【作者】 赵炎波；

【导师】 傅国胜；

【作者基本信息】 浙江大学 ， 内科学， 2011， 博士

【摘要】 血管内皮损伤在心血管疾病中的重要性已被广泛认同。成熟内皮细胞能够修复损伤内皮,但是其再生能力有限。因此越来越多的科学研究者关注内皮祖细胞(endothelial progenitor cells, EPCs)在血管修复、重构和血管新生中的作用。尽管目前对于EPCs的确切来源和功能定义仍存有争议,但大量新兴研究表明,EPCs在缺血组织的血管新生、内皮损伤的修复等方面扮演重要角色,然而,多种心血管疾病或心血管疾病危险因素诸如老龄、高血压、高胆固醇血症、吸烟、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。既然异体输注健康人捐献的EPCs要面临免疫排斥问题,因此我们有理由相信功能修饰(如促进EPCs动员、归巢、存活或分泌保护性生长因子等)自体EPCs可能是将来EPCs-细胞治疗心血管疾病的重要策略。胸腺素β4 (thymosinβ4, TB4),一种由43个氨基酸残基组成的小分子量蛋白,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,目前国内外尚缺乏Tβ4对循环EPCs的作用及机制的系统研究。我们近期研究发现Tβ4能够通过激活PI3K/Akt/eNOS信号通路促进EPCs迁移,但是Tβ4对于EPCs凋亡、增殖、衰老等功能的影响仍有待进一步研究。基于上述考虑,我们提出假设：Tβ4能增强循环EPCs的功能,促进血管内皮损伤的修复,维持血管内皮的完整性,从而增强循环EPCs的心血管保护作用。由于目前国内外对Tβ4影响EPCs的功能及其机制方面的研究甚少,我们首先观察了Tβ4对EPCs活性、增殖、粘附、集落形成和衰老等功能的影响；其次我们研究了Tβ4对EPCs凋亡的作用,并探讨了可能参与其中的信号转导机制；最后我们还观察了Tβ4对EPCs旁分泌作用的影响。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对循环内皮祖细胞功能的影响目的：观察外源性Tβ4体外干预对循环EPCs功能的影响。方法：采用密度梯度离心法从健康人外周血获取单个核细胞,接种于人纤维连接蛋白包被的培养板,培养7d后,采用激光共聚焦显微镜检测FITC-UEA-I及DiI-acLDL双染色鉴定EPCs,采用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 (1、10、100釉1000ng/mL)干预不同时间后。分别采用MTT比色法、Brdu细胞增殖试剂盒、粘附能力测定、集落形成实验实验和SA-β-半乳糖苷酶染色试剂盒观察EPCs的活性、增殖、粘附、集落形成和衰老。结果：Tβ4呈浓度依赖地增加EPCs的活性、增殖、集落形成和粘附能力,在1000ng/mL组达到最大效应(与对照组比较,细胞活性0.410±0.052 vs 0.264±0.035,P<0.05；增殖能力0.802±0.081 vs 0.593±0.042,P<0.05；粘附能力44.4±3.4 vs 17.8±4.3,P<0.05；集落形成能力17.3±3.1 vs 5.7±2.1,P<0.05)。此外,Tβ4干预还显著抑制EPCs的衰老,且呈一定的量效关系,在1000ng/mL时达到最大效应(与对照组比较,20.67±3.06% vs 45.33±3.51%,P<0.05)。结论：Tβ4可增强EPCs的细胞活性、增殖和粘附等功能,同时抑制EPCs的衰老,且Tβ4对EPCs功能的作用具有一定的浓度依赖性。2胸腺素p4对循环内皮祖细胞凋亡的作用及机制目的：观察Tβ4对循环EPCs凋亡的作用并探讨其信号转导机制。方法：从健康人外周血分离、培养循环EPCS。细胞培养至7d后,采用去血清培养48h以诱导EPCs凋亡。加入不同浓度的Tβ4处理后,western blot检测EPCs Akt/p-Akt Ser473、eNOS/p-eNOS Ser1177、PTEN/PTEN Ser380、JNK/p-JNK、P38/p-P38釉ERK1/2/p-ERK1/2等信号通路蛋白以及凋亡相关蛋白caspase-3^ caspase-9、Bax、Bcl-2和细胞色素C等凋亡相关蛋白的表达。随后,在分别加入磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase, PI3K)抑制剂、内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)抑制剂、c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)抑制剂等各种信号通路抑制剂预处理EPCs 30min后,再予以1000ng/mL Tβ4干预24h,采用Annexin-V/PI凋亡检测试剂盒检测EPCs的凋亡率。结果：Tβ4干预显著抑制去血清诱导的EPCs凋亡和胞浆细胞色素C水平,且呈浓度依赖性。P13K抑制(LY294002和Wortmannin)或JNK抑制剂SP600125能够拮抗Tβ4的抗凋亡作用。此外,Tβ4还抑制了caspase-3、caspase-9的表达和活性,同时上调Bcl-2/Bax比值。Tβ4能够在EPCs能与ILK形成免疫复合物,增加ILK的活性而不影响其表达。ILK-siRNA转染后完全抑制Tβ4对ILK-Akt的激活,从而最终拮抗了Tβ4对EPCs凋亡的作用。结论：Tβ4抑制去血清诱导的EPCs凋亡与ILK-Akt的激活有关,此外,JNK MAPK也可能参与了Tβ4对EPCs凋亡作用的调节。3胸腺素p4对循环内皮祖细胞旁分泌作用的影响目的：观察Tβ4对循环EPCs旁分泌作用的影响。方法：从健康人外周血分离、培养循环EPCs。EPCs条件培养液(EPCs-derivedconditioned medium, EPCs-CM)是循环EPCs用不含血清的EBM-2培养24h获得。采用Ki-67免疫荧光检测、Trans-well细胞迁移检测和Martigel胶毛细血管样结构形成分别检测脐静脉内皮细胞(Human umbilical vein endothelial cells, HUVECs)体外增殖、迁移和血管形成能力。荧光定量PCR检测EPCs内VEGF.SDF-1、IGF-1、HGF-1、FGF-2、Tβ4和IL-8的表达。结果：EPCs-CM能够显著增强内皮细胞增殖、迁移和毛细血管样结构形成能力。Tβ4干预能够进一步增强EPCs-CM对内皮细胞功能的影响。此外,Tβ4干预增加EPCs内VEGF、FGF-2、IL-8的表达,而对内源性Tβ4、SDF-1α等细胞因子的表达无明显作用。结论：Tβ4能够促进EPCs的旁分泌作用,Tβ4增加EPCs内VEGF、FGF-2、IL-8等细胞因子的表达。更多还原

