血管紧张素II对大鼠骨髓源内皮祖细胞生物学特性的影响

发布时间：2018-01-17 22:02

  本文关键词：血管紧张素II对大鼠骨髓源内皮祖细胞生物学特性的影响　出处：《第四军医大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 内皮祖细胞 血管紧张素 缬沙坦 增殖 凋亡 一氧化氮 粘附 血管内皮生长因子受体

【摘要】： 实验背景及目的 内皮祖细胞(endothelial progenitor cells,EPCs),又称成血管细胞,存在于骨髓(bone marrow,BM)、脐带血(umbilical cord blood,UCB)及外周血(peripheral blood,PB)中,可以分化为成熟的内皮细胞(endothelial cells,ECs),是血管ECs的前体细胞。研究表明,EPCs不仅参与胚胎时期血管生成,而且在缺血、血管损伤等刺激下,可动员并迁移至病变局部参与出生后的血管发生及受损内皮的修复,在缺血组织的血运重建方面发挥着重要作用,给缺血性疾病的治疗带来了新的希望。但对EPCs的生物学特性及其影响因素尚缺乏全面了解,而这是对于更好的应用EPCs为人类造福所必需的。 最新的研究发现,EPCs的数量、功能等生物学特性与心血管疾病的危险因素如高脂血症、糖尿病、C-反应蛋白(C-reactive protein,CRP)等成负相关。血管紧张素II(AngII)是一种与多种心血管疾病的病理生理过程密切相关的致病因素,它的水平是否也会影响EPCs的生物学特性尚不清楚。另一方面,近些年不断有研究报道AngII可以促进新生血管的形成,提示它可能与EPCs存在一定的相互关系。 AngII可以促进ECs的增殖,抑制凋亡,上调其粘附分子的表达,增加其内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)mRNA的合成,从而增加一氧化氮(nitric oxide,NO)的分泌。因此,我们假设AngII对EPCs也具有类似作用。本实验在体外培养大鼠BM-EPCs的基础上,观察一定浓度的AngII对其增殖、凋亡、NO分泌及粘附能力等生物学特性的影响,并进一步探讨其可能机制,以期为临床更好地利用EPCs治疗缺血性疾病提供思路。 实验方法 第一部分:大鼠BM-EPCs的体外分离、诱导分化及鉴定。无菌分离大鼠股骨及胫骨,冲洗骨髓腔,收集细胞悬液,利用Ficoll密度梯度离心法分离BM单个核细胞(mononuclear cells,MNCs),接种在含有特定浓度血管内皮生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的培养基中进行诱导分化。对原代细胞的表面标志分子CD133和血管内皮生长因子2型受体(vascular endothelial growth factor type 2 receptor,VEGFR-2/Flk-1)进行双染,激光共聚焦鉴定,双阳性为EPCs。 第二部分:AngII对BM-EPCs增殖能力的影响及机制探讨。取原代培养的EPCs,分成若干组,在其培养基中添加不同浓度的AngII,至培养终点,四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)比色法测定细胞增殖活性。利用逆转录聚合酶链反应(reverse transcription-polymerase chain raction,RT-PCR)对不同实验组的Flk-1 mRNA进行扩增,半定量分析其表达量的差异。 第三部分:AngII对BM-EPCs凋亡、NO分泌及粘附能力的影响。取原代培养的EPCs,不同浓度的AngII干预后,Hoechst染色测定凋亡率,试剂盒测定NO分泌量,结晶紫染色测定粘附能力。 实验结果 1、体外成功培养出大鼠BM-EPCs,并经鉴定证实。 2、与空白对照组比较,在VEGF存在的前提下,不同浓度的AngII可以使EPCs的增殖活性增强(P0.05),且呈浓度依赖性;AngII可以促进EPCs Flk-1 mRNA的表达。血管紧张素II 1型受体(angiotensin II type 1 receptor,AT1R)拮抗剂缬沙坦、蛋白激酶C(protein kinase C,PKC)抑制剂均可以拮抗这种作用(P0.05)。 3、与空白对照组比较,不同浓度的AngII可以使无血清条件下EPCs的凋亡率下降(P0.05);不同浓度的AngII可以使EPCs NO的分泌量增加(P0.05);不同浓度的AngII可以使短期内EPCs的贴壁细胞数量增加(P0.05)。作用均呈浓度依赖性,缬沙坦可拮抗AngII的上述作用(P0.05)。 结论 一定浓度的AngII可以改善BM-EPCs的生物学特性,表现为细胞增殖活性增强、凋亡率下降、NO分泌量增加、粘附能力增强,而AngII的上述作用是通过AT1R介导的。结果提示AngII可能是通过对EPCs的作用产生促进血管生成的效应。
[Abstract]:Experimental background and purpose
Endothelial progenitor cells (endothelial progenitor cells, EPCs), also known as vascular cells in bone marrow (bone, marrow, BM), umbilical cord blood (umbilical cord blood, UCB) and peripheral blood (peripheral, blood, PB) can differentiate into mature endothelial cells (endothelial, cells, ECs), is a former somatic cell vascular ECs. The results show that EPCs is not only involved in embryonic angiogenesis, but also in ischemia, vascular damage and other stimuli, can mobilize and migrate to the lesion involved in postnatal neovascularization and repair of damaged endothelium, plays an important role in ischemic tissue revascularization, brings new hope for the treatment of ischemic diseases. But the factors of biological characteristics and its effect on EPCs is still lack of comprehensive understanding, which is better for the application of EPCs for the benefit of mankind is necessary.
