| 1. Background and ObjectiveSince 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. Method2.1 Relations of age and mice Kl expression and EPCs function2.1.1 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 miceUsing 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 mice2.2.1 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. ResultsPart 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 expressionPart 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. Conclusion4.1 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;...
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