胸腺素β4优化内皮祖细胞功能的基础和应用研究

发布时间：2018-05-01 08:09

  本文选题：内皮祖细胞 + 胸腺素β4　； 参考：《浙江大学》2015年博士论文

【摘要】：目前,缺血性心血管疾病的发病率和致死率逐年上升,严重危害人类健康。其发病制十分复杂,其中内皮细胞功能障碍是导致缺血性心血管病的重要环节。虽然成熟内皮细胞能够修复损伤内皮,但是其再生能力有限。内皮祖细胞(endothelial progenitor cells, EPCs)是一类血管内皮细胞的前体细胞,具有向内皮细胞分化的潜能。大量研究表明,EPCs在血管修复和新生中发挥多种作用。但是,目前EPCs移植在临床应用仍面临诸多问题,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、糖尿病等会减少循环EPCs数目,并损害EPCs的功能,这很大程度上限制了EPCs在缺血性心血管疾病中的应用。因此,改善EPCs功能将是未来EPCs移植治疗的重要策略。胸腺素P4 (thymosin β4, Tβ4),是一种由43个氨基酸残基组成的小分子量蛋白,它介导了多种生物学反应,如血管新生、创伤愈合、炎症控制等。我们此前的研究发现Tβ4能够增强人外周血EPCs的增殖、迁移,并抑制人外周血EPCs的凋亡和衰老,但是Tβ4对于循环EPCs的成血管功能、旁分泌功能以及Tβ4预处理EPCs后进行移植是否能进一步提高EPCs移植治疗的疗效目前尚不明确。基于上述考虑,我们首先观察了体外实验中Tβ4对EPCs成血管功能的影响及相关机制；其次我们研究了Tβ4对EPCs旁分泌的作用；最后我们进行了动物实验探究了Tβ4预处理EPCs后进行移植是否能进一步提高EPCs移植治疗的疗效。以下分三部分对本研究的方法、结果及结论分别简述。1胸腺素β4对循环内皮祖细胞成血管功能的影响目的：观察Tβ4体外干预对外周血内皮祖细胞成血管功能的影响及相关机制方法：采用密度梯度离心法从人外周血获取单个核细胞,接种于人纤维连接蛋白(HFN)包被的培养板,培养7d后,收集贴壁细胞,采用FITC-UEA-I和DiI-acLDL染色,在激光共聚焦显微镜下观察,双染阳性细胞鉴定为正在分化的EPCs。加入不同浓度的Tβ4 (10ng/mL, 100ng/mL和1000ng/mL)干预,采用Matrigel胶体外成血管能力测定实验观察EPCs的成血管能力。Western blot检测EPCs Akt Ser473和eNOS Ser1177的磷酸化水平。随后,在分别加入Akt siRNA 及 eNOS siRNA干扰后,再予1000ng/mL Tβ4干预,再次采用Matrigel胶体外成血管能力测定实验观察不同实验组EPCs的成血管能力。结果：Tβ4能明显增加EPCs的成血管能力,并呈一定的量效关系,在1000ng/mL达到最大效应(与对照组比较,33.33±1.86 vs 18.34±2.02,P0.05)。Western blot检测显示Tβ4能促进Akt Ser473及eNOS Ser1177的磷酸化,且呈一定的量效关系。Akt siRNA及eNOS siRNA均显著抑制了Tβ4促进EPCs成血管的作用。结论：Tβ4可增强EPCs的体外成血管功能,且呈一定的量效关系。Akt/eNOS信号转导途径参与对Tβ4促进EPCs体外成血管能力的调控。2胸腺素β4对循环内皮祖细胞旁分泌作用的研究目的：观察Tβ4影响外周血内皮祖细胞旁分泌功能的影响及相关机制方法：分离和获取外周血内皮祖细胞(EPCs)方法同前。加入不同浓度的Tβ4(10ng/mL, 100ng/mL和1000ng/mL)干预,采用荧光定量PCR检测EPCs内VEGF和IL-8的表达,采用ELISA试剂盒检测EPCs条件培养基(EPCs-derived conditioned medium,EPCs-CM)中VEGF和IL-8的表达。采用Transwell细胞迁移检测和Matrigel胶毛细血管样结构形成分别检测EPCs-CM、Tβ4-EPCs-CM及Tβ4-EPCs-CM加入中和性抗体(VEGF和IL-8中和性抗体)干预之后人脐静脉内皮细胞(Human umbilical vein endothelial cells, HUVECs)体外迁移和成血管能力。在分别加入Akt siRNA及eNOS siRNA干扰后,再予1000ng/mL Tβ4干预,采用ELISA试剂盒检测EPCs-CM中VEGF和IL-8的表达。结果： Tβ4干预能明显增加EPCs内和EPCs-CM中VEGF和IL-8的表达,EPCs-CM能够显著增加内皮细胞迁移和成血管作用,Tβ4干预能进一步增强EPCs-CM对内皮功能的影响,而VEGF和IL-8的中和性抗体可以分别拮抗T04对EPCs-CM促内皮迁移和成血管的增强效应。Akt siRNA可以显著降低Tβ4-EPCs-CM中VEGF和IL-8的表达；eNOS siRNA可以显著降低Tβ4-EPCs-CM中、VEGF而非IL-8的表达。结论：Tβ4能够促进EPCs的旁分泌作用,增加1EPCs内和EPCs-CM中VEGF和IL-8的表达,且这一作用与Akt/eNOS信号转导途径有关。3胸腺素β4对循环内皮祖细胞移植作用的影响目的：观察Tβ4对外周血内皮祖细胞移植作用的影响方法：分离和获取外周血内皮祖细胞(EPCs)方法同前。采用前降支结扎法建立大鼠心肌梗死模型,按不同分组分别将150μL培养基、1×106个EPCs及相同数量的经Tβ4预处理24小时后的EPCs采用心肌注射的方法移植于心脏缺血区。心梗4周后采用超声心动图检测大鼠的心脏功能,组织切片免疫荧光检测心梗缺血边缘区血管密度及移植EPCs驻留和参与血管修复情况；组织切片免疫组化及组织蛋白电泳检测缺血区VEGF的表达及组织纤维化的程度。结果：Tβ4预处理能明显提高EPCs在体驻留,并参与血管的修复,较单纯EPCs移植进一步增加了缺血区血管密度,减低纤维化程度,最终改善大鼠心梗4周后的心脏功能。结论：Tβ4能明显提高EPCs的移植效果,增加缺血区血管密度,降低纤维化程度,改善心功能。
[Abstract]:At present, the incidence and mortality rate of ischemic cardiovascular disease are increasing year by year, seriously endangering human health. Its pathogenesis is very complex, and endothelial cell dysfunction is an important part of ischemic cardiovascular disease. Although mature endothelial cells can repair damaged endothelium, but its regeneration ability is limited. Endothelial progenitor cells (endothelial Progenitor cells, EPCs, a kind of precursor cells of vascular endothelial cells, has the potential to differentiate into endothelial cells. A large number of studies have shown that EPCs plays a variety of