Klotho抑制EPCs衰老促进老龄小鼠损伤动脉再内皮化的研究
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摘要
1.背景与目的:
自从1904年德国莱比锡病理学家Marchand首次提出动脉粥样硬化(Atherosclerosis, AS)一词以来,历经一个多世纪,人们对AS的认识已逐步深入。然而,目前动脉粥样硬化发病率和致死率仍然很高,AS导致的心脑血管疾病仍是当今人类死亡的首位原因之一。AS是一种退行性和增生性病变,与衰老有着密切的关系。而我国拥有世界上最庞大的老年人群,AS的迁延性和难愈性不仅严重危害了老年患者的健康,还导致医学资源连续消耗。研究衰老在AS发生发展中的机制对于预防和治疗AS有着重要的意义,有较大的学术价值和社会效应。
AS的发病机制十分复杂,目前认为,血管内皮结构和功能损伤可能是动脉粥样硬化的病理生理基础,内皮损伤己被证明是AS最早期的改变,而损伤血管有效地再内皮化可以维持血管内皮细胞单层的完整性,预防AS的发生。目前促进损伤血管修复的措施主要有以下几种:戒烟等生活方式的改善;应用药物如他汀类药物、血管紧张素转换酶抑制剂、沙坦类药物、胰岛素增敏剂等抗氧化药物保护血管内皮细胞功能;血管内皮生长因子抑制内皮细胞凋亡;血管内皮祖细胞(Endothelial Progenitor Cells,EPCs)移植。
以EPCs为基础的细胞疗法修复损伤血管已成为大家关注的方向。EPCs由造血干细胞分化而来,是血管内皮细胞的前体细胞,表达干细胞及内皮细胞相关抗原,主要来源于骨髓、胎肝、脐血和脾脏。传统观点认为血管损伤后修复主要依赖于邻近成熟内皮细胞的迁移和生长增殖。而新近的研究表明骨髓和外周血中EPCs是血管内皮损伤修复的最重要的机制之一。在血管内皮损伤后,EPCs从骨髓中动员并归巢于损伤血管内皮局部,分化为成熟内皮细胞,加快损伤血管再内皮化,抑制病理性新生内膜的形成,在血管内皮损伤修复中起着重要的作用。
EPCs的数量和活性受到多种生理和病理因素的影响,包括年龄、高血压、高糖、氧化LDL、慢性肾功能衰竭等等。研究表明单纯的年龄增加会使大鼠EPC动员减少,增殖、黏附和迁移能力降低,并且发现EPCs参与损伤血管再内皮化的过程具有年龄依赖性,即年轻供体EPCs移植促进血管内皮损伤再内皮化的作用更明显,老龄供体EPCs该效应不明显。衰老导致EPCs的功能受损、数量减少,可能是老年患者血管损伤后再内皮化能力下降的原因。如何有效抑制EPCs衰老,促进老年患者损伤血管有效再内皮化成为目前急需解决的问题。
Klotho ( Kl)基因是Kuro等在1997年发现的与衰老相关的新基因。Kl基因在小鼠中的表达缺失导致类似于人类衰老的各种表型变化,例如动脉内膜增厚、中膜钙化、动脉硬化和骨质疏松、肺气肿、糖和能量代谢异常等。Kl基因主要在肾远曲小管上皮细胞上表达。
目前,Kl预防衰老的机制尚不十分清楚,有研究证实在Kl基因变异的小鼠,随着机体的逐渐衰老,骨髓、外周血EPCs数量会明显降低,提示Kl基因表达水平与EPCs关系密切。本课题拟研究小鼠增龄对Kl表达的影响,探讨Kl对EPCs数量、生物学功能及其参与血管损伤后再内皮化的影响,初窥Kl抑制EPCs衰老的可能的信号通路。
2.方法:
2.1小鼠增龄与Kl表达水平及EPCs功能的关系
2.1.1检测不同月龄小鼠EPCs数量和功能雄性C57BL/6小鼠,按照月龄分为2月龄、12月龄、20月龄三组。采用内皮细胞选择性培养基培养和诱导分化密度梯度离心法获的小鼠骨髓源性单个核细胞,以获得小鼠骨髓源性EPCs,并分别通过acLDL-DiI和FITC-UEA-I荧光双染鉴定、流式细胞仪检测细胞表面干细胞抗原及内皮细胞特异性抗原鉴定,和用免疫组化法检测细胞表面内皮细胞特异性抗原来鉴定。分别采用acLDL-DiI和FITC-UEA-I荧光双阳性细胞计数、MTT分析、改良的Boyden小室、黏附细胞计数来检测不同年龄EPCs数量、增殖、迁移和黏附能力。
2.1.2检测不同月龄小鼠肾脏Kl mRNA和外周血Kl蛋白水平采用RT-PCR法检测不同月龄小鼠肾脏Kl mRNA的表达,用Western-blot法检测不同月龄小鼠外周血Kl蛋白水平,分析不同月龄小鼠Kl水平与EPCs数量及功能的相关性。
2.2 Kl对老龄小鼠EPCs部分生物学功能的影响
2.2.1观察Kl对体外培养的老龄小鼠EPCs部分生物学功能的影响20月龄小鼠骨髓源性EPCs与不同浓度的Kl蛋白孵育,分别采用acLDL-DiI和FITC-UEA-I荧光双阳性细胞计数、MTT分析和改良的Boyden小室法来检测其数量、增殖、迁移能力的变化。
2.2.2初步探讨Kl抑制EPCs衰老的可能的信号通路采用Western-blot法检测不同浓度Kl孵育老龄小鼠p53、p21的表达和Akt的磷酸化水平,分析Kl影响EPCs衰老的机制。
2.3 Kl促进老龄小鼠损伤血管再内皮化的研究主要观察Kl对老龄小鼠血管损伤后EPCs动员及修复损伤内皮的影响。采用非显微外科法建立小鼠(20月龄)颈动脉损伤模型。在小鼠颈动脉损伤后各时点(术前、术后1天、术后3天、术后7天和术后14天)获取外周血,采用流式细胞仪检测外周血EPCs数量。在小鼠颈动脉损伤后第3天,采用流式细胞仪检测Kl处理组、Kl抗体处理组外周血EPCs的数量;14天后通过Evans blue染色检测损伤动脉再内皮化情况,通过HE染色检测损伤动脉新生内膜增生情况。
3.结果