【Abstract】 The essential role of vascular endothelium in cardiovascular disorders is increasingly recognized. Mature endothelial cells contribute to the repair of endothelial injury, whereas they possess limited regenerative capacities. This has led to growing interest in circulating endothelial progenitor cells (EPCs) among scientific researchers, especially into their roles in vascular repair, remodeling and postnatal neovascularization. Although the exact origin and functional definition of EPCs remains rather controversial, emerging evidence demonstrated that EPCs contribute to neoangiogenesis after tissue ischemia. However, these positive effects after autologous transfusion or mobilization of EPCs are significantly limited by low survival rate, insufficient cell number, and impaired funcition in the patients with cardiovascular risk factors and cardiovascular diseases. Indeed, there is increasing evidence for reduced availability and impaired EPCs function in the presence of cardiovascular risk factors as well as various cardiovascular disease states, such as aging, hypertension, hypercholesterolemia, smoking, and diabetes. Since allogeneic transfusion of EPCs from healthy donors bears the problem of immunologic incompatibilities, it is quite reasonable to regard functional enhancement (such as enhancing mobilization, homing, survival, and secretion of growth factors in a paracrine/autocrine manner) of autologous EPCs as the future strategy for EPCs-based therapy in cardiovascular diseases.Thymosinβ4 (Tβ4), a small ubiquitous protein containing 43 amino acids, as the major actin-sequestering molecule in eukaryotic cells, plays pleiotropic roles in tissue development, maintenance, repair, and pathology. Previous study found that Tβ4 can stimulate migration of cardiomyocytes and endothelial cells and promote survival of cardiomyocytes. Despite the extensive identification of multiple biological activities for Tβ4, little progress is actually available for identifying the effects of Tβ4 on circulating EPCs. Our recent study demonstrated that Tβ4 can induce EPCs migration via PI3K/Akt/eNOS signal transduction pathway, but the effect of T(34 on circulating EPCs apoptosis, proliferation and senescence remains unexplored.Based on these considerations, we hypothesized that Tβ4 enhanced EPCs functional activity, subsequently promoted endothelial repair process and maintained the integrity of endothelium to perform its cardiovascular protection. To test this hypothesis, we examined the cell viability, proliferation activity, colonies formation, adhesive capacities and senescence of EPCs exposed to Tβ4, and then we studied the effect of Tβ4 on EPCs apoptosis and the signal transduction pathways involved in this process. Finally, we investigated the effects of Tβ4 on paracrine effects of EPCs.Part 1:Effects of exogenous thymosinβ4 on activity of circulating endothelial progenitor cellsEmerging evidence indicates that circulating endothelial progenitor cells (EPCs) contribute to neoangiogenesis after tissue ischemia. Here we aimed to investigate the effects of exogenous thymosin (34 (TβM) on circulating EPCs from healthy volunteers. EPCs, isolated from peripheral blood, were cultured on fibronectin-coated dishes. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding under a laser scanning confocal microscope. They were further documented by demonstrating the expression of VE-cadherin, KDR, CD34 and AC133 by flow cytometry. Cells were treated with Tββ4 (lng/mL, 10ng/mL, 100ng/mL and 1000ng/mL) or vehicle control. Cell viability, proliferation, adhesion, colony-formation and sensence of EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Brdu incorporation, adhesion assay, colony-forming assay and acidicβ-galactosidase