roles in vascular repair and regeneration. However, there are still many problems in the clinical application of EPCs transplantation. Many cardiovascular and cardiovascular diseases such as aging and high risk factors are high. Blood pressure, hypercholesterolemia, diabetes and so on can reduce the number of circulating EPCs and damage the function of EPCs, which largely limits the use of EPCs in ischemic cardiovascular disease. Therefore, the improvement of EPCs function will be an important strategy for the future EPCs transplantation treatment. Thymosin P4 (thymosin beta 4, T beta 4) is a form of 43 amino acid residues. Small molecular weight protein, which mediates a variety of biological reactions, such as angiogenesis, wound healing, and inflammatory control. Our previous study found that T beta 4 could enhance the proliferation, migration, and apoptosis of human peripheral blood EPCs, and inhibit the apoptosis and senescence of human peripheral blood EPCs, but T beta 4 for the vascular function of circulating EPCs, paracrine function, and T beta 4 preconditioning EP It is not clear whether transplanting after Cs can further improve the therapeutic effect of EPCs transplantation. Based on the above considerations, we first observed the effect of T beta 4 on the vascular function of EPCs in vitro and related mechanisms; secondly, we studied the effect of T beta 4 on the paracrine secretion of EPCs; finally, we conducted an animal experiment to explore the preposition of T beta 4. Whether the transplantation of EPCs can further improve the therapeutic effect of EPCs transplantation. The following three parts give a brief account of the effects of.1 thymosin beta 4 on the vascular function of circulating endothelial progenitor cells: the effect of T beta 4 on the vascular function of peripheral blood progenitor cells in vitro and the related mechanism Methods: a density gradient centrifugation method was used to obtain mononuclear cells from human peripheral blood, inoculated with human fibronectin (HFN) coated culture plate, and cultured 7d to collect adherent cells. FITC-UEA-I and DiI-acLDL staining were used to observe with laser confocal microscopy. Double staining positive cells were identified as being differentiated EPCs. added to different concentrations of T. The intervention of beta 4 (10ng/mL, 100ng/mL and 1000ng/mL) was used to observe the vascular ability of Matrigel colloid to observe the vascular ability of EPCs..Western blot was used to detect the phosphorylation level of EPCs Akt Ser473 and eNOS Ser1177. The angiogenic ability of EPCs in different experimental groups was observed. Results: T beta 4 could significantly increase the vascular ability of EPCs, and showed a certain dose effect relationship. The maximum effect was achieved in 1000ng/mL (compared with the control group, 33.33 + 1.86 vs 18.34 + 2.02, P0.05).Western blot detection showed that T beta 4 could promote Akt Ser473 and eNOS eNOS. Phosphorylation and a certain dose effect relationship between.Akt siRNA and eNOS siRNA significantly inhibit the role of T beta 4 in promoting EPCs angiogenesis. Conclusion: T beta 4 can enhance the vascular function of EPCs in vitro, and is involved