第一部分:培养7天的小鼠骨髓单个核细胞呈梭形或纺锤状,与内皮细胞形态相似,绝大多数细胞可摄取acLDL-DiI和与UEA-I结合,流式细胞仪检测显示这些细胞既有干细胞抗原Sca-1的高表达,又有内皮特异性标记物VEGFR-2的高表达,免疫组化检测显示这些细胞还高表达内皮特异性标记物vWF。荧光双染计数2M、12M、20M月龄小鼠EPCs数量,发现随着月龄的增加,EPCs数量明显减少。采用MTT法检测不同月龄小鼠骨髓源性EPCs增殖能力,用Boyden迁移小室法测定其迁移能力,用粘附细胞计数测量其粘附能力,结果发现,随着月龄的增加,小鼠骨髓源性EPCs的粘附功能、迁移功能、增殖功能明显下降。分别采用RT-PCR和Western-blot法检测不同月龄小鼠肾脏Kl mRNA和外周血Kl蛋白水平,结果显示Kl mRNA和外周血Kl蛋白水平随着月龄的增加而显著性降低。同时分析显示Kl水平与EPCs的数量和粘附功能、迁移功能和增殖功能具有相关性。
第二部分:体外细胞培养实验中,荧光双染计数显示,Kl刺激浓度依赖性增加老龄小鼠骨髓单个核细胞分化成EPCs的数量;MTT分析显示,Kl刺激浓度依赖性增强EPCs的增殖能力;改良的Boyden小室分析显示,Kl浓度依赖性增加迁移的EPCs数量。
采用Western-blot法测定不同浓度的Kl蛋白处理后的EPCs细胞的p53/p21表达水平,发现p53/p21呈Kl蛋白浓度依赖性下降;以P-Akt与Akt的比值来代表EPCs Akt磷酸化的水平,发现Akt的磷酸化水平随着Kl蛋白浓度的增加而逐渐下降。第三部分:非显微外科手术方法损伤小鼠颈动脉后,取损伤动脉Evans blue染色观察,损伤动脉内皮完全剥脱。小鼠颈动脉损伤后,采用流式细胞仪检测外周血EPCs数量显示,小鼠颈动脉损伤后1天、3天和7天,外周血EPCs数量明显高于假手术组,外周血EPCs数量在动脉损伤后3天达到高峰。采集术后第3天Kl处理组和Kl抗体处理组小鼠的外周血,检测其EPCs数量,结果显示:与颈动脉损伤组相比较,Kl处理组的循环Kl明显升高,而Kl抗体处理组无明显上升。术后第14天,损伤血管Evans blue染色显示,在Kl处理组,损伤血管再内皮化的面积明显高于对照颈动脉损伤组,Kl抗体处理组的再内皮化的面积与颈动脉损伤组相比无明显差异。HE染色后应用Image ProPlus软件用来测量新生内膜、中膜面积,利用内膜面积和中膜面积的比值来评价内膜新生,结果显示,Kl处理组内膜增生程度较对照组及Kl抗体处理组明显降低。
4.结论
4.1不同月龄小鼠肾脏Kl mRNA表达、外周血Kl蛋白水平与EPCs的数量、迁移、增殖、黏附功能相关,且随小鼠月龄的增加下降;
4.2在本研究浓度范围内,Kl蛋白可能参与体外培养的老龄小鼠骨髓单个核细胞分化,促进骨髓单个核细胞分化成为EPCs,呈浓度依赖性提高其迁移能力和增殖能力;
4.3在本研究浓度范围内,Kl蛋白可浓度依赖性降低体外培养的EPCs的p53/p21的表达以及Akt的磷酸化水平,可能部分通过下调Akt活性,降低p53/p21水平,抑制细胞衰老;
4.4在本研究浓度及时间范围内,Kl蛋白可以升高老龄小鼠动脉损伤后EPCs的循环数量,增强EPCs的动员,促进老年小鼠损伤动脉再内皮化,降低新生内膜增生。
1. Background and Objective
Since Atherosclerosis was first presented by Marchand, a pathologist of Leipzig of Germany, more than one century before, it has been understood gradually. However, at present the incidence and lethality rate of atherosclerosis are still high, and the heart cerebrovascular disease caused by atherosclerosis is still one of the first place reasons of death now. Atherosclerosis is one kind of degenerative and proliferative lesion, and has close relationship with aging. Because of our country has the hugest senile population in the world, the persistence and chronicity of atherosclerosis not only severely impair aging patients health, but also cause the medicine resources to be consumed continuously. Studying on aging mechanism in the development of atherosclerosis has vital significance regarding to prevent and treat atherosclerosis, and also has great academic value and social profile as well.
