【作者】 徐其渊；

【导师】 王建安； Hong Yu； Keith A. Webster；

【作者基本信息】 浙江大学 ， 内科学， 2010， 博士

【摘要】 实验背景：随着社会经济的发展,心脑血管疾病的发病率日益增高。动脉闭塞作为冠心病、脑血管意外和下肢缺血的共同病因,其发病率在老年人群中渐增。利用人体自身的细胞来生成新血管的细胞替代治疗作为一种低成本、低风险的治疗方案已越来越受重视。不幸的是,可能从中受益最大的老年患者体内可用的祖细胞数量及其血管生成能力均已极度衰减。骨髓源性干细胞(BMCs)具有多向分化能力,并能提供支持血管生长和成熟的蛋白质,从而促进新生血管生成。基质细胞衍生因子-1(SDF-1)是动员骨髓细胞和促进细胞向靶组织归巢的过程中的关键性趋化因子。SDF-1通过与靶细胞表面受体CXCR4结合并激活CXCR4而起作用。不同类型的细胞表面CXCR4的表达各不相同,而通过适当的体外培养可以增加其表达。我们过去的研究表明,钙处理可提高对骨髓细胞表面CXCR4的表达。BMCs表面CXCR4的表达水平决定了细胞向靶组织归巢及随后血管生成的效率。MSCs或CD34+细胞CXCR4的过度表达,在体外实验中会增强这些细胞向SDF-1的迁移,在体内实验中则能增加骨髓移植的疗效,这些都很可能归功于细胞存活和归巢的增加。但是,老年人骨髓中所含有的干细胞祖细胞在治疗和修复功能方面有所缺陷。无论动物或人类,其EPC水平和功能均因衰老、糖尿病、缺血性心脏病和动脉粥样硬化而受到不利影响,血管生成功能也因衰老而受损,然而这些因素在分子层面的影响尚不明确。实验目的：研究骨髓细胞CXCR4表达是否因衰老而有所缺陷,从而使骨髓中CXCR4+祖细胞的数量减少,并进一步导致骨髓细胞向缺血组织的归巢能力及促血管新生能力的受损。并对可能的机制进行探讨。实验方法：应用流式细胞术检测全骨髓细胞表面及细胞内CXCR4的表达,分别根据骨髓细胞表面抗原的不同和细胞大小的不同对其进行分群,并对不同细胞亚群表面CXCR4的表达进行检测；应用real-time PCR技术检测CXCR4的mRNA表达；从基因转录、蛋白表达及蛋白内移能力等不同水平、不同细胞亚群研究衰老对CXCR4表面表达的影响。通过1mM氯化钙作为细胞外刺激因子短期处理骨髓细胞,研究BMCold CXCR4对细胞外刺激因子的反应是否与BMCyoung有所区别。通过测定骨髓细胞与氯化钙混合后细胞内钙离子浓度的升高对钙离子内流进行分析,研究衰老对细胞钙内流的影响,从而解释衰老细胞CXCR4对钙刺激不敏感的原因。应用Western Blot技术检测SDF-1/CXCR4的下游信号通路AKT和ERK1／2的磷酸化水平,以研究衰老对SDF-1/CXCR4的下游信号通路的影响。应用改进的Boyden小室法检测BMC的迁移能力；又以细胞粘附试验检测SDF-1介导的BMC向HUVEC单细胞层的粘附能力,以垂直胶原凝胶侵袭试验检测骨髓细胞穿越内皮细胞的迁移能力,从而在体外模拟CXCR4+的BMCs在体内先被SDF-1捕获,紧密粘附于血管内皮细胞表面,再完成跨内皮细胞迁移至受损组织的过程；建立小鼠后肢缺血模型,经尾静脉注射输入骨髓细胞,再分别通过免疫组化和激光多普勒灌注成像术(LDPI)检测在缺血组织内不同骨髓细胞的增殖、归巢和血管生成,从而研究衰老引起CXCR4表达受损是否进一步影响细胞的功能。通过骨髓细胞移植对接受致死量的放射线照射后的骨髓重建研究年轻和老年微环境对骨髓细胞CXCR4表达的影响。实验结果：小鼠后肢缺血模型中老年小鼠在2和3周时缺血肢体灌注的恢复明显低于年轻小鼠,并且SDF-1治疗也没有提高灌注量。相较于年轻小鼠,老年小鼠中向缺血部位归巢的祖细胞更少,且对SDF-1处理更不敏感,导致其血管新生反应受损。通过流式细胞术分析发现,BMCold表面表达CXCR4的细胞数量更少,且每一个细胞表面的CXCR4表达更少。钙离子处理可以增强BMCyoung中CXCR4的表达,但老年小鼠细胞表面CXCR4的表达却对钙离子的刺激并不敏感。而其原因可能是BMCold的钙内流缺陷使CXCR4基因转录水平降低,从而导致钙诱导CXCR4表达反应受损。在不同的BMC亚群中,除了BMCyoung中的Lin-细胞明显多于BMCold外,其余亚群细胞量在BMCyoung和BMCold间没有显著差异。Lin-Sca-1+亚群表面CXCR4的表达,年轻小鼠显著高于老年小鼠,而两者之间CD34+Flk1+亚群、Gr-1+CD11b+亚组的CXCR4表达没有显著差异。而根据细胞大小对BMC进行分组后,“小细胞”亚群总是具有更高的表面CXCR4表达量。钙处理所致亚群CXCR4表达的增长在BMCold亚群中也明显要少很多。SDF-1与其受体CXCR4的结合会使CXCR4向细胞内移位。BMCold CXCR4的这种内移反应正常,其与BMCyoung的主要区别在于基础CXCR4表达的降低及其对钙处理应答的缺乏。在SDF-1/CXCR4的下游信号通路的反应方面,BMCyoung对钙刺激提高细胞内通过SDF-1/CXCR4相互作用的信号通路活性更为敏感,而BMCold中SDF-1／Akt通路受到了抑制。另一方面,ERK1／2信号转导通路则未发生改变。在体外试验中,BMCold的粘附能力、向SDF-1的迁移能力和跨内皮细胞的侵袭能力均受损。而通过相互的骨髓细胞移植发现,体内微环境的改变并不能改变骨髓细胞CXCR4表达和对SDF-1的迁移能力。在体内试验中,由SDF-1介导的BMCold向缺血肌肉归巢的能力受损,血管生成能力受损,且不因钙处理而提高。实验结论：相较于BMCyoung,BMCold：(1)表面CXCR4表达及细胞内CXCR4的浓度均降低了；(2)对细胞外辅助剂如钙对细胞表面CXCR4表达的刺激的敏感性降低；(3)明显降低的钙内流和钙刺激不能使CXCR4 mRNA表达量增加；(4)SDF-1介导的CXCR4内移对钙刺激的反应极大地减少；(5)相应于SDF-1处理的Akt磷酸化几乎完全受抑。而衰老对骨髓细胞CXCR4表达的这些影响也转化成了以下的功能障碍：(1)骨髓细胞未能向SDF-1浓度梯度迁移；(2)钙不能加强这方面的迁移能力；(3)骨髓细胞在体内缺血后肢中未能响应SDF-1的化学诱导作用向缺血组织归巢；(4)尽管向缺血肌肉中注射了SDF-1,血管新生能力还是明显受损。总之,BMCold的CXCR4/SDF-1轴缺陷极大地导致了衰老相关的血管生成反应的丧失。更多还原

【Abstract】 Background:Bone marrow derived cells (BMCs) contribute to angiogenesis by differentiating into endothelial cells (ECs), smooth muscle cells, and pericytes, and by delivering angiogenic proteins that support vessel growth and maturation. The chemotactic cytokine stromal cell derived factor-1 (SDF-1) plays a key role in mobilizing cells from the bone marrow and promoting homing of the cells to target tissues. SDF-1 acts by binding and activating the cell surface receptor CXCR4 on target cells. CXCR4 is essential for homing and maintenance of hematopoietic stem cells (HSCs) in distinct stromal cell niches within the marrow. CXCR4 surface expression varies between cell types. Most mesenchymal stem cells (MSCs) do not express CXCR4 but endothelial progenitor cells (EPCs) express relative high surface CXCR4. Only 5% of freshly isolated CD34+ cells from peripheral blood express CXCR4 