金黄色葡萄球菌超抗原样蛋白-5对内皮祖细胞功能的影响

发布时间：2018-08-14 12:22

【摘要】：近年,随着社会经济的发展和人口的老龄化,动脉粥样硬化已经成为严重危害人类健康的一大类疾病,它是造成心脏、脑及肢体缺血的主要原因。其发生、发展与炎症反应激活以及内皮损伤有关;同时机体内又具有以内皮祖细胞为代表的修复机制。血管病变的发生则是由损伤与修复的失衡所导致。动脉粥样硬化(atherosclerosis)的发生与多种心血管病危险因素相关,在当今积极控制已知心血管病危险因素(吸烟、血脂、血压和血糖等)的情况下,以动脉粥样硬化病变为基础的心脑血管病在我国仍然呈迅速上升的趋势。这提示,在常见的心血管病危险因素之外,还可能有一些危险因素没有被重视或控制。目前,感染因素与血栓和血管病变形成之间的关系日益受到重视,“感染负荷”已被认定为ATS新的危险因素和防治靶向1。例如,金黄色葡萄球菌是心血管系统感染的常见致病菌,是导致感染性心内膜炎和糖尿病足患者动脉血管病变的常见病原体之一。若能阐明某些感染因素在血管损伤与修复两方面的作用,将有利于提高动脉粥样硬化及其并发症的防治水平。金黄色葡萄球菌超抗原样蛋白-5(staphylococcal superantigen -like protein-5, SSL5)是金黄色葡萄球菌(S. aureus)家族中的一员,属分泌型蛋白。本研究旨在进一步阐明金黄色葡萄球菌感染促进血管病变形成的作用和机制,从而为预防感染所致血管病变的发生奠定基础。目的:本研究在构建SSL5蛋白表达载体的基础上从人脐血中提取EPCs,并对其进行培养及鉴定。研究SSL5与EPCs表面PSGL-1的特异性结合情况,以及SSL5对EPCs粘附、迁移、增殖等生物学特性的影响。材料和方法:(1)构建SSL5蛋白表达载体,从金黄色葡萄球NCTC8325菌株中扩增出ssl5基因,将ssl5基因克隆于原核表达载体pET32a中,再转染于大肠杆菌BL21中扩增表达,采用SDS-PAGE电泳检测SSL5蛋白纯度;(2)采取密度梯度离心联合贴壁培养法分离、培养人脐血源性内皮祖细胞,在内皮系选择性培养基(EGM-2)中培养,采用免疫荧光对其表面标志物进行检测和鉴定。(3)采用激光共聚焦和流式细胞术(FACS)检测EPCs表面PSGL-1表达;以FACS检测SSL5与EPCs的结合能力,以及SSL5对“抗人PSGL-1单抗(KPL-1)与EPCs结合”的竞争性抑制作用。采用粘附实验、Millicell-PCF培养小室和CCK-8试剂盒分别检测不同浓度SSL5 (0、1.0、3.0、10、30mg/L)对EPCs粘附、迁移、增殖活性的影响。结果:(1)重组SSL5蛋白经SDS-PAGE电泳检测其纯度90%。(2)分离获得的人脐血源性EPCs具有干细胞集落形成能力,在内皮选择性培养基条件下,培养30天后能够向成熟内皮细胞分化,表达内皮细胞标志物CD31。(3)流式细胞仪检测结果显示, PSGL-1在EPCs表面有较丰富的表达,阳性细胞率为76.6%。激光共聚焦显微镜检测结果显示,PE-KPL-1和FITC-UEA-1双染阳性细胞约占细胞总数的66.3%。随着SSL5浓度的增加,其与EPCs的结合量逐渐增加,同时,SSL5可竞争性抑制KPL-1与EPCs的结合。SSL5还可抑制EPCs在P-选择素表面的黏附,并可对EPCs的迁移和增殖能力产生抑制作用。结论:(1)人脐血源性EPCs表面有较丰富的PSGL-1表达;(2) SSL5可与EPCs表面的PSGL-1结合并抑制EPCs在P-选择素表面的黏附。这提示,SSL5可能通过抑制EPCs在损伤内皮部位(有丰富的E-/P-选择素表达)的黏附,以及对EPCs迁移和增殖能力的抑制,最终导致EPCs修复功能下降,促进血管病变的形成。
[Abstract]:In recent years, with the development of social economy and the aging of the population, atherosclerosis has become a serious threat to human health, which is the main cause of heart, brain and limb ischemia. The occurrence of atherosclerosis is associated with a variety of cardiovascular risk factors, and is based on atherosclerosis when known cardiovascular risk factors (smoking, blood lipids, blood pressure, blood sugar, etc.) are actively controlled. Cardiovascular and cerebrovascular diseases are still on the rise in China, which suggests that besides the common cardiovascular risk factors, some risk factors may not be taken seriously or controlled. Staphylococcus aureus, for example, is a common pathogen of cardiovascular system infections and is one of the common pathogens causing arterial lesions in patients with infective endocarditis and diabetic foot. It is beneficial to improve atherosclerosis and to clarify the role of certain infectious factors in vascular injury and repair. The level of prevention and treatment of complications.