The pathogenesis of atherosclerosis is very complex, mainly centers on four theories: fatty infiltration theory, blood platelet accumulation and thrombus form theory, smooth muscle cell clone theory and damage response theory. And damage response theory is supported by majority of scholar. This theory believes that the vascular endothelial structure and function damage are the pathologic and physiologic foundation of atherosclerosis. And it has been prooved that endothelial damage is the early change of atherosclerosis, and damaged vascular endothelials’effective re-endothelialization can maintain the integrity of blood endothelial cell monolayer, and prevent the occurrence of atherosclerosis. At present, pathways to repair damaged vascular endothelial have the following several kinds: life style improvement, for example no-smoking and so on; using medicine like statins,angiotensin converting enzyme inhibitor ect to protect vascular endothelial cell function; using vascular endothelial growth factor to suppress endothelial cell apoptosis; endothelial progenitor cells transplantation.
Cytotherapy based on EPCs has become one of the newest research direction of repairing vascular endothelial damage. EPCs are produced by hematopoietic stem cell differentiation, are the precursor cells of vascular endothelial cells, and mainly come from bone marrow, embryo liver, umbilical cord blood and spleen. Conventional viewpoint states that the reparation of vascular endothelial damage mainly relies on the migration and growth proliferation of neighbor nature endothelial cell surrounding the damage. But recent researches indicate that in bone marrow and peripheral blood EPCs is one of most important mechanisms of vascular endothelial damage reparation. After vascular endothelial damage, EPCs mobilized from bone marrow homing to damaged vascular endothelial, and differentiate to mature endothelial cell, speed up the re-endothelialization of damaged area, suppress the formation of pathological new intimae, play very important role in vascular endothelial damage reparation.
There are many kinds of factors can influence the quantity and activity of EPCs, including age, hypertension, high glucose, oxidized LDL, chronic renal failure and so on. The research indicates that pure age increasing may reduce EPCs mobilization, proliferation and adhesion and migration ability. And also discovered that the process which EPCs participating in the repairation of damaged area has age dependence, EPCs transplantation of young donors can promote the re-endothelialization, but the EPC function of aging ones are not obvious. Therefore aging causes EPCs function impairment and quantity decrease, possibly is the reason of senile patients re-endothelialization ability. So how to suppress EPCs aging effectively and promote re-endothelialization of aging patients’damaged blood vessel comes to be urgent.
Klotho discovered by Kuro ect in 1997 is one kind of new genes has relationship with aging. Kl gene expression deletion in mice can cause various morbodity changes similar to human, for example: intimae thickening, medial calcification, arteriosclerosis, osteoporosis, emphysema, abnormality of glucose and energy metabolism, ect. Kl is mainly expressed by epithelial cells of distal convoluted tubule of the kidney.
At present, the mechanism of Kl preventing aging is still unclear, some researches confirmed that in Kl gene mutation mice, the EPCs quantity of bone marrow and peripheral blood would reduce obviously with body aging gradually, prompting Kl expression and EPCs related closely. We investigate the study to research aging influence on EPCs function and Kl expression, observe Kl influence on EPCs quantity and biological function and participation in re-endothelialization of damaged vascular endothelial, probe into the possibility of suppressing EPCs aging by via p53/ p21signaling pathway.
2. Method
2.1 Relations of age and mice Kl expression and EPCs function
2.1.1 Detecting EPCs quantity and function in different month mice Male C57BL/6 mice were divided into three groups according to month, two-month old, twelve-month old, and twenty-month old. Using endothelial cells selective medium culture and differentiation and density gradient centrifugation to isolate bone marrow derived single nuclear cell of mice, and acquire bone marrow derived EPCs, and using acLDL-DiI and FITC-UEA-I direct fluorescent staining under an inverted fluorescent microscope and flow cytometry to detect stem cell surface specific antigen and endothelial cell specific antigen, and using immunohistochemical method to identify cell surface endothelial cell antigen expression. Using acLDL-DiI and FITC-UEA-I direct fluorescent staining under an inverted fluorescent microscope and MTT analysis and improved boyden chamber and adhesion cell counting method to detect EPCs quantity, proliferation, migration, and adhesion ability .
