia is often observed in patients with obesity,impaired glucose tolerance,insulin resistance,and type 2 diabetes.Recent studies have showed that adiponectin's overexpression could decrease atherosclerosis by attenuating the endothelial inflammatory response and macrophage-to-foam cell transformation in vivo.Therefore,adiponectin has anti-atherogenic properties through anti-inflammatory effects.Vascular endothelial functions are closely related to cardiovascular diseases.Endothelial function's disorders happen in the situation of high blood glucose and endothelial injury.Low plasma adiponectin level was also observed in patients with cardiovascular diseases such as hypertension and CAD.In a recent study,adiponectin affected plaque components,and low plasma adiponectin,a well-known independent risk factor for CAD,might enhance the vulnerability of atherosclerotic plaques and vessels,which could lead to acute coronary syndrome(ACS).Therefore,plasma adiponectin level is now considered as one of the indicators of its prognosis prediction.
Endothelial progenitor cells(EPCs) are a cell population that have the capacity to circulate,proliferate,and differentiate into mature endothelial cells,but have neither acquired characteristic mature endothelial markers nor formed a lumen.EPCs have been shown to be incorporated into sites of physiological and pathological neovascularization in vivo.More recently,it has been suggested that circulating EPCs may also be a marker of endothelial function and cardiovascular risk.Endothelial dysfunction is usually one of the earlier markers of atherosclerosis,and predisposes the vasoconstriction and thrombosis.EPCs may exert an important function as an endogenous repair mechanism to maintain the intergrity of the endothelial monolayer by replacing denuded parts of the artery.These beneficial propertities of EPCs are attractive for cell therapy that targets endothelial regeneration for treatment of ischemic diseases and wound healing,Therefore,how to promote expansion,proliferation,and differentiation of EPCs effectively is a current research hotspot.Recent research has shown that adiponectin receptors expressed in mouse hematopoietic stem cells(HSCs) and adiponectin could promote HSCs proliferation in vitro /vivo and improve their functions.EPCs are also precursor cells and have some biological characteristics of both endothelial cells and progenitors cells.Whether adiponectin receptors are expressed in EPCs or adiponectin affects proliferation and differentiation of EPCs and its mechanism is unclear.
On the basis of these considerations,we hypothesized that adiponectin affected EPCs number and functional activity,thus influenced endothelial repair process and maintained the balance between the magnitide of injury and the capacity for repair, which prevented atherosclerotic lesion developing.Therefore,the aim of this study was to investigate the expression of adiponectin receptors in EPCs,and whether adiponectin affect the proliferation and differentiation of EPCs and the possible mechanism.The following 2 Parts would describe the methods,results and conclusions of the study briefly.
Part 1 Isolation,Culture and Identification of EPCs from Human Umbilical Blood and Expression of Adiponectin Receptors in EPCs.
Objective:To isolate,culture and identify EPCs from human umbilical blood,and investigate the expression of adiponectin receptors in EPCs.
Methods:Total mononuclear cells(MNCs) were isolated from human umbilical blood by Ficoll density gradient centrifugation,and plated on fibronection-coated culture dishes.EPCs were characterized as adherent cells double positive for DiI-Ac-LDL-uptake and FITC-UEA-I lectin binding by direct fluorescent staining under a fluorescent microscope.EPCs were further documented by demonstrating the expression of VEGFR2,CD34,and AC133 with flow cytometry.The expression of adiponectin receptors were measured using RT-PCR,Western blot and immunocytochenistry.
Results:Adherent cells isolated and cultured from human umbilical blood DiI-Ac-LDL uptake and FITC-UEA-I lectin binding were assessed under direct fluorescent microscope,and the percentage of double positive cells were 81.4±11.7%. Analysis of VEGFR2,CD34 and AC133-expression by flow cytometry.Ex vivo EPCs were positive by 76.6±7.0%for VEGFR2,42.9±8.5%for CD34,and 19.8±3.9%for AC133.Adoponectin receptors' mRNA and protein were expressed in EPCs.
Conclusions:EPCs can be isolated from human umbilical blood and cultured by Ficoll density gradient centrifugation and adherence to fibronectin-coated culture dishes. Both AdopR1 and AdopR2 are expressed in EPCs.
Part 2 Effects of Adiponectin on Proliferation and Differentiation of Endothelial Progenitor Cells and the Possible Mechanism
Objective:To investigate the effects of adiponectin on proliferation and differentiation of EPCs and the possible signaling pathway.
Methods:After isolated EPCs were cultured 15-20 days,EPCs' proliferation was assayed with MTT assay.EPCs' differentiation was determined by Real-time PCR by measureing relative expression of surface markers including vWF,CD31,CD34,and AC133.The expression of p-JNK,JNK,p-p38,p38,ERK,p-ERK were measured by Western blot.MAPK's inhibitor SB203580 and SP600125 were also used for testing the signaling pathway.
Results:Adiponectin increased the number of EPCs with dose and time dependence, with maximum at 30μg/ml,48h.Adiponectin significantly increased vWF and CD31 expression of EPCs with maximum at 30μg/ml,48h,but decreased CD34 and AC133 expression.Western blot analysis showed that adiponectin induced activation of p38 and JNK,but not activation of ERK1/2 in EPCs.These effects were blocked by JNK inhibitor(SB600125) and p38 inhibitor(SB203580).Pretreatment of EPCs with p38 inhibitor(SB203580) and JNK inhibitor(SB600125) partly diminished adiponectin-induced cell proliferation and the expression of vWF,CD31,CD34,and AC133.
Conclusions:Adiponectin can promote EPCs proliferation and differentiation by dose-and time-dependent manner.Adiponectin enhances EPCs proliferation and differentiation through MAPK signaling pathway.
引文
[1]Ouchi N,Kihara S,Funahashi T,et al.Obesity,adiponectin and vascular inflammatory disease.Curr Opin Lipidol.2003;14(6):561-6.
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