【作者】 邱福宇；

【导师】 傅国胜；

【作者基本信息】 浙江大学 ， 临床医学， 2009， 博士

【摘要】 内皮祖细胞（endothelial progenitor cells,EPCs）是一类血管内皮细胞的前体细胞,它可向血管内皮细胞分化,但尚未完全表达成熟血管内皮细胞表型。EPCs兼具造血干细胞及内皮细胞的部分表面标志,如CD34、CD133、VEGFR-2等。大量研究表明,EPCs在缺血组织的血管新生、损伤血管的再内皮化、内皮功能障碍的逆转等方面扮演重要角色,因而在心血管系统的修复中发挥重要作用。然而,多种冠心病危险因素诸如老龄、吸烟、高血压、高胆固醇血症、糖尿病等会使循环EPCs的功能受损,从而限制了基于EPCs的细胞治疗手段在临床中的应用。因此,探求有效途径增强EPCs功能至关重要。胸腺素（thymosins）是一类多肽分子,最初从小牛胸腺中提取得到,根据其等电点的不同而分为三个亚家族（α,β和γ亚家族）。胸腺素β4（thymosinβ4,Tβ4）是胸腺素β家族中的一员,由43个氨基酸残基组成,广泛分布于人体内多种组织与细胞中。Tβ4介导了多种生物学反应,如血管新生、创伤愈合等。此前研究表明,Tβ4能增强多种干/祖细胞的增殖、迁移、分化等功能,进而促进血管新生及心肌修复。但是,目前尚未明确Tβ4是否可通过增强循环EPCs的功能而发挥心血管保护作用。基于上述理论基础,我们提出假设:Tβ4能增强EPCs的功能,促进其修复血管内皮,维持内皮的完整性,从而发挥心血管保护作用。由于目前对Tβ4影响EPCs的功能及其机制方面知之甚少,我们首先观察了Tβ4对EPCs增殖、迁移、粘附等功能的影响,并探讨了可能参与其中的信号转导途径,然后观察了Tβ4对EPCs凋亡及衰老的作用。以下分三部分对本研究的方法、结果及结论作一简述。1胸腺素β4对外周血内皮祖细胞功能的影响目的:观察Tβ4体外干预对外周血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,将其接种于人纤维连接蛋白包被的培养板,培养7d后,收集贴壁细胞,予FITC-UEA-I及DiI-acLDL染色,激光共聚焦显微镜下观察,双染色阳性细胞鉴定为正在分化的EPCs,流式细胞仪检测其表面标志（VEGFR-2,CD34,CD133）,进一步鉴定EPCs。加入不同浓度Tβ4（1ng/mL,10ng/mL,100ng/mL和1000ng/mL）干预,并进行细胞功能学实验。分别采用MTT比色法、集落形成实验、Transwell迁移实验和粘附能力测定实验观察EPCs的增殖能力、集落形成能力、迁移能力和粘附能力。结果:Tβ4能明显增加EPCs的增殖、集落形成、迁移和粘附能力,并呈一定的量效关系,在1000ng/mL组达到最大效应（与对照组比较,增殖能力0.409±0.020 vs0.257±0.015,P<0.05;集落形成能力16.7±2.6 vs 6.0±1.0,P<0.05;迁移能力79.6±5.6 vs 31.5±3.4,P<0.05;粘附能力45.3±4.7 vs 18.8±3.0,P<0.05）。结论:Tβ4可增强EPCs的功能,且Tβ4对EPCs功能的影响呈一定的量效关系。2胸腺素β4影响外周血内皮祖细胞功能的信号转导机制探讨目的:观察Tβ4影响外周血内皮祖细胞功能的信号转导机制方法:采用密度梯度离心法从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,收集贴壁细胞,予FITC-UEA-I及DiI-acLDL染色,激光共聚焦显微镜下观察,双染色阳性细胞鉴定为正在分化的EPCs,流式细胞仪检测其表面标志（VEGFR-2,CD34,CD133）,进一步鉴定EPCs。加入Tβ4干预,western blot检测EPCs Akt Ser⁴⁷³、eNOS Ser¹¹⁷⁷和ERK1/2的磷酸化水平。随后,在分别加入磷脂酰肌醇3激酶（phosphatidylinositol 3-kinase,PI3K）抑制剂、内皮型-氧化氮合酶（endothelial nitric oxide synthase,eNOS）抑制剂、细胞外信号调节激酶（extracellular signal-regulated protein kinase,ERK）1/2抑制剂预处理EPCs30min后,再予1000ng/mLTβ4干预,采用MTT比色法、Transwell迁移实验和粘附能力测定实验分别观察EPCs的增殖能力、迁移能力和粘附能力。结果:Westernblot检测显示Tβ4能促进Akt ser⁴⁷³、eNOS Ser¹¹⁷⁷和ERK1/2的磷酸化,且均呈一定的时效和量效关系。PI3K抑制剂（LY294002,Wortmannin）及eNOS抑制剂（L-NAME）均显著抑制了Tβ4促进EPCs增殖、迁移和粘附的作用,而ERK1/2抑制剂（PD98059）则均无明显影响。结论:PI3K/Akt/eNOS而非ERK1/2信号转导途径参与对Tβ4促进EPCs增殖、迁移、粘附的调控。3胸腺素β4对外周血内皮祖细胞凋亡和衰老的影响目的:观察Tβ4对外周血内皮祖细胞凋亡和衰老的影响方法:采用密度梯度离心法从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,收集贴壁细胞,予FITC-UEA-I及DiI-acLDL染色,激光共聚焦显微镜下观察,双染色阳性细胞鉴定为正在分化的EPCs,流式细胞仪检测其表面标志（VEGFR-2,CD34,CD133）,进一步鉴定EPCs。贴壁细胞培养7d后,采用去血清培养以诱导EPCs凋亡。在去血清培养EPCs 24h后加入不同浓度Tβ4（1ng/mL,10ng/mL,100ng/mL和1000ng/mL）干预,继续培养24h,Alexa Fluor^（?）488-Annexin V/PI流式细胞仪检测细胞凋亡。贴壁细胞培养4d后,加入不同浓度Tβ4（1ng/mL,10ng/mL,100ng/mL,1000ng/mL）干预,继续培养7d后,采用SA-β-半乳糖苷酶染色试剂盒检测细胞衰老。结果:Tβ4能明显抑制EPCs的凋亡和衰老,且均呈一定的量效关系,在1000ng/mL组达到最大效应（与对照组比较,细胞凋亡率6.67±0.72% vs 16.58±1.56%,P<0.05;SA-β-半乳糖苷酶阳性细胞数量19.3±2.6% vs 42.7±4.3%,P<0.05）。结论:Tβ4能明显抑制EPCs的凋亡和衰老,且Tβ4对EPCs凋亡和衰老的影响均呈一定的量效关系。更多还原