2.1.2 Detecting renal Kl mRNA and peripheral blood Kl protein level in different month mice
Using RT-PCR to detect renal Kl mRNA, using Western-blot to detect peripheral blood Kl protein level of different age mice, and analyzing relevance between Kl level and EPCs quantity and ability in different month mice.
2.2 The influence of Kl on EPCs function of aging mice
2.2.1 Observing Kl influence on biological function of EPCs in aging mice in vitro Incubating bone marrow derived EPCs of twenty-month old mice with different concentrations Kl, and using sing acLDL-DiI and FITC-UEA-I direct fluorescent staining under an inverted fluorescent microscope and MTT analysis and improved boyden chamber method to detect EPCs quantity, proliferation and migration activity.
2.2.2 Try probe into the possible mechanism of suppressing EPCs aging by Kl protein Using Western-blot method to detect p53,p21and p-Akt expression, and analyzing the mechanism of Kl affecting EPCs aging.
2.3 In vivo study of Kl promoting senile mice re-endothelialization of damaged vascular endothelial.
Using non-microsurgery method to establish mice(twenty-month old) carotid artery injury model, and using pathology method to examine mice carotid artery injury model whether to establish successfully. At the different time after mice carotid artery injury(before operation,and the 1st day,3rd day, 7th day and 14th day after operation ) obtaining peripheral blood, using flow cytometry to detect peripheral blood EPCs quantity. The 3rd day after mice carotid artery injury, using flow cytometry to detect peripheral blood EPCs of Kl treated group and Kl antibody treated group .Fourteen days later using Evans blue method to detect re-endothelialization of damaged vascular endothelial,and using HE staining to detect the degree of new-born tunica intimae.
3. Results
Part one:
After 7 days cultured in endothelial cell selection medium,bone marrow mononuclear cells turned into spindle-shaped,endothelial cell-like cells. These cells were characterized further by demonstrating the expression of the mouse stem-cell marker Sca-1 as well as the endothelial cell lineage antigen VEGFR-2 by flow cytometry.Endothelial cell lineage antigen vWF was also expressed by most of these cells in immunohistochemical analysis. Fluorescence double staining counting 2M,12M, 20M mice, discovering along with age growth, EPCs quantity obviously reduced. Using MTT method to detect mice bone marrow derived EPCs proliferative ability, using Boyden chamber method to detect EPCs migrating ability, using adherent cells counting to detect EPCs adhesion ability, the results showed that along with age increasing, mice bone marrow derived EPCs adhesion ability, migrating ability and proliferating function obviously dropped. Using RT-PCR and Western-blot to detect renal Kl mRNA and peripheral blood Kl protein level separately, the results showed that along with month increasing, Kl mRNA, peripheral blood Kl protein level obviously dropped. Simultaneously analyzing demonstrated that the Kl level and EPCs quantity and adhesion ability, migrating ability and proliferating function has closely relevance.
Part two:
In culturing experiment in Vitro, Fluorescence double staining counting demonstrating that Kl protein increases aging mice bone marrow mononuclear cell differentiating has concentration dependence . MTT analyzing demonstrating that Kl protein strengthenes proliferating function of EPCs; and Improved boyden chamber analyzing demonstrating Kl protein strengthened migrated EPCs quantity by concentration dependence. Western-blot method demonstrating Kl protein decreased p53 ,p21 and p-Akt expression
Part three:
Obtaining complete removal of endothelium with a non-microscopical surgery method, and detecting it with HE staining.At different time after mice carotid artery injury(before operation,and the 1st day,3rd day, 7th day and 14th day after operation) obtaining peripheral blood , EPCs quantity detected by flow cytometry demonstrating that at different time after mice carotid artery injury: the 1st day,3rd day and 7th day after operation, peripheral blood EPCs quantity obviously higher than sham-operation group, and 3rd achieved the peak. Gathering 3rd peripheral blood of Kl treated group and Kl antibody treaded group ,detecting EPCs quantity, discovering that comparing with carotid artery injury group, Kl treated group peripheral blood amount og EPCs elevated obviously, but Kl antibody treaded group had no significance difference. 14th day injured blood vessel dying discovering that in Kl treated group the re-endothelialization area of damaged vascular endothelial was higher than control group, but Kl antibody treaded group had no obviouse significance difference.
4. Conclusion
4.1 Mice renal Kl mRNA and peripheral blood Kl ptotein level and age assuming inverse correlation;Mice EPCs quantity, proliferation, migration, adhesion ability and age assuming inverse correlation.
4.2 Kl protein promoting bone marrow stem cells of aging mice cultured in vitro to differentiate into EPC. And Kl protein strengthening EPCs proliferating function and migrating ability by concentration dependence.