staining respectively. Incubation of EPCs with Tβ4 significantly increased the cell viability, proliferation, adhesion, and colony-formation of EPCs, reached a maximum at 1000ng/mL. In addition, Tβ4 significantly inhibited EPCs senescence in a dose dependent manner. Together, these results revealed that T04 can dose-dependently improve cell activities of EPCs including cell viability, proliferation, adhesive capacities and colony-formation of EPCs, meanwhile inhibit EPCs senescence in a dose dependent manner.Part 2:Effects and mechanisms of thymosinβ4 on circulating endothelial progenitor cells apoptosisThymosinβ4 (Tβ4) has been suggested to regulate multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival and angiogenesis. Here, we investigated the effect of Tβ4 on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the corresponding signal transduction pathways involved in this process. Circulating EPCs, isolated from healthy volunteers, were cultured in the absence or presence of T04 and various signal cascade inhibitors. Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Incubation of EPCs with Tβ4 caused a concentration dependent increase in cell viability and proliferation activity. It also caused an inhibitory effect on EPCs apoptosis, which was abolished by PI3K inhibitors (either LY294002 or Wortmannin) or JNK MAPK inhibitor SP600125. In addition, the expression and activity of caspase-3 and-9 were decreased by T(34, which markedly increased the Bcl-2/Bax ratio within EPCs. Furthermore, Tβ4 was immunoprecipitated with integrin-linked kinase (ILK), accompanied by augmentation of ILK activity. Transfection of EPCs with ILK-siRNA resulted in abolishment of the activation of ILK-Akt and the ameliorative effect on apoptosis by Tβ4. Together, T(34 mediated inhibitory effect on EPCs apoptosis under serum deprivation can be attributed, at least in part, to ILK-Akt activation. The activation of JNK MAPK might also be involved in this process.Part 3:Effects of thymosinβ4 on paracrine effects of circulating endothelial progenitor cellsEmerging evidence suggests that paracrine effects of EPCs play a pivotal role in various processes of tissue repair. We previously demonstrated that thymosinβ4 (Tβ4) can increase the cell viability, proliferation, adhesive capacities and colony formation of EPCs. In present study, we aimed to investigate the roles of Tβ4 on EPCs paracrine effects. EPCs, isolated from healthy volunteers, were cultured and characterized as described previously. EPCs-derived conditioned medium (EPCs-CM) was obtained from culture EPCs subjected to trophic deprivation 24 h. HUVECs proliferation, migration and in vitro angiogenesis were assayed with Ki-67 immunofluorescence assay, trans-well migration assay and capillary-like tube formation on Matrigel gel respectively. EPCs-CM significantly improved endothelial function in vitro including proliferation, migration and capillary-like tube formation, which can be further enhanced by treatment with exogenous Tβ4. Furthermore, Tβ4 increased the expression of pro-angiogenic factors (such as VEGF, FGF-2 and IL-8) within EPCs. Taken together, Tβ4 can significantly enhace the paracrine effects of EPCs on endothelial cells, increased secretion of pro-mitogenic cytokines (such as VEGF, FGF-2 and IL-8) from EPCs may involve in this process.更多还原