but the expression can be increased> 10-fold by exposing cells to appropriate culture in vitro. Our previous study shows that calcium treatment can increase the surface expression of CXCR4 on BMCs.Accumulating evidence indicates that the level of CXCR4 surface expression on BMCs determines the efficiency of homing and the subsequent angiogenic response within target tissues. Over-expression of CXCR4 in MSCs or CD34+ cells enhances the migration of these cells towards SDF-1 in vitro and increases the efficiency of bone marrow transplant in vivo, probably due to increased cell survival and homing. Infusions of autologous progenitor cells have been shown to promote revascularization of muscle in animal models of peripheral ischemia but this has not been translated well into clinical applications. One reason may be that bone marrow of aged or diseased subjects contain stem cell progenitors that are defective for therapy and repair. EPC level and function are reported to be adversely impacted by aging, diabetes, ischemic heart disease and atherosclerosis in animals and humans. Angiogenesis is also impaired in aging. The molecular aspects of these defects are not known.We hypothesize that defective CXCR4 expression develops during aging, decreasing the number of CXCR4-positive progenitor cells of both the bone marrow and the circulation. This results in reduced progenitor cell presentation to the ischemic tissue and reduces angiogenesis.Objectives:The objectives of this study is to determine that aging defects bone marrow cells both on the decreased population of CXCR4+ progenitor cells and on the impaired homing capacity and angiogenetic ability and to explore possible mechanisms.Methods:To evaluate the aging impact on CXCR4 expression from gene transcription to protein synthesis and internalization, surface and intracellular CXCR4 expression on BMCs from old or young mice were analyzed with FCM. And multi-color FCM was used to examine the CXCR4 expression on the surface of different subpopulations of BMCs. The mRNA of CXCR4 was detected by real-time PCR and normalized to hypoxanthine phosphoribosyl transferase 1(HPRT1) mRNA levels. To compare the inducibility of CXCR4 expression in response to environmental change between BMCold and BMCyoung,1 mM CaCl2 was used as an extracellular stimuli to treat BMCs for 4 hours. To examine how BMCold are defect on calcium-induced CXCR4 surface expression, calcium influx was analyzed by measuring the increase of intracellular calcium after BMCs were mixed with CaCl2. Western blot analysis was performed to quantify the SDF-1-mediated phosphorylation of Akt kinase and Extracellular signal-regulated kinases 1 and 2 (ERK1/2). To imitate the in vivo process of BMCs homing from the CXCR4+ BMCs capture by SDF-1, close adhesion to vascular endothelial cell, to the trans-endothelial cell migration to injured tissue in vitro, cell-cell adhesion assays were used to detect the SDF-1 mediated adhesion of BMCs to the HUVEC monolayer, boyden chamber assays and vertical Collagen Gel invasion assay were used respectively to quantify the migration and trans-endothelial migration of BMCs from young and old mice in response to a gradient of SDF-1. We also established a mouse hindlimb ischemia model, injected BMCs via the tail vein, then detect proliferation, homing, and angiogenesis of the BMCs in ischemic tissue using immunohistochemistry and laser Doppler perfusion imaging (LDPI), to study whether aging impaired CXCR4 expression on BMCs would impact the cell functions. To study the effect of the micro-environment on BMC CXCR4 expression, BMCs of young