4.3 Kl inhibiting EPCs p53, p21 and p-Akt expression by concentration dependence.
4.4 Kl promoting EPCs mobilization after aging mice carotid artery injury and re-endothelialization of mice carotid artery injuried. decreasing the proliferation of new-born tunica intimae;
自从1904年德国莱比锡病理学家Marchand首次提出动脉粥样硬化(Atherosclerosis, AS)一词以来,历经一个多世纪,人们对AS的认识已逐步深入。然而,目前动脉粥样硬化发病率和致死率仍然很高,AS导致的心脑血管疾病仍是当今人类死亡的首位原因之一。AS是一种退行性和增生性病变,与衰老有着密切的关系。而我国拥有世界上最庞大的老年人群,AS的迁延性和难愈性不仅严重危害了老年患者的健康,还导致医学资源连续消耗。研究衰老在AS发生发展中的机制对于预防和治疗AS有着重要的意义,有较大的学术价值和社会效应。
AS的发病机制十分复杂,目前认为,血管内皮结构和功能损伤可能是动脉粥样硬化的病理生理基础,内皮损伤己被证明是AS最早期的改变,而损伤血管有效地再内皮化可以维持血管内皮细胞单层的完整性,预防AS的发生。目前促进损伤血管修复的措施主要有以下几种:戒烟等生活方式的改善;应用药物如他汀类药物、血管紧张素转换酶抑制剂、沙坦类药物、胰岛素增敏剂等抗氧化药物保护血管内皮细胞功能;血管内皮生长因子抑制内皮细胞凋亡;血管内皮祖细胞(Endothelial Progenitor Cells,EPCs)移植。
以EPCs为基础的细胞疗法修复损伤血管已成为大家关注的方向。EPCs由造血干细胞分化而来,是血管内皮细胞的前体细胞,表达干细胞及内皮细胞相关抗原,主要来源于骨髓、胎肝、脐血和脾脏。传统观点认为血管损伤后修复主要依赖于邻近成熟内皮细胞的迁移和生长增殖。而新近的研究表明骨髓和外周血中EPCs是血管内皮损伤修复的最重要的机制之一。在血管内皮损伤后,EPCs从骨髓中动员并归巢于损伤血管内皮局部,分化为成熟内皮细胞,加快损伤血管再内皮化,抑制病理性新生内膜的形成,在血管内皮损伤修复中起着重要的作用。
EPCs的数量和活性受到多种生理和病理因素的影响,包括年龄、高血压、高糖、氧化LDL、慢性肾功能衰竭等等。研究表明单纯的年龄增加会使大鼠EPC动员减少,增殖、黏附和迁移能力降低,并且发现EPCs参与损伤血管再内皮化的过程具有年龄依赖性,即年轻供体EPCs移植促进血管内皮损伤再内皮化的作用更明显,老龄供体EPCs该效应不明显。衰老导致EPCs的功能受损、数量减少,可能是老年患者血管损伤后再内皮化能力下降的原因。如何有效抑制EPCs衰老,促进老年患者损伤血管有效再内皮化成为目前急需解决的问题。
Klotho ( Kl)基因是Kuro等在1997年发现的与衰老相关的新基因。Kl基因在小鼠中的表达缺失导致类似于人类衰老的各种表型变化,例如动脉内膜增厚、中膜钙化、动脉硬化和骨质疏松、肺气肿、糖和能量代谢异常等。Kl基因主要在肾远曲小管上皮细胞上表达。
目前,Kl预防衰老的机制尚不十分清楚,有研究证实在Kl基因变异的小鼠,随着机体的逐渐衰老,骨髓、外周血EPCs数量会明显降低,提示Kl基因表达水平与EPCs关系密切。本课题拟研究小鼠增龄对Kl表达的影响,探讨Kl对EPCs数量、生物学功能及其参与血管损伤后再内皮化的影响,初窥Kl抑制EPCs衰老的可能的信号通路。
2.方法:
2.1小鼠增龄与Kl表达水平及EPCs功能的关系
2.1.1检测不同月龄小鼠EPCs数量和功能雄性C57BL/6小鼠,按照月龄分为2月龄、12月龄、20月龄三组。采用内皮细胞选择性培养基培养和诱导分化密度梯度离心法获的小鼠骨髓源性单个核细胞,以获得小鼠骨髓源性EPCs,并分别通过acLDL-DiI和FITC-UEA-I荧光双染鉴定、流式细胞仪检测细胞表面干细胞抗原及内皮细胞特异性抗原鉴定,和用免疫组化法检测细胞表面内皮细胞特异性抗原来鉴定。分别采用acLDL-DiI和FITC-UEA-I荧光双阳性细胞计数、MTT分析、改良的Boyden小室、黏附细胞计数来检测不同年龄EPCs数量、增殖、迁移和黏附能力。
2.1.2检测不同月龄小鼠肾脏Kl mRNA和外周血Kl蛋白水平采用RT-PCR法检测不同月龄小鼠肾脏Kl mRNA的表达,用Western-blot法检测不同月龄小鼠外周血Kl蛋白水平,分析不同月龄小鼠Kl水平与EPCs数量及功能的相关性。
2.2 Kl对老龄小鼠EPCs部分生物学功能的影响
2.2.1观察Kl对体外培养的老龄小鼠EPCs部分生物学功能的影响20月龄小鼠骨髓源性EPCs与不同浓度的Kl蛋白孵育,分别采用acLDL-DiI和FITC-UEA-I荧光双阳性细胞计数、MTT分析和改良的Boyden小室法来检测其数量、增殖、迁移能力的变化。
2.2.2初步探讨Kl抑制EPCs衰老的可能的信号通路采用Western-blot法检测不同浓度Kl孵育老龄小鼠p53、p21的表达和Akt的磷酸化水平,分析Kl影响EPCs衰老的机制。
2.3 Kl促进老龄小鼠损伤血管再内皮化的研究主要观察Kl对老龄小鼠血管损伤后EPCs动员及修复损伤内皮的影响。采用非显微外科法建立小鼠(20月龄)颈动脉损伤模型。在小鼠颈动脉损伤后各时点(术前、术后1天、术后3天、术后7天和术后14天)获取外周血,采用流式细胞仪检测外周血EPCs数量。在小鼠颈动脉损伤后第3天,采用流式细胞仪检测Kl处理组、Kl抗体处理组外周血EPCs的数量;14天后通过Evans blue染色检测损伤动脉再内皮化情况,通过HE染色检测损伤动脉新生内膜增生情况。
