高糖对内皮祖细胞功能的影响及相关机制的研究
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摘要
内皮祖细胞(EPCs)作为血管内皮细胞的前体细胞,在成体血管内皮修复和血管新生过程中发挥重要作用,糖尿病可以导致外周血EPCs数量的减少及功能障碍,糖尿病血管病变的发生发展与EPCs功能紊乱密切相关。糖尿病视网膜病变(DR)是最为常见的糖尿病微血管眼部并发症,以早期的视网膜微循环障碍以及晚期的视网膜新生血管形成为主要病理特征,血管内皮功能障碍、微血管损伤是DR的主要病理基础,而EPCs在生理及病理条件下的视网膜血管损伤修复过程中发挥重要作用,因此推断DR的发生发展与糖尿病导致的EPCs功能障碍密切相关,但有关EPCs参与DR进展的具体机制尚无明确报道。临床上,糖尿病的主要标志是血糖的升高,高糖可能是导致糖尿病EPCs功能异常的主要原因,因此探讨高糖对EPCs生物学特性的影响及其相关机制,对研究DR等糖尿病血管并发症的病因及其防治非常重要。
研究目的
研究体外高糖环境对EPCs增殖、凋亡、粘附、摄取LDL能力等生物学特性的影响及相关机制,为进一步探讨EPCs参与DR发生发展的可能机制及调控途径奠定基础,同时为DR等眼内新生血管性疾病的治疗寻找新的靶点。
研究方法
1.密度梯度离心法分离人脐带血单个核细胞,体外诱导分化为EPCs并鉴定。
2.高糖及正常条件下培养EPCs,MTT法检测高糖对细胞增殖能力的影响;流式细胞仪检测高糖对EPCs凋亡数量的影响;RT-PCR法检测高糖对EPCs凋亡相关基因Bcl-2、Bax在mRNA表达水平的影响;共聚焦显微镜检测高糖对EPCs摄取acLDL能力的影响;Fluo3/AM负载EPCs,共聚焦显微镜下检测各组细胞细胞内游离钙离子水平([Ca2+]i),细胞外加入氯化钙(CaCl2),动态检测各组EPCs [Ca2+]i的变化;膜片钳技术研究EPCs的电生理特性并研究髙糖对EPCs电生理特性的影响;Western-blot检测体外高糖环境对EPCs内p-eNOS以及p-AKT蛋白表达的影响。
3.光动力法建立大鼠视网膜血管损伤模型,眼底照相及荧光眼底造影判定视网膜血管损伤情况,RT-PCR法检测视网膜血管损伤后视网膜SDF-1 mRNA表达水平的变化。
结果
1.脐带血单个核细胞经体外诱导分化,3d内可形成小集落,10d左右增殖旺盛,可见纺锤形细胞,于2-3w时呈现EPCs典型的铺路石样外观。流式细胞学结果显示EPCs体外培养10d时CD133、CD34、VEGFR2、CD31均有一定程度的表达,大部分细胞DiI-acLDL、FITC-UEA-I双染色阳性。免疫细胞化学染色结果显示EPCs内有VEGF的阳性表达。
2.①髙糖培养1d时EPCs增殖活力较正常对照组增强( P<0.05),3d时与正常对照组比较无明显差异,髙糖培养7d时EPCs增殖活力较正常对照组降低( P<0.05);②髙糖培养1d及3d时,EPCs粘附能力与正常对照组比较均明显降低( P<0.05);③高糖培养7d,与正常对照组比较,EPCs凋亡细胞数增加,Bax mRNA表达上调,而Bcl-2 mRNA表达降低。④高糖培养3d时EPCs[Ca2+ ]i水平较正常对照组无显著差别;在无钙介质中,各组EPCs在加入氯化钙(CaCl2)后均出现快速大幅度的[Ca2+]i升高,高糖组[Ca2+]i升高的幅度较正常对照组小;两组细胞[Ca2+]i在快速升高后迅速下降,正常组EPCs的[Ca2+]i均恢复至静息水平,而高糖组部分EPCs的[Ca2+]i未能恢复至静息水平。⑤正常条件下EPCs的静息膜电位维持在-50mv左右,在TEA作用下膜电流幅度降低,表明存在钙依赖性钾电流参与膜电位的构成。髙糖培养下EPCs静息膜电位水平及膜电流幅度无明显差别。⑥Western-blot结果显示高糖培养7d,eNOS及AKT的磷酸化水平较正常组比较均明显降低(P<0.05)。3.光动力法光凝视网膜静脉可以造成大鼠视网膜血管损伤,出现视网膜血管闭塞、渗漏、视网膜出血等眼底表现,视网膜血管损伤后3d及14d时,视网膜组织SDF-1 mRNA表达水平较正常组增高。
结论
1.高糖可能通过抑制PI3K/AKT/eNOS通路影响EPCs的增殖、粘附、动员等生物学功能,并可能通过在mRNA水平上调Bax表达并抑制Bcl-2表达而促进EPCs凋亡。2.高糖可以影响EPCs钙稳态,使细胞易于发生钙超载,高糖导致的EPCs功能紊乱可能与钙稳态失调导致的细胞内钙信号通路异常有关。3.EPCs细胞膜表面存在钙依赖性钾通道(KCa),KCa参与了EPCs膜电位的维持。高糖对EPCs膜电位水平及膜电流幅度无显著影响。4.视网膜血管损伤可导致视网膜局部SDF-1水平的增高,SDF-1/CXCR4轴可能在EPCs参与视网膜血管损伤修复及新生血管的形成过程中发挥重要作用。
Endothelial progenitor cells (EPCs), as the precursor cells of vascular endothelial cells, play an important role in the repair of adult vascular endothelium and angiogenesis. It has been suggested that the number and function of circulating EPCs could be reduced in diabetic, and there is a significant relationship between the development of diabetic vasculopathy and the dysfunction of EPCs. Diabetic retinopathy (DR) is the most common ocular diabetic microvascular complication, which is characterized by the disorder of retinal microcirculation in early stage and the formation of retinal neovascularization in late stage. The main pathological basis of DR is the dysfunction of vascular endothelium and the injury of