胸腺素β4对外周血内皮祖细胞功能的影响及机制探讨

发布时间：2018-05-20 00:24

本文选题：内皮祖细胞 + 胸腺素β4　；参考：《浙江大学》2009年博士论文

【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,P0.05;集落形成能力16.7±2.6 vs 6.0±1.0,P0.05;迁移能力79.6±5.6 vs 31.5±3.4,P0.05;粘附能力45.3±4.7 vs 18.8±3.0,P0.05)。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 Ser~(473)、eNOS Ser~(1177)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 ser~(473)、eNOS Ser~(1177)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%,P0.05;SA-β-半乳糖苷酶阳性细胞数量19.3±2.6% vs 42.7±4.3%,P0.05)。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。
[Abstract]:Endothelial progenitor cells (EPCs) is a kind of precursor cells of a kind of vascular endothelial cells. It can differentiate into vascular endothelial cells, but it has not completely expressed the partial surface markers of the mature vascular endothelial cell phenotype.EPCs with both hematopoietic stem cells and endothelial cells, such as CD34, CD133, VEGFR-2 and so on. A large number of studies have shown that EPCs is in ischemia. It plays an important role in vascular regeneration, endothelialization of blood vessels, the reversal of endothelial dysfunction, and thus plays an important role in the repair of the cardiovascular system. However, multiple coronary risk factors such as aging, smoking, hypertension, hypercholesterolemia, diabetes, etc. can damage the function of circulatory EPCs, thus limiting the function of circulation. The application of cell therapy based on EPCs in clinical practice, therefore, it is very important to explore effective ways to enhance EPCs function.
Thymosin (thymosins) is a class of polypeptide molecules, originally extracted from the calf thymus and divided into three subfamilies (alpha, beta and gamma subfamilies) according to their isoelectric points. Thymosin beta 4 (thymosin beta 4, T beta 4) is a member of the thymosin beta family, consisting of 43 amino acid residues and widely distributed in a variety of tissues and cells in the human body and.T beta 4. A variety of biological reactions, such as angiogenesis and wound healing, have been introduced. Previous studies have shown that T beta 4 can enhance the proliferation, migration, differentiation and other functions of multiple stem / progenitor cells to promote angiogenesis and myocardial repair. However, it is not clear whether T beta 4 can play a role in cardiovascular protection by enhancing the function of cyclic EPCs.
Based on the above theoretical basis, we propose that T beta 4 can enhance the function of EPCs, promote the repair of vascular endothelium, maintain endothelium integrity, and play the role of cardiovascular protection. As we know little about the function and mechanism of T beta 4 affecting EPCs, we first observed the effects of T beta 4 on EPCs proliferation, migration, adhesion and other functions, The signal transduction pathway may be involved, and the effect of T beta 4 on EPCs apoptosis and senescence is observed. The following three parts give a brief account of the methods, results and conclusions of this study.
Effect of 1 thymosin beta 4 on peripheral blood endothelial progenitor cells
Objective: To observe the effect of T beta 4 intervention on peripheral blood EPCs function in vitro.
Methods: density gradient centrifugation was used to obtain mononuclear cells from human peripheral blood and inoculated into the culture plate of human fibronectin package. After culture 7d, the adherent cells were collected and stained with FITC-UEA-I and DiI-acLDL. The two staining positive fine cells were identified as the differentiated EPCs, and the flow cytometry was used to detect the cells. The surface markers (VEGFR-2, CD34, CD133), further identified EPCs. intervention with different concentrations of T beta 4 (1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL), and carried out cell functional experiments. The MTT colorimetric assay, colony formation experiment, Transwell Migration Experiment and adhesion ability test were used to observe the proliferation, colony forming ability and migration ability of EPCs. And adhesion ability.