3.结果
第一部分:培养7天的小鼠骨髓单个核细胞呈梭形或纺锤状,与内皮细胞形态相似,绝大多数细胞可摄取acLDL-DiI和与UEA-I结合,流式细胞仪检测显示这些细胞既有干细胞抗原Sca-1的高表达,又有内皮特异性标记物VEGFR-2的高表达,免疫组化检测显示这些细胞还高表达内皮特异性标记物vWF。荧光双染计数2M、12M、20M月龄小鼠EPCs数量,发现随着月龄的增加,EPCs数量明显减少。采用MTT法检测不同月龄小鼠骨髓源性EPCs增殖能力,用Boyden迁移小室法测定其迁移能力,用粘附细胞计数测量其粘附能力,结果发现,随着月龄的增加,小鼠骨髓源性EPCs的粘附功能、迁移功能、增殖功能明显下降。分别采用RT-PCR和Western-blot法检测不同月龄小鼠肾脏Kl mRNA和外周血Kl蛋白水平,结果显示Kl mRNA和外周血Kl蛋白水平随着月龄的增加而显著性降低。同时分析显示Kl水平与EPCs的数量和粘附功能、迁移功能和增殖功能具有相关性。
第二部分:体外细胞培养实验中,荧光双染计数显示,Kl刺激浓度依赖性增加老龄小鼠骨髓单个核细胞分化成EPCs的数量;MTT分析显示,Kl刺激浓度依赖性增强EPCs的增殖能力;改良的Boyden小室分析显示,Kl浓度依赖性增加迁移的EPCs数量。
采用Western-blot法测定不同浓度的Kl蛋白处理后的EPCs细胞的p53/p21表达水平,发现p53/p21呈Kl蛋白浓度依赖性下降;以P-Akt与Akt的比值来代表EPCs Akt磷酸化的水平,发现Akt的磷酸化水平随着Kl蛋白浓度的增加而逐渐下降。第三部分:非显微外科手术方法损伤小鼠颈动脉后,取损伤动脉Evans blue染色观察,损伤动脉内皮完全剥脱。小鼠颈动脉损伤后,采用流式细胞仪检测外周血EPCs数量显示,小鼠颈动脉损伤后1天、3天和7天,外周血EPCs数量明显高于假手术组,外周血EPCs数量在动脉损伤后3天达到高峰。采集术后第3天Kl处理组和Kl抗体处理组小鼠的外周血,检测其EPCs数量,结果显示:与颈动脉损伤组相比较,Kl处理组的循环Kl明显升高,而Kl抗体处理组无明显上升。术后第14天,损伤血管Evans blue染色显示,在Kl处理组,损伤血管再内皮化的面积明显高于对照颈动脉损伤组,Kl抗体处理组的再内皮化的面积与颈动脉损伤组相比无明显差异。HE染色后应用Image ProPlus软件用来测量新生内膜、中膜面积,利用内膜面积和中膜面积的比值来评价内膜新生,结果显示,Kl处理组内膜增生程度较对照组及Kl抗体处理组明显降低。
4.结论
4.1不同月龄小鼠肾脏Kl mRNA表达、外周血Kl蛋白水平与EPCs的数量、迁移、增殖、黏附功能相关,且随小鼠月龄的增加下降;
4.2在本研究浓度范围内,Kl蛋白可能参与体外培养的老龄小鼠骨髓单个核细胞分化,促进骨髓单个核细胞分化成为EPCs,呈浓度依赖性提高其迁移能力和增殖能力;
4.3在本研究浓度范围内,Kl蛋白可浓度依赖性降低体外培养的EPCs的p53/p21的表达以及Akt的磷酸化水平,可能部分通过下调Akt活性,降低p53/p21水平,抑制细胞衰老;
4.4在本研究浓度及时间范围内,Kl蛋白可以升高老龄小鼠动脉损伤后EPCs的循环数量,增强EPCs的动员,促进老年小鼠损伤动脉再内皮化,降低新生内膜增生。
1. Background and Objective
Since Atherosclerosis was first presented by Marchand, a pathologist of Leipzig of Germany, more than one century before, it has been understood gradually. However, at present the incidence and lethality rate of atherosclerosis are still high, and the heart cerebrovascular disease caused by atherosclerosis is still one of the first place reasons of death now. Atherosclerosis is one kind of degenerative and proliferative lesion, and has close relationship with aging. Because of our country has the hugest senile population in the world, the persistence and chronicity of atherosclerosis not only severely impair aging patients health, but also cause the medicine resources to be consumed continuously. Studying on aging mechanism in the development of atherosclerosis has vital significance regarding to prevent and treat atherosclerosis, and also has great academic value and social profile as well.