retinal capillary. However, EPCs play an important role in the restore of damaged retinal blood vessels in physiological and pathological conditions. Therefore, we consider that the development of DR is closely related to the dysfunction of EPCs caused by diabetes, but the definite mechanism is not clear so far. Hyperglycemia is the main sign of diabetes, it is very important to explore the effect of high glucose on the biological characteristics of EPCs and further to study the related mechanisms, which may play important roles in the study of the etiology and prevention of DR.
Objective To investigate the effects of high glucose on the proliferation, apoptosis, adhesion and acLDL uptake ability of EPCs in vitro, and further to study the related mechanisms.The study may lay a foundation for the study of the pathogenesis and the regulating pathways of DR, and may provide new targets for the treatment of DR.
Methods①EPCs were isolated by density gradient centrifugation from human umbilical cord blood and were cultured in vitro, then EPCs were identified by flow cytometry and confocal microscope.②EPCs were cultured in normal and high glucose condition respectively. The effects of high glucose on the proliferation and adhesion capacity of EPCs were detected by MTT assay and cell counts respectively. Flow cytometry was used to detect apoptosis of EPCs in each group. RT-PCR was used to detect the mRNA levels of Bcl-2 and Bax. The effect of high glucose on the acLDL uptake ability of EPCs was detected by confocal microscope. Fluo3/AM staining was used to detect the [Ca2+]i level in each group, as well as the dynamic variation of [Ca2+]i level after dropping of CaCl2. Patch clamp technology was introduced to analysis the effect of glucose on electrophysiological properties of EPCs. Western Blot was used to detect the influence of high glucose on the expression of p-eNOS and p-AKT on protein level.③Photodynamic method was used to establish retinal vascular injury model in rat. Fundus photography and fundus fluorescein angiography were used to investigate the degree of retinal vascular damage. RT-PCR was used to detecte the SDF-1 mRNA expression after retinal vascular injury.
Results
1. Mononuclear cells were isolated from human umbilical cord blood, which were then cultured and induced into EPCs in vitro. After culturing for 3 days, EPCs formed small colonies. After culture about 10 days, the cells were highly proliferative and presented spindle shape. The EPCs exhibited cobblestone morphology on about 2-3w. Flow cytometry analysis showed that EPCs expressed CD133, CD34, VEGFR2 and CD31 at different levels on 10d, and most of the cells were double positive to DiI-acLDL and FITC-UEA-I. Immunocytochemistry stain showed that VEGF was expressed in the EPCs.