【Abstract】 Endothelial progenitor cells (EPCs) are a specific cell population that have the potential to differentiate into mature endothelial cells, but have not acquired characteristic markers of mature endothelial cells. EPCs express various combinations of antigens traditionally associated with hematopoietic stem cells as well as endothelial cells, such as CD34, CD133 and vascular endothelial growth factor receptor (VEGFR) -2. Accumulating evidence indicates the impact of EPCs in cardiovascular repair since these cells have been shown to contribute to angiogenesis in response to ischemia, reendothelialization after vascular injury, and reversal of endothelial dysfunction. However, various risk factors for coronary artery disease, such as aging, smoking, hypertension, hypercholesterolemia, and diabetes may affect the activity of circulating EPCs and restrict the application of EPCs-based cell therapy. Therefore, it is important to improve activity of EPCs.Thymosins comprise a family of polypeptides originally isolated from calf thynius, and are divided into three groups (α,βandγ) based on their isoelectric point. The most abundant member ofβ-thymosins is thymosinβ4 (Tβ4), a 43 amino acid polypeptide. Tβ4 has been found highly expressed in a number of tissues and cells, suggesting various functions. Previous studies have implicated Tβ4 in a number of physiological and pathological events, such as wound healing and angiogenesis. Recently, it showed that Tβ4 may play roles in various stem/progenitor cells proliferation, migration and differentiation to promote angiogenesis and cardiac repair. However, little is known about the effects of Tβ4 on circulating EPCs.On the basis of these considerations, we hypothesized that Tβ4 enhanced EPCs functional activity, thus promoted endothelial repair process and maintained the integrity of endothelium to perform its cardiovascular protection. To test this hypothesis, we measured the proliferative, migratory and adhesive capacities of EPCs exposed to Tβ4 and investigated the signal transduction pathways involved in these processes, and then we studied the effects of Tβ4 on EPCs apoptosis and senescence. Part 1: Effects of thymosinβ4 on activity of endothelial progenitorcells from peripheral bloodThe aim of this study is to investigate the effects of thymosinβ4 (Tβ4) on functional activity of endothelial progenitor cells (EPCs) from healthy volunteers. Peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation with Ficoll separating solution, and then the cells were plated on culture dishes coated with fibronectin. Under a laser scanning confocal microscope, EPCs were characterized as adherent cells double positive for lectin binding and DiI-acLDL-uptake by direct fluorescent staining after 7 days in culture. EPCs were further documented by showing the expression of VEGFR-2, CD34 and CD133 with flow cytometry. Attached cells were stimulated with TβM (1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL) or