The pathogenesis of atherosclerosis is very complex, mainly centers on four theories: fatty infiltration theory, blood platelet accumulation and thrombus form theory, smooth muscle cell clone theory and damage response theory. And damage response theory is supported by majority of scholar. This theory believes that the vascular endothelial structure and function damage are the pathologic and physiologic foundation of atherosclerosis. And it has been prooved that endothelial damage is the early change of atherosclerosis, and damaged vascular endothelials’effective re-endothelialization can maintain the integrity of blood endothelial cell monolayer, and prevent the occurrence of atherosclerosis. At present, pathways to repair damaged vascular endothelial have the following several kinds: life style improvement, for example no-smoking and so on; using medicine like statins,angiotensin converting enzyme inhibitor ect to protect vascular endothelial cell function; using vascular endothelial growth factor to suppress endothelial cell apoptosis; endothelial progenitor cells transplantation.
Cytotherapy based on EPCs has become one of the newest research direction of repairing vascular endothelial damage. EPCs are produced by hematopoietic stem cell differentiation, are the precursor cells of vascular endothelial cells, and mainly come from bone marrow, embryo liver, umbilical cord blood and spleen. Conventional viewpoint states that the reparation of vascular endothelial damage mainly relies on the migration and growth proliferation of neighbor nature endothelial cell surrounding the damage. But recent researches indicate that in bone marrow and peripheral blood EPCs is one of most important mechanisms of vascular endothelial damage reparation. After vascular endothelial damage, EPCs mobilized from bone marrow homing to damaged vascular endothelial, and differentiate to mature endothelial cell, speed up the re-endothelialization of damaged area, suppress the formation of pathological new intimae, play very important role in vascular endothelial damage reparation.
There are many kinds of factors can influence the quantity and activity of EPCs, including age, hypertension, high glucose, oxidized LDL, chronic renal failure and so on. The research indicates that pure age increasing may reduce EPCs mobilization, proliferation and adhesion and migration ability. And also discovered that the process which EPCs participating in the repairation of damaged area has age dependence, EPCs transplantation of young donors can promote the re-endothelialization, but the EPC function of aging ones are not obvious. Therefore aging causes EPCs function impairment and quantity decrease, possibly is the reason of senile patients re-endothelialization ability. So how to suppress EPCs aging effectively and promote re-endothelialization of aging patients’damaged blood vessel comes to be urgent.
Klotho discovered by Kuro ect in 1997 is one kind of new genes has relationship with aging. Kl gene expression deletion in mice can cause various morbodity changes similar to human, for example: intimae thickening, medial calcification, arteriosclerosis, osteoporosis, emphysema, abnormality of glucose and energy metabolism, ect. Kl is mainly expressed by epithelial cells of distal convoluted tubule of the kidney.
At present, the mechanism of Kl preventing aging is still unclear, some researches confirmed that in Kl gene mutation mice, the EPCs quantity of bone marrow and peripheral blood would reduce obviously with body aging gradually, prompting Kl expression and EPCs related closely. We investigate the study to research aging influence on EPCs function and Kl expression, observe Kl influence on EPCs quantity and biological function and participation in re-endothelialization of damaged vascular endothelial, probe into the possibility of suppressing EPCs aging by via p53/ p21signaling pathway.
2. Method
2.1 Relations of age and mice Kl expression and EPCs function
2.1.1 Detecting EPCs quantity and function in different month mice Male C57BL/6 mice were divided into three groups according to month, two-month old, twelve-month old, and twenty-month old. Using endothelial cells selective medium culture and differentiation and density gradient centrifugation to isolate bone marrow derived single nuclear cell of mice, and acquire bone marrow derived EPCs, and using acLDL-DiI and FITC-UEA-I direct fluorescent staining under an inverted fluorescent microscope and flow cytometry to detect stem cell surface specific antigen and endothelial cell specific antigen, and using immunohistochemical method to identify cell surface endothelial cell antigen expression. Using acLDL-DiI and FITC-UEA-I direct fluorescent staining under an inverted fluorescent microscope and MTT analysis and improved boyden chamber and adhesion cell counting method to detect EPCs quantity, proliferation, migration, and adhesion ability .