2.①The proliferation activity of EPCs was enhanced after cultivation with high glucose for 1d compared with the normal condition, and the proliferation activity of EPCs in high glucose group had no significant difference with normal group at 3d, while the proliferation activity decreased obviously after treatment with high glucose for 7d.②The adhesion property of EPCs decreased by treatment with high glucose for 1d and 3d compared with normal condition.③After high glucose treatment for 7d, the number of pro-apoptotic EPCs increased, and the mRNA level of Bax raised, while the Bcl-2 mRNA level decreased.④After high glucose culture for 3d, the [Ca2+]i in the two groups had no significant difference. In calcium free medium, the [Ca2+]I in EPCs presented an rapid increase in both group after administration of CaCl2, the [Ca2+]i in normal group return to the basal level gradually, while the [Ca2+]i in some cells of high glucose group failed to recover to the basal line.⑤The resting membrane potential of EPCs remained at about -50mv in normal conditions, and there was no significant difference on the membrane potential between the high glucose group and the control group. The membrane current of EPCs decreased under the action of TEA, indicating the presence of calcium-dependent potassium current in the composition of membrane potential. There was no significant difference between high glucose group and control group on membrane currents.⑥Western blot showed that the phosphorylation level of eNOS and AKT decreased obviously after high glucose culture compared with normal circumstance.
3. Rat retinal microvascular damage can be established by photodynamic coagulation of retinal vein. Occlusion, leakage and hemorrhage of retina can be observed. The SDF-1 mRNA level increased after retinal vascular injury.
Conclusions①High glucose can affect EPCs on proliferation, adhersion and mobilization, which may be caused by inhibition of PI3K/AKT/eNOS pathway. High glucose may promote apoptosis by upregulation of Bax gene expression and downregulation of Bcl-2 gene expression on mRNA level.②High glucose may affect calcium homeostasis, the EPCs were more likely to suffer calcium overloading under high glucose condition, the abnormal calcium signal transduction pathway caused by calcium homeostasis disorder may be responsible for the dysfunction of EPCs.③Calcium-dependent potassium channel (Kca) exist on the membrane of EPCs, and Kca may take part in the maintaining of membrane potential of EPCs. High glucose had no notable effect on the level of membrane potential and the amplitude of membrane current flow.④Retinal vascular injury can cause increased SDF-1 expression in the retina, the SDF-1/CXCR4 axis may related to the retinal vascular repair and angiogenesis mediated by EPCs.
研究目的
研究体外高糖环境对EPCs增殖、凋亡、粘附、摄取LDL能力等生物学特性的影响及相关机制,为进一步探讨EPCs参与DR发生发展的可能机制及调控途径奠定基础,同时为DR等眼内新生血管性疾病的治疗寻找新的靶点。
研究方法
1.密度梯度离心法分离人脐带血单个核细胞,体外诱导分化为EPCs并鉴定。