in a certain amount of.Akt/eNOS signal transduction pathway involved in the regulation of.2 thymosin beta 4 in the circulation of T beta 4. The objective of paracrine paracrine effect: To observe the effect of T beta 4 on the paracrine function of peripheral blood endothelial progenitor cells and the related mechanism methods: separation and acquisition of peripheral blood endothelial progenitor cells (EPCs) method in the same front. Adding different concentrations of T beta 4 (10ng/mL, 100ng/mL and 1000ng/mL) intervention, using fluorescence quantitative PCR to detect VEGF and IL-8 in EPCs The expression of VEGF and IL-8 in the EPCs conditioned medium (EPCs-derived conditioned medium, EPCs-CM) was detected by ELISA kit. Transwell cell migration detection and Matrigel gel capillary like structure were used to detect EPCs-CM. The human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs) were migrated and vasculogenic in vitro. After the interference of Akt siRNA and eNOS siRNA respectively, the intervention of 1000ng/mL T beta 4 was given. The expression of IL-8, EPCs-CM can significantly increase endothelial cell migration and angiogenesis, T beta 4 intervention can further enhance the effect of EPCs-CM on endothelial function, while VEGF and IL-8 neutralizing antibodies can antagonize T04 to EPCs-CM promoting endothelial migration and angiogenesis..Akt siRNA can significantly reduce T beta 4-EPCs-CM. ENOS siRNA can significantly reduce the expression of VEGF but not IL-8 in T beta 4-EPCs-CM. Conclusion: T beta 4 can promote the paracrine effect of EPCs, increase the expression of VEGF and IL-8 in 1EPCs and EPCs-CM, and this effect is related to the effect of thymosin beta 4 on the transplantation of endogenous progenitor cells. The effect of beta 4 on the transplantation of peripheral endothelial progenitor cells: isolation and acquisition of peripheral blood endothelial progenitor cells (EPCs) method. The model of rat myocardial infarction was established by anterior descending ligature. The 150 L medium, 1 x 106 EPCs and the same number of EPCs after 24 hours of T beta 4 were injected into the myocardium. Methods the cardiac function of rats was detected by echocardiography after 4 weeks of myocardial infarction. The density of blood vessels in the marginal zone of myocardial infarction, EPCs residing and vascular repair were detected by tissue section immunofluorescence. The expression of VEGF in ischemic area and tissue fibrosis were detected by immunohistochemistry and tissue protein electrophoresis. Results: T beta 4 preconditioning can significantly improve the retention of EPCs in body, and participate in the repair of blood vessels. Compared with simple EPCs transplantation, the density of blood vessels in the ischemic area, the degree of fibrosis and the cardiac function after 4 weeks of myocardial infarction can be improved. Conclusion: T beta 4 can obviously improve the effect of EPCs transplantation, increase the density of blood vessels in ischemic area, and reduce fiber. The degree of maintenance and improvement of heart function.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543

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