VDR基因对EPCs生物学功能的影响

发布时间：2018-06-08 04:16

  本文选题：内皮祖细胞 + 维生素D　； 参考：《中南大学》2014年硕士论文

【摘要】：背景：肾脏是机体供血量最丰富的器官,其多种功能均通过血管得到实现,如肾小球滤过、肾小管重吸收等重要生理过程,且目前研究证实血管损害的严重程度与肾脏疾病的预后关系密切,因此,维持血管正常结构及功能对实现肾脏功能至关重要。早期血管损害表现为VEC受损、平滑肌细胞迁移及基质增生等,其中VEC受损是始动环节,而晚期血管损害则表现为解剖结构的改变如血管局部畸形、狭窄、断裂甚至完全阻塞进而影响血供。血管损伤治疗方法包括病因治疗、药物治疗和手术治疗等。药物主要有抗氧化剂、降压药、血管紧张素转换酶抑制剂、血管紧张素受体抑制剂等,但药物疗效甚微仅能够缓解血管损害进展而并不能从根本上修复血管。临床上可通过支架置入、血管成形术等外科手段治疗血管晚期损害,但手术治疗创伤大、风险高,且干预时机晚。研究者发现机体内VEC一旦受损,骨髓中EPCs就可被动员、迁移、归巢、分化为成熟VEC,以取代损伤的VEC进而修复血管。研究者提出EPCs移植治疗法,且多项动物和人体干预实验均证实EPCs移植对组织缺血疗效良好。因其疗效好,创伤小,干预时机早,EPCs移植治疗已成为血管性疾病的新希望。但该技术目前仍面临一些问题如血管修复需大量EPCs,而骨髓中数量少,体外培养扩增不能满足需求,且体外成血管能力不稳定。因此,关于EPCs增殖和成血管能力的研究必将受到关注。 研究显示VD缺乏与心血管疾病明显关联,VD干预对心血管相关疾病有一定益处,但作用机制尚不清楚。EPCs中也存在VDR表达,利用VD干预可促进EPCs增殖及成血管能力,并呈VDR数量依赖性,提示VDR与EPCs增殖及成血管能力有关。VDR作为核受体与配体-VD结合可调节细胞周期,影响EPCs的增殖、凋亡及衰老,但近期有研究表明VDR可作为第二信使对细胞发挥调控作用。那么,VDR在没有VD干预的情况下,能否独立地对EPCs的生物学功能进行调控?VDR基因提前转染至EPCs中是否会有利于EPCs移植的治疗效果?目前并无相关研究。 目的：初步研究不同水平VDR基因对EPCs增殖、凋亡、衰老、细胞周期及迁移的影响,为EPCs移植治疗提供新的视角和理论基础。 方法：(1)分离大鼠骨髓源单核细胞,定向诱导分化EPCs,并应用免疫荧光染色鉴定。(2)构建Ad-VDR和Ad-VDR shRNA。分别转染Ad-VDR/Ad-VDR shRNA至EPCs中,以未处理EPCs为对照组,检测各组VDR mRNA(RT-PCR法)和VDR蛋白质(WB法)相对表达水平。(3)设立6个组：正常水平组即EPCs未处理组、高水平组即转染Ad-VDR至EPCs中、低水平组即转染Ad-VDR shRNA至EPCs中、正常水平+Rapamycin组、高水平+Rapamycin组及低水平-+Rapamycin组。分别检测各组EPCs增殖(MTT法和BrdU法)、凋亡(EACS法和WB法)、衰老(p-半乳糖苷酶染色法)、细胞周期(FACS法)和迁移(侵袭小室法)。 结果：(1)成功分离大鼠骨髓源单核细胞,经定向诱导分化,免疫荧光染色鉴定细胞为CD34+/CD133+/VEGFR2+细胞,是目前公认的EPCs。(2)转染Ad-VDR至EPCs组的VDR mRNA及VDR蛋白质表达水平均明显高于对照组,而转染Ad-VDR shRNA至EPCs组的VDR mRNA及VDR蛋白质表达量均明显低于对照组。(3)相对于对照组别,高水平组别OD值和BrdU LI均增高,而低水平组别OD值和BrdU LI均降低。高水平组别Caspase3表达量降低,而低水平组别FACS所检测凋亡率和Caspase3表达量均增高。在衰老检测中,低水平组别中衰老细胞数增多,高水平组别和正常水平组别对比无显著差异。高水平组别中G1期细胞比例降低,S期细胞比例增高,而低水平组别S期细胞比例降低,甚至在低水平+Rapamycin组无S期细胞,G2期细胞比例增高。在迁移检测中,高水平组别中EPCs迁移细胞数明显增多,低水平组别与正常水平组别对比无显著差异。 结论：一定水平的VDR基因对维持EPCs正常功能是必需的；VDR基因呈低表达水平时,EPCs的增殖受抑、凋亡和衰老增加、G2/M期过渡受阻。高表达水平的VDR基因可促进EPCs增殖,抑制凋亡,促进G1/S期过渡,对EPCs的迁移有显著促进作用,但对衰老无明显影响。
[Abstract]:Background: the kidney is the most abundant organ supplying blood in the body, and its various functions are realized through blood vessels, such as glomerular filtration, reabsorption of renal tubules and other important physiological processes, and the present study confirms that the severity of vascular damage is closely related to the prognosis of renal diseases. Therefore, the normal structure and function of the blood vessels are maintained to achieve the function of the kidney. The early vascular damage is VEC damage, smooth muscle cell migration and matrix hyperplasia, in which VEC damage is the starting link, and late vascular damage is manifested by changes in anatomical structure such as vascular local malformation, stenosis, fracture and even complete obstruction that affect blood supply. The treatment of vascular injury includes etiological treatment, drugs The drugs mainly include antioxidants, antihypertensive drugs, angiotensin converting enzyme inhibitors, angiotensin receptor inhibitors, but the drug has little effect on vascular damage and can not be radically repaired. However, the researchers found that once the VEC was damaged, the EPCs in the bone marrow could be mobilized, migrated, and returned to mature VEC to replace the damaged VEC and then repair the blood vessels. The researchers proposed a EPCs transplantation therapy, and a number of animal and human intervention tests confirmed EPCs transplant. The effect of tissue ischemia is good. Because of its good curative effect, small trauma and early intervention, EPCs transplantation has become a new hope for vascular diseases. However, the technology still faces some problems, such as a large number of EPCs in vascular repair, while the number of bone marrow is few, in vitro culture expansion can not meet the requirement, and in vitro the vascular ability is unstable. Therefore, about EPCs The study of proliferation and angiogenesis will be concerned.
