低氧对外周血内皮祖细胞功能的影响及机制探讨

发布时间：2018-04-15 13:02

本文选题：内皮祖细胞 + 低氧　；参考：《浙江大学》2008年博士论文

【摘要】： 越来越多的证据表明损伤器官的修复不仅包括受损部位邻近细胞的增值和迁移还包括其他部位干细胞的参与。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提细胞,其特征性的表达cD34、KDR及CD133。研究表明EPCs在缺血组织的血管新生(revascularizatioa)及受损血管的再内皮化(re-endothelialization)中起到了重要的作用。当受到如缺血组织释放的各种细胞因子的刺激,内皮祖细胞从骨髓进入血液循环(mobilization),到达需要组织修复的部位或受损血管(homing)分化成为内皮细胞(differentiation),并释放多种可以促进局部内皮细胞的迁移和增殖的细胞因子。EPCs移植在心血管疾病治疗中有广泛的应用前景,其可以改善心肌梗死患者的心功能,减少心肌重塑,增加下肢缺血病人的运动耐量。低氧与心绞痛,心肌梗死,心力衰竭及外周动脉疾病密切相关,低氧和组织缺血多由于血管闭塞或由于高血压所致功能及解剖性微血管稀疏所致。降低的氧浓度刺激机体产生适应性反应,例如内皮细胞的增殖,血管生成(angiogenesis)等,从而缓解局部缺氧。为适应低氧刺激,细胞内多种信号分子被激活,其中低氧诱导分子(Hypoxia-inducible factor-1,HIF-1)是细胞适应低氧微环境的关键信号分子。晚近研究表明,低氧微环境(如缺血组织,受损血管)通过HIF-1路径刺激局部细胞释放内皮生长因子(Vascular endothelial growth factor,VEGF)及基质细胞衍生因子-1α(stromal cell-derived factor-1α,SDF-1α)从而促进EPCs的动员及归巢。但低氧微环境对EPCs迁移及凋亡的影响,目前还不清楚。基于上述理论,我们提出假设:低氧可以影响EPCs的功能,继而影响受损血管内皮修复能力及血管生成,进而影响一系列心血管疾病的发生、发展。目前国内外有关低氧对EPCs功能的影响及其机制的研究尚很少。为此,我们首先观察了体外低氧对外周血EPCs功能的影响,随后进一步探讨了低氧影响EPCs功能的可能机制。以下分两部分对本研究的方法、结果以及结论作一简述。第一部分低氧对外周血内皮祖细胞功能的影响目的:观察体外低氧对外周血EPCs功能的影响。方法:采用密度梯度离心法从人外周血获取单个核细胞,将其接种在人纤维连接蛋白包被的培养板,培养7d,细胞同步化后放入低氧微环境(2%O_2)干预。多波长激光共聚焦显微镜鉴定FITC-UEA-I和DiI-acLDL双染色阳性细胞为正在分化的EPCs,流式细胞仪检测其表面标志进一步鉴定EPCs。采用改良的Boyden小室、MTT比色法、FITC-Annexin V/PI流式细胞仪检测、TUNEL染色法分别观察EPCs的迁移能力、细胞活力和细胞凋亡情况。结果:低氧24h能显著增强外周血EPCs向SDF-1a(100ng/mL)的迁移能力。去血清48h,EPCs细胞活力明显下降,细胞凋亡明显增多,低氧24h可以延缓去血清诱导的细胞活力降低,抑制去血清诱导的EPCs凋亡(与对照组比较,迁移能力58.57±10.49 vs 38.57±7.48,细胞活力0.74±0.13 vs 0.62±0.09,凋亡情况27.87%±2.03%vs 32.43%±3.11%)。结论:低氧可增强EPCs迁移能力,抑制EPCs活力下降及凋亡。第二部分低氧对内皮祖细胞功能影响的机制探讨目的:研究低氧增强EPCs迁移及抑制其凋亡的机制。方法:采用密度梯度离心法从人外周血获取单个核细胞,将其接种在人纤维连接蛋白包被的培养板,培养7d后,多波长激光共聚焦显微镜鉴定FITC-UEA-I和DiI-acLDL双染色阳性细胞为EPCs,流式细胞仪检测其表面标志进一步鉴定EPCs。培养7d后,细胞同步化后,EPC s低氧培养24h干预或先予以磷脂酰肌醇3激酶(Phosphoinositide 3-kinase,PI3K)、胞外信号调节激酶(Extracellular signal-regulated protein kinase1/2,ERK1/2)抑制剂预处理1h后干预,RT-PCR检测SDF-1α受体CXCR4 mRNA的表达;流式细胞仪测定CXCR4表达率、改良的Boyden小室测量EPCs向SDF-1α(100ng/mL)的迁移能力。FITC-Annexin V/PI流式细胞仪检测细胞凋亡。Western blot检测EPCs Akt、ERK1/2、GSK-3β(Glycogen synthasekina se-3β)磷酸化水平及HIF-1α蛋白水平。采用MTT比色法观察EPCs的活力。结果:低氧24h,EPCs向SDF-1a(100ng/mL)的迁移能力明显增强,SDF-1α受体CXCR4 mRNA及蛋白表达水平明显高于对照组,CXCR4特异性阻断剂AMD3100和PI3K抑制剂(LY294002)可以近乎完全抑制增强的迁移能力,并且后者可以明显抑制低氧诱导的CXCR4表达增高。而ERK1/2抑制剂(PD98059)则对CXCR4的表达及EPCs的迁移能力没有明显作用。研究还发现PI3K抑制剂(LY294002)可以近乎完全的抵消低氧抑制的去血清培养诱导的EPCs活力下降及凋亡,而PD98059则无明显作用。western blot检测显示低氧可以促进EPCs Akt、ERK1/2、GSK-3β的磷酸化和HIF-1α蛋白水平的增加。结论:低氧可增加EPCs受体CXCR4的表达,抑制EPCs凋亡;PI3K/Akt而非ERK1/2信号途径参与低氧诱导的CXCR4表达上调及抑制的细胞凋亡。
[Abstract]:Endothelial progenitor cells ( EPCs ) are a kind of prerequisite cells which can proliferate and differentiate into vascular endothelial cells . The study shows that EPCs can be differentiated into endothelial cells and release a variety of cytokines which can promote the migration and proliferation of local endothelial cells .