mice were replaced with BMCold and vice versa after receiving lethal irradiation. After a 2-month reconstitution, mice were sacrificed to recover BMCs from femurs and tibias. The BMC surface CXCR4 expression before infusion and after recovery was determined by flow cytometry.Results:BMCold have less number of cells expressing CXCR4 and less CXCR4 on the surface of each cell as compared to BMCyoung. CXCR4 expression on BMCyoung could be enhanced by calcium, but CXCR4 surface expression in cells from old mice increased significantly less then BMCyoung. And it partly because of the defective calcium influx in BMCold which reduced the CXCR4 gene transcription, consequently lead to impaired responses to calcium-induced CXCR4 surface expression. There were no significant differences in the different BMC subpopulations, except the lineage negative (Lin-) subset, was observed between BMCyoung and BMCold. BMCs from young mice have significantly more Lin-cells than old mice. Surface CXCR4 expression in Lin-Sca-1+ subpopulation from young mice is significantly higher than that of old mice, while CXCR4 expression on CD34+Flkl+ subset and Gr-1+CD11b+ subset was not significantly different between BMCs from old and young mice. When the subpopulations of BMCs were grouped according to cell size, the "small cell" subpopulations from both young and old mice have higher CXCR4 surface expression than the other cells with bigger size. In cells from young mice, CXCR4 expression in all subpopulations was significantly increased by calcium treatment. However, such increase was significant less in the subpopulations of BMCs from old mice.SDF-1 binding causes internalization of the CXCR4 receptor. In BMCs from old mice, this CXCR4 receptor internalization responds normally to SDF-1, and the main difference is lower basal CXCR4 expression and absence of a response to calcium. Furthermore, BMCyoung are more sensitive to calcium stimulation to enhance intracellular signaling through SDF-1/CXCR4 interaction, suggesting that the SDF-1/Akt pathway is repressed in BMCs from old mice. On the other hand, the ERK1/2 signaling pathway is not altered in BMCold. BMCold showed weaker adhesion, lower mobility and lower trans-endothelial migration in vitro, and this was not enhanced by calcium pretreatment. After reciprocal bone marrow transplants, we found the changes due to transplantation were not significantly different in either case. Similarly transplantation did not change basal or calcium-stimulated mobility toward SDF-1. And also, aged BMCs have the impaired SDF-1-mediated homing capacity and angiogenic response to ischemia in vivo, with the defective calcium response. Conclusions:Compared to BMCs from young mice, BMCs from aged mice had:(1) reduced both surface CXCR4 expression and intracellular CXCR4 concentration. (2) diminished stimulation of CXCR4 surface expression by extracellular adjuvant like calcium; (3) significant lower Ca-influx and loss of calcium-stimulated CXCR4 mRNA accumulation; (4) dramatic reduction of SDF-1-mediated CXCR4 internalization both in the presence and absence of calcium stimulation; (5) Nearly complete abrogation of Akt phosphorylation in response to SDF-1 treatment.These effects of aging on CXCR4 expression in BMCs translated into the following functional impairments:(1) BMCs failed to migrate in an SDF-1 gradient; (2) Calcium failed to enhance this migration; (3) BMCs failed to home in vivo to the ischemic tissue in response to SDF-1 chemoattraction in the ischemic hindlimb; (4) Angiogenesis was markedly impaired despite injected SDF-1 in the ischemic muscle.更多还原