Studies have shown that VD deficiency is significantly associated with cardiovascular disease, VD intervention has certain benefits for cardiovascular related diseases. However, the mechanism of action is still unclear in the presence of VDR expression in.EPCs. VD intervention can promote EPCs proliferation and angiogenesis, and the VDR quantitative dependence. It suggests that VDR is associated with EPCs proliferation and vascular ability as a nuclear receptor and a nuclear receptor. Ligand -VD binding can regulate cell cycle and influence the proliferation, apoptosis and senescence of EPCs, but recent studies have shown that VDR can play a role in regulating cells as second messengers. Then, can VDR regulate the biological function of EPCs independently without VD intervention? Whether the VDR gene is transfected to EPCs in advance will be beneficial to the EPCs shift. The effect of plant therapy? There is no related research at present.
Objective: To study the effects of different levels of VDR gene on the proliferation, apoptosis, senescence, cell cycle and migration of EPCs, and provide a new perspective and theoretical basis for the treatment of EPCs transplantation.
Methods: (1) isolated rat bone marrow mononuclear cells were isolated and induced to differentiate EPCs. (2) Ad-VDR and Ad-VDR shRNA. were transfected into Ad-VDR/Ad-VDR shRNA to EPCs respectively, and the untreated EPCs as the control group, and the relative expression level of VDR mRNA (RT-PCR method) and VDR protein was detected in each group. (3) 6 groups were set up: positive The normal level group was EPCs untreated group, and the high level group was transfected into Ad-VDR to EPCs, and the low level group was transfected into Ad-VDR shRNA to EPCs, normal level +Rapamycin group, high level +Rapamycin group and low level -+Rapamycin group. The EPCs proliferation of EPCs (MTT method and BrdU method), the dying method and the aging (galactosidase staining method) were detected in each group. Cell cycle (FACS) and migration (invasive chamber method).
Results: (1) the mononuclear cells of bone marrow derived from rats were successfully separated and differentiated by directed induction. The immunofluorescence staining was used to identify the CD34+/CD133+/VEGFR2+ cells. It was recognized that the expression level of VDR mRNA and VDR protein in the EPCs. (2) transfected Ad-VDR to EPCs group was significantly higher than that of the control group, while the VDR mRNA and protein transfected from the Ad-VDR shRNA to EPCs group were transfected. The quantity of qualitative expression was significantly lower than that of the control group. (3) the high level group O value and BrdU LI were all higher than the control group, while the low level group O and BrdU LI decreased. The high level group Caspase3 expression decreased, while the low level group FACS detected the apoptosis rate and the Caspase3 expression. In the aging test, the low level group senescence. There was no significant difference in the number of cells in the high level group and the normal level group. The proportion of G1 cells in the high level group decreased, the proportion of S cells increased, while the proportion of S phase cells in the low level group decreased, even in the low level +Rapamycin group, there was no S phase cells, and the proportion of cells in the G2 phase increased. In the migration detection, the EPCs migration in the high-level group was fine. EPCs migration was fine in the high level group. The number of cells increased significantly, and there was no significant difference between low level group and normal group.
Conclusion: a certain level of VDR gene is necessary for the maintenance of normal function of EPCs. When the VDR gene is low expression level, the proliferation of EPCs is inhibited, apoptosis and senescence are increased, and the transition of G2/M phase is blocked. The high expression of VDR gene can promote EPCs proliferation, inhibit apoptosis, promote the transition of G1/S phase, and promote the migration of EPCs, but not to senescence. Obviously.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

【共引文献】

相关硕士学位论文 前1条

1 夏晶;DDAH2/ADMA通路在高糖诱导内皮祖细胞线粒体功能障碍中的作用及机制[D];中南大学;2013年

，

本文编号：1994416