vehicle control. EPCs proliferation, colony-formation, migration and adhesion were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony-forming assay, transwell migration assay and adhesion assay respectively. We showed that Tβ4 significantly enhanced the proliferative, colony-forming, migratory and adhesive capacities of EPCs, maximum at 1000ng/ml (to compare with that of control subjects, proliferative capacities 0.409±0.020 vs 0.257±0.015, P＜0.05; colony-forming capacities 16.7±2.6 vs 6.0±1.0, P＜0.05; migratory capacities 79.6±5.6 vs 31.5±3.4, P＜0.05; adhesive capacities 45.3±4.7 vs 18.8±3.0, P＜0.05). These results suggest that Tβ4 can increase the proliferative, colony-forming, migratory and adhesive capacities of EPCs. Part 2: Signal transduction pathways involved in effects of thymosinβ4 on activity of endothelial progenitor cells from peripheral bloodOur previous study demonstrated that thymosinβ4 (Tβ4) can enhance the proliferative, migratory and adhesive capacities of endothelial progenitor cells (EPCs). In present study, we aimed to determine the possible signal transduction pathways involved in effects of Tβ4 on functional activity of EPCs. EPCs were isolated from peripheral blood and characterized as described previously. Using western blot analysis, we showed that treatment of EPCs with thymosinβ4 resulted in time and concentration-dependent phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK)1/2. Functional analysis showed that thymosinβ4-induced EPCs proliferation, migration and adhesion were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) or eNOS inhibitor (Nω-Nitro-L-arginine methyl ester), but were not significantly attenuated by ERK1/2 inhibitor (PD98059). These findings suggest that thymosinβ4 stimulates EPCs proliferation, migration and adhesion via PI3K/Akt/eNOS, rather than via ERK1/2 signal transduction pathway. Part 3: Effects of thymosinβ4 on apoptosis and senescence of endothelial progenitor cells from peripheral bloodAccumulating evidence has showed that reduced activity of endothelial progenitor cells (EPCs) was associated with EPCs apoptosis and senescence. We previously demonstrated that thymosinβ4 (Tβ4) can increase the proliferative, migratory and adhesive capacities of EPCs. In present study, we aimed to investigate the roles of Tβ4 in EPCs apoptosis and senescence. EPCs were isolated from peripheral blood and characterized as described previously. Using an apoptosis assay, we showed that Tβ4 decreased EPCs apoptosis induced by serum deprivation in a dose-dependent manner. After ex-vivo prolonged cultivation, EPCs became senescence as determined by acidicβ-galactosidase staining. Tβ4 dose-dependently inhibited the onset of EPCs senescence in culture. In conclusion, Tβ4 can significantly inhibit EPCs apoptosis and senescence, which may lead to the enhancement of EPCs activity.更多还原