2.高糖及正常条件下培养EPCs,MTT法检测高糖对细胞增殖能力的影响;流式细胞仪检测高糖对EPCs凋亡数量的影响;RT-PCR法检测高糖对EPCs凋亡相关基因Bcl-2、Bax在mRNA表达水平的影响;共聚焦显微镜检测高糖对EPCs摄取acLDL能力的影响;Fluo3/AM负载EPCs,共聚焦显微镜下检测各组细胞细胞内游离钙离子水平([Ca2+]i),细胞外加入氯化钙(CaCl2),动态检测各组EPCs [Ca2+]i的变化;膜片钳技术研究EPCs的电生理特性并研究髙糖对EPCs电生理特性的影响;Western-blot检测体外高糖环境对EPCs内p-eNOS以及p-AKT蛋白表达的影响。
3.光动力法建立大鼠视网膜血管损伤模型,眼底照相及荧光眼底造影判定视网膜血管损伤情况,RT-PCR法检测视网膜血管损伤后视网膜SDF-1 mRNA表达水平的变化。
结果
1.脐带血单个核细胞经体外诱导分化,3d内可形成小集落,10d左右增殖旺盛,可见纺锤形细胞,于2-3w时呈现EPCs典型的铺路石样外观。流式细胞学结果显示EPCs体外培养10d时CD133、CD34、VEGFR2、CD31均有一定程度的表达,大部分细胞DiI-acLDL、FITC-UEA-I双染色阳性。免疫细胞化学染色结果显示EPCs内有VEGF的阳性表达。
2.①髙糖培养1d时EPCs增殖活力较正常对照组增强( P<0.05),3d时与正常对照组比较无明显差异,髙糖培养7d时EPCs增殖活力较正常对照组降低( P<0.05);②髙糖培养1d及3d时,EPCs粘附能力与正常对照组比较均明显降低( P<0.05);③高糖培养7d,与正常对照组比较,EPCs凋亡细胞数增加,Bax mRNA表达上调,而Bcl-2 mRNA表达降低。④高糖培养3d时EPCs[Ca2+ ]i水平较正常对照组无显著差别;在无钙介质中,各组EPCs在加入氯化钙(CaCl2)后均出现快速大幅度的[Ca2+]i升高,高糖组[Ca2+]i升高的幅度较正常对照组小;两组细胞[Ca2+]i在快速升高后迅速下降,正常组EPCs的[Ca2+]i均恢复至静息水平,而高糖组部分EPCs的[Ca2+]i未能恢复至静息水平。⑤正常条件下EPCs的静息膜电位维持在-50mv左右,在TEA作用下膜电流幅度降低,表明存在钙依赖性钾电流参与膜电位的构成。髙糖培养下EPCs静息膜电位水平及膜电流幅度无明显差别。⑥Western-blot结果显示高糖培养7d,eNOS及AKT的磷酸化水平较正常组比较均明显降低(P<0.05)。3.光动力法光凝视网膜静脉可以造成大鼠视网膜血管损伤,出现视网膜血管闭塞、渗漏、视网膜出血等眼底表现,视网膜血管损伤后3d及14d时,视网膜组织SDF-1 mRNA表达水平较正常组增高。
结论
1.高糖可能通过抑制PI3K/AKT/eNOS通路影响EPCs的增殖、粘附、动员等生物学功能,并可能通过在mRNA水平上调Bax表达并抑制Bcl-2表达而促进EPCs凋亡。2.高糖可以影响EPCs钙稳态,使细胞易于发生钙超载,高糖导致的EPCs功能紊乱可能与钙稳态失调导致的细胞内钙信号通路异常有关。3.EPCs细胞膜表面存在钙依赖性钾通道(KCa),KCa参与了EPCs膜电位的维持。高糖对EPCs膜电位水平及膜电流幅度无显著影响。4.视网膜血管损伤可导致视网膜局部SDF-1水平的增高,SDF-1/CXCR4轴可能在EPCs参与视网膜血管损伤修复及新生血管的形成过程中发挥重要作用。
Endothelial progenitor cells (EPCs), as the precursor cells of vascular endothelial cells, play an important role in the repair of adult vascular endothelium and angiogenesis. It has been suggested that the number and function of circulating EPCs could be reduced in diabetic, and there is a significant relationship between the development of diabetic vasculopathy and the dysfunction of EPCs. Diabetic retinopathy (DR) is the most common ocular diabetic microvascular complication, which is characterized by the disorder of retinal microcirculation in early stage and the formation of retinal neovascularization in late stage. The main pathological basis of DR is the dysfunction of vascular endothelium and the injury of retinal capillary. However, EPCs play an important role in the restore of damaged retinal blood vessels in physiological and pathological conditions. Therefore, we consider that the development of DR is closely related to the dysfunction of EPCs caused by diabetes, but the definite mechanism is not clear so far. Hyperglycemia is the main sign of diabetes, it is very important to explore the effect of high glucose on the biological characteristics of EPCs and further to study the related mechanisms, which may play important roles in the study of the etiology and prevention of DR.
Objective To investigate the effects of high glucose on the proliferation, apoptosis, adhesion and acLDL uptake ability of EPCs in vitro, and further to study the related mechanisms.The study may lay a foundation for the study of the pathogenesis and the regulating pathways of DR, and may provide new targets for the treatment of DR.