Staphylococcal superantigen-like protein-5 (SSL5) is a secretory protein of Staphylococcus aureus (S. aureus). The purpose of this study is to further elucidate the role and mechanism of Staphylococcal infection in promoting angiopathy and thus preventing the blood vessels from infection. Lay the foundation for the occurrence of the disease.
Objective: To isolate and identify EPCs from human umbilical cord blood on the basis of constructing SSL5 protein expression vector, and to study the specific binding of SSL5 to PSGL-1 on the surface of EPCs, and the effects of SSL5 on the biological characteristics of EPCs such as adhesion, migration and proliferation.
Materials and Methods: (1) SSL5 protein expression vector was constructed, ssl5 gene was amplified from grape globe NCTC8325 strain, ssl5 gene was cloned into prokaryotic expression vector pET32a, and then transfected into E. coli BL21 for amplification and expression, and the purity of SSL5 protein was detected by SDS-PAGE electrophoresis; (2) density gradient centrifugation combined with adherent culture was used for isolation. Human umbilical cord blood-derived endothelial progenitor cells were cultured in the selective medium of endothelial cell line (EGM-2), and their surface markers were detected and identified by immunofluorescence. (3) The expression of PSGL-1 on the surface of EPCs was detected by confocal laser scanning and flow cytometry (FACS); the binding ability of SSL5 to EPCs was detected by FACS; and the anti-human PSGL-1 monoclonal antibody (KP) was detected by SSL5. The effects of different concentrations of SSL5 (0,1.0,3.0,10,30mg/L) on the adhesion, migration and proliferation of EPCs were studied by adhesion test, Millicell-PCF culture chamber and CK-8 kit.
Results: (1) The purity of recombinant SSL5 protein was detected by SDS-PAGE electrophoresis. (2) The isolated human umbilical cord blood-derived EPCs could differentiate into mature endothelial cells after 30 days of culture in endothelial selective medium, and expressed CD31. (3) Flow cytometry showed that PSGL-1 was present in EP. The results of confocal laser microscopy showed that PE-KPL-1 and FITC-UEA-1 double-stained positive cells accounted for 66.3% of the total cells. With the increase of SSL5 concentration, the binding of EPCs to PE-KPL-1 increased gradually. At the same time, SSL5 could competently inhibit the binding of KPL-1 and EPCs. SSL5 could also inhibit the binding of EPCs in P. - the adhesion of selectin surface can inhibit the migration and proliferation of EPCs.
Conclusion: (1) Human umbilical cord blood-derived EPCs have abundant PSGL-1 expression; (2) SSL5 can bind to PSGL-1 on the surface of EPCs and inhibit the adhesion of EPCs to P-selectin. This suggests that SSL5 may inhibit the adhesion of EPCs to the damaged endothelium by inhibiting the expression of E-/P-selectin and the migration and proliferation of EPCs. The repair function of EPCs decreased, and the formation of vascular disease was promoted.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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