Hypoxia and angina pectoris , myocardial infarction , heart failure and peripheral artery disease are closely related , hypoxia and ischemia are caused by vascular occlusion or due to hypertension .

On the basis of the above theory , we put forward that hypoxia can affect the function of EPCs , then affect the repair ability of injured vascular endothelium and angiogenesis , and then influence the development of a series of cardiovascular diseases . At present , we first observed the effect of hypoxia on EPCs function and the possible mechanism of hypoxia on EPCs function .

Effect of first part hypoxia on the function of endothelial progenitor cells in peripheral blood

Objective : To observe the effect of hypoxia on the function of EPCs in peripheral blood .

Methods : Single nuclear cells were obtained from human peripheral blood by density gradient centrifugation . The cells were inoculated into cultured plates coated with human fibronectin . After 7 days of incubation , the cells were synchronized and then placed in hypoxic microenvironment ( 2 % O2 ) . The surface markers of FITC - UEA - I and DiI - acLDL were detected by multi - wavelength laser confocal microscope . The migration ability , cell viability and apoptosis of EPCs were observed by using modified Boyden chamber , MTT colorimetric method , FITC - annexing V / PI flow cytometry .

Results : After 24 hours of hypoxia , the cell viability of EPCs decreased significantly , and the apoptosis of EPCs decreased significantly . The apoptosis of EPCs induced by serum was decreased and the apoptosis of EPCs induced by serum was decreased ( 58.57 卤 10.49 vs 38.57 卤 7.48 , cell viability was 0.74 卤 0.13 vs 0.62 卤 0.09 , and apoptosis was 27.87 % 卤 2 . 03 % vs 32.43 % 卤 3.11 % ) .

Conclusion : Hypoxia can enhance EPCs migration ability , inhibit EPCs activity decline and apoptosis .

Study on the mechanism of hypoxia on endothelial progenitor cell function in the second part

Objective : To study the mechanism of hypoxia - enhanced EPCs migration and the inhibition of apoptosis .

Methods : Human peripheral blood mononuclear cells were isolated from human peripheral blood by density gradient centrifugation . After 7 days of culture , FITC - UEA - I and DiI - acLDL double staining positive cells were identified as EPCs . After 7 days of incubation , the expression of CXCR4 mRNA was detected by flow cytometry . The expression rate of CXCR4 mRNA was detected by flow cytometry . The phosphorylation level and HIF - 1伪 protein level of EPCs were detected by flow cytometry . The activity of EPCs was observed by MTT assay .

Results : The expression level of CXCR4 mRNA and protein in EPCs was significantly higher than that of control group . The expression of CXCR4 mRNA and protein in EPCs was significantly higher than that of control group .

Conclusion : Hypoxia can increase the expression of CXCR4 in EPCs , inhibit the apoptosis of EPCs , inhibit the expression of CXCR4 in hypoxia - induced CXCR4 expression and inhibit the apoptosis of EPCs .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

【共引文献】