Methods①EPCs were isolated by density gradient centrifugation from human umbilical cord blood and were cultured in vitro, then EPCs were identified by flow cytometry and confocal microscope.②EPCs were cultured in normal and high glucose condition respectively. The effects of high glucose on the proliferation and adhesion capacity of EPCs were detected by MTT assay and cell counts respectively. Flow cytometry was used to detect apoptosis of EPCs in each group. RT-PCR was used to detect the mRNA levels of Bcl-2 and Bax. The effect of high glucose on the acLDL uptake ability of EPCs was detected by confocal microscope. Fluo3/AM staining was used to detect the [Ca2+]i level in each group, as well as the dynamic variation of [Ca2+]i level after dropping of CaCl2. Patch clamp technology was introduced to analysis the effect of glucose on electrophysiological properties of EPCs. Western Blot was used to detect the influence of high glucose on the expression of p-eNOS and p-AKT on protein level.③Photodynamic method was used to establish retinal vascular injury model in rat. Fundus photography and fundus fluorescein angiography were used to investigate the degree of retinal vascular damage. RT-PCR was used to detecte the SDF-1 mRNA expression after retinal vascular injury.
Results
1. Mononuclear cells were isolated from human umbilical cord blood, which were then cultured and induced into EPCs in vitro. After culturing for 3 days, EPCs formed small colonies. After culture about 10 days, the cells were highly proliferative and presented spindle shape. The EPCs exhibited cobblestone morphology on about 2-3w. Flow cytometry analysis showed that EPCs expressed CD133, CD34, VEGFR2 and CD31 at different levels on 10d, and most of the cells were double positive to DiI-acLDL and FITC-UEA-I. Immunocytochemistry stain showed that VEGF was expressed in the EPCs.
2.①The proliferation activity of EPCs was enhanced after cultivation with high glucose for 1d compared with the normal condition, and the proliferation activity of EPCs in high glucose group had no significant difference with normal group at 3d, while the proliferation activity decreased obviously after treatment with high glucose for 7d.②The adhesion property of EPCs decreased by treatment with high glucose for 1d and 3d compared with normal condition.③After high glucose treatment for 7d, the number of pro-apoptotic EPCs increased, and the mRNA level of Bax raised, while the Bcl-2 mRNA level decreased.④After high glucose culture for 3d, the [Ca2+]i in the two groups had no significant difference. In calcium free medium, the [Ca2+]I in EPCs presented an rapid increase in both group after administration of CaCl2, the [Ca2+]i in normal group return to the basal level gradually, while the [Ca2+]i in some cells of high glucose group failed to recover to the basal line.⑤The resting membrane potential of EPCs remained at about -50mv in normal conditions, and there was no significant difference on the membrane potential between the high glucose group and the control group. The membrane current of EPCs decreased under the action of TEA, indicating the presence of calcium-dependent potassium current in the composition of membrane potential. There was no significant difference between high glucose group and control group on membrane currents.⑥Western blot showed that the phosphorylation level of eNOS and AKT decreased obviously after high glucose culture compared with normal circumstance.
3. Rat retinal microvascular damage can be established by photodynamic coagulation of retinal vein. Occlusion, leakage and hemorrhage of retina can be observed. The SDF-1 mRNA level increased after retinal vascular injury.
Conclusions①High glucose can affect EPCs on proliferation, adhersion and mobilization, which may be caused by inhibition of PI3K/AKT/eNOS pathway. High glucose may promote apoptosis by upregulation of Bax gene expression and downregulation of Bcl-2 gene expression on mRNA level.②High glucose may affect calcium homeostasis, the EPCs were more likely to suffer calcium overloading under high glucose condition, the abnormal calcium signal transduction pathway caused by calcium homeostasis disorder may be responsible for the dysfunction of EPCs.③Calcium-dependent potassium channel (Kca) exist on the membrane of EPCs, and Kca may take part in the maintaining of membrane potential of EPCs. High glucose had no notable effect on the level of membrane potential and the amplitude of membrane current flow.④Retinal vascular injury can cause increased SDF-1 expression in the retina, the SDF-1/CXCR4 axis may related to the retinal vascular repair and angiogenesis mediated by EPCs.
引文
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