2.1.2 Detecting renal Kl mRNA and peripheral blood Kl protein level in different month mice
Using RT-PCR to detect renal Kl mRNA, using Western-blot to detect peripheral blood Kl protein level of different age mice, and analyzing relevance between Kl level and EPCs quantity and ability in different month mice.
2.2 The influence of Kl on EPCs function of aging mice
2.2.1 Observing Kl influence on biological function of EPCs in aging mice in vitro Incubating bone marrow derived EPCs of twenty-month old mice with different concentrations Kl, and using sing acLDL-DiI and FITC-UEA-I direct fluorescent staining under an inverted fluorescent microscope and MTT analysis and improved boyden chamber method to detect EPCs quantity, proliferation and migration activity.
2.2.2 Try probe into the possible mechanism of suppressing EPCs aging by Kl protein Using Western-blot method to detect p53,p21and p-Akt expression, and analyzing the mechanism of Kl affecting EPCs aging.
2.3 In vivo study of Kl promoting senile mice re-endothelialization of damaged vascular endothelial.
Using non-microsurgery method to establish mice(twenty-month old) carotid artery injury model, and using pathology method to examine mice carotid artery injury model whether to establish successfully. At the different time after mice carotid artery injury(before operation,and the 1st day,3rd day, 7th day and 14th day after operation ) obtaining peripheral blood, using flow cytometry to detect peripheral blood EPCs quantity. The 3rd day after mice carotid artery injury, using flow cytometry to detect peripheral blood EPCs of Kl treated group and Kl antibody treated group .Fourteen days later using Evans blue method to detect re-endothelialization of damaged vascular endothelial,and using HE staining to detect the degree of new-born tunica intimae.
3. Results
Part one:
After 7 days cultured in endothelial cell selection medium,bone marrow mononuclear cells turned into spindle-shaped,endothelial cell-like cells. These cells were characterized further by demonstrating the expression of the mouse stem-cell marker Sca-1 as well as the endothelial cell lineage antigen VEGFR-2 by flow cytometry.Endothelial cell lineage antigen vWF was also expressed by most of these cells in immunohistochemical analysis. Fluorescence double staining counting 2M,12M, 20M mice, discovering along with age growth, EPCs quantity obviously reduced. Using MTT method to detect mice bone marrow derived EPCs proliferative ability, using Boyden chamber method to detect EPCs migrating ability, using adherent cells counting to detect EPCs adhesion ability, the results showed that along with age increasing, mice bone marrow derived EPCs adhesion ability, migrating ability and proliferating function obviously dropped. Using RT-PCR and Western-blot to detect renal Kl mRNA and peripheral blood Kl protein level separately, the results showed that along with month increasing, Kl mRNA, peripheral blood Kl protein level obviously dropped. Simultaneously analyzing demonstrated that the Kl level and EPCs quantity and adhesion ability, migrating ability and proliferating function has closely relevance.
Part two:
In culturing experiment in Vitro, Fluorescence double staining counting demonstrating that Kl protein increases aging mice bone marrow mononuclear cell differentiating has concentration dependence . MTT analyzing demonstrating that Kl protein strengthenes proliferating function of EPCs; and Improved boyden chamber analyzing demonstrating Kl protein strengthened migrated EPCs quantity by concentration dependence. Western-blot method demonstrating Kl protein decreased p53 ,p21 and p-Akt expression
Part three:
Obtaining complete removal of endothelium with a non-microscopical surgery method, and detecting it with HE staining.At different time after mice carotid artery injury(before operation,and the 1st day,3rd day, 7th day and 14th day after operation) obtaining peripheral blood , EPCs quantity detected by flow cytometry demonstrating that at different time after mice carotid artery injury: the 1st day,3rd day and 7th day after operation, peripheral blood EPCs quantity obviously higher than sham-operation group, and 3rd achieved the peak. Gathering 3rd peripheral blood of Kl treated group and Kl antibody treaded group ,detecting EPCs quantity, discovering that comparing with carotid artery injury group, Kl treated group peripheral blood amount og EPCs elevated obviously, but Kl antibody treaded group had no significance difference. 14th day injured blood vessel dying discovering that in Kl treated group the re-endothelialization area of damaged vascular endothelial was higher than control group, but Kl antibody treaded group had no obviouse significance difference.
4. Conclusion
4.1 Mice renal Kl mRNA and peripheral blood Kl ptotein level and age assuming inverse correlation;Mice EPCs quantity, proliferation, migration, adhesion ability and age assuming inverse correlation.
4.2 Kl protein promoting bone marrow stem cells of aging mice cultured in vitro to differentiate into EPC. And Kl protein strengthening EPCs proliferating function and migrating ability by concentration dependence.
4.3 Kl inhibiting EPCs p53, p21 and p-Akt expression by concentration dependence.
4.4 Kl promoting EPCs mobilization after aging mice carotid artery injury and re-endothelialization of mice carotid artery injuried. decreasing the proliferation of new-born tunica intimae;
引文
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