Results: T beta 4 could significantly increase the proliferation of EPCs, colony formation, migration and adhesion, and the maximum effect in 1000ng/mL group. Compared with the control group, the proliferation ability was 0.409 + 0.020 vs0.257 + 0.015, P0.05; the colony forming ability was 16.7 + 2.6 vs 6 + 1, P0.05; migration ability was 79.6 + 5.6 vs 31.5 + 3.4, P0.05; adhesion energy The force was 45.3 + 4.7 vs 18.8 + 3, P0.05).
Conclusion: T beta 4 can enhance the function of EPCs, and T beta 4 has a dose-dependent relationship with EPCs function.
Effect of 2 thymosin beta 4 on signal transduction mechanism of endothelial progenitor cells in peripheral blood
Objective: To observe the signal transduction mechanism of T beta 4 on the function of endothelial progenitor cells in peripheral blood.
Methods: density gradient centrifugation was used to obtain mononuclear cells from peripheral blood and inoculated into the culture plate of human fibronectin package. After culture 7d, the adherent cells were collected and stained with FITC-UEA-I and DiI-acLDL. The two staining positive cells were identified as the differentiated EPCs, and the flow cytometry was used to detect the cells. VEGFR-2 (CD34, CD133), further identification of T beta 4 intervention by EPCs., Western blot to detect EPCs Akt Ser~ (473), eNOS Ser~ (1177) and phosphorylation level. OS) inhibitor, extracellular signal regulated kinase (extracellular signal-regulated protein kinase, ERK) 1/2 inhibitor pretreated EPCs30min, and then 1000ng/mLT beta 4 intervention. MTT colorimetric assay, Transwell Migration Experiment and adhesion assay test were used to observe the proliferation, migration and adhesion of EPCs.
Results: Westernblot test showed that T beta 4 could promote the phosphorylation of Akt ser~ (473), eNOS Ser~ (1177) and ERK1/2, and all of them showed a certain aging and dose effect relationship. Both.PI3K inhibitor (LY294002, Wortmannin) and eNOS inhibitor (L-NAME) all significantly inhibited the effect of beta 4 on promoting proliferation, migration and adhesion. Influence.
Conclusion: PI3K/Akt/eNOS, rather than ERK1/2 signal transduction pathway, is involved in the regulation of T beta 4 on EPCs proliferation, migration and adhesion.
Effects of 3 thymosin beta 4 on apoptosis and senescence of peripheral blood endothelial progenitor cells
Objective: To observe the effect of T beta 4 on apoptosis and aging of endothelial progenitor cells in peripheral blood.
Methods: density gradient centrifugation was used to obtain mononuclear cells from peripheral blood and inoculated into the culture plate of human fibronectin package. After culture 7d, the adherent cells were collected and stained with FITC-UEA-I and DiI-acLDL. The two staining positive cells were identified as the differentiated EPCs, and the flow cytometry was used to detect the cells. VEGFR-2, CD34, CD133), after further identification of EPCs. adherent cell culture, 7d was cultured to induce the apoptosis of EPCs. After the serum culture of EPCs 24h, EPCs 24h was cultured with different concentrations of T beta 4 (1ng/mL, 10ng/mL, 100ng/mL, etc.) to detect cell apoptosis. After the cells were cultured for 4D, the cell senescence was detected by SA- beta galactosidase staining kit after adding different concentrations of T beta 4 (1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL) and continuing to cultivate 7d.
Results: T beta 4 could significantly inhibit the apoptosis and senescence of EPCs, and all showed a certain dose effect relationship. The maximum effect was achieved in the 1000ng/mL group (compared with the control group, the apoptosis rate was 6.67 + 0.72% vs 16.58 + 1.56%, P0.05; the number of SA- beta galactosidase positive cells was 19.3 + 2.6% vs 42.7 + 4.3%, P0.05).
Conclusion: T beta 4 can significantly inhibit the apoptosis and senescence of EPCs, and the effect of T beta 4 on EPCs apoptosis and senescence is dose effect relationship.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【共引文献】