The latest study found that the number of EPCs, such as hyperlipidemia and diabetes risk factors, biological function and cardiovascular disease, C- reactive protein (C-reactive protein, CRP) and a negative correlation. Angiotensin II (AngII) is closely related to a variety of pathogenic factors and cardiovascular disease pathophysiology the process, it will also affect the level of the biological characteristics of EPCs is not clear. On the other hand, in recent years it has been reported that AngII can promote the formation of new blood vessels, suggesting that it may have a certain correlation with EPCs.
AngII can inhibit apoptosis, promote the proliferation of ECs by up regulating the expression of adhesion molecules, increase the endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS) mRNA synthesis, thereby increasing nitric oxide (nitric oxide, NO) secretion. Therefore, we suppose that AngII had a similar effect on EPCs in this experiment. In vitro cultured rat BM-EPCs based on the observation of a certain concentration of AngII on the proliferation, apoptosis, affect the biological characteristics of NO secretion and adhesion ability, and further explore its possible mechanism, in order to make better use of EPCs for the clinical treatment of ischemic diseases to provide ideas.
Experimental method
The first part: isolation of rat BM-EPCs in vitro, differentiation and identification. The separation of aseptic femoral and tibial bone marrow cavity of rats, flushing, cell suspension was collected, separated BM mononuclear cells by Ficoll density gradient centrifugation (mononuclear, cells, MNCs), growth factor in vascular endothelial specific inoculation concentration (vascular endothelial growth factor, VEGF), basic fibroblast growth factor (basic fibroblast, growth factor, bFGF) of the culture medium to induce the differentiation of primary cells. The surface markers of CD133 and vascular endothelial growth factor receptor 2 (vascular endothelial growth factor type 2 receptor, VEGFR-2/Flk-1) by double staining, laser scanning confocal microscope double positive for EPCs.
The second part: To investigate the effect of AngII on the proliferation of BM-EPCs and its mechanism. The primary cultured EPCs, divided into several groups, in the culture medium with different concentrations of AngII, to train end point, four methyl thiazolyl tetrazolium salt (methyl thiazolyl, tetrazolium, MTT) determination of cell proliferation activity by reverse transcriptase polymerase assay. Chain reaction (reverse transcription-polymerase chain raction, RT-PCR) of the different groups of Flk-1 mRNA were amplified, the expression difference and semi quantitative analysis.
The third part: the effect of AngII on the apoptosis, NO secretion and adhesion of BM-EPCs. After primary culture of EPCs and AngII with different concentrations, the apoptosis rate was determined by Hoechst staining, the NO secretion was determined by kit, and the adhesion ability was determined by crystal violet staining.
experimental result
1, rat BM-EPCs was successfully cultured in vitro and confirmed by identification.
2, compared with the control group, in the premise of the existence of VEGF under different concentrations of AngII can make the EPCs proliferation enhancement (P0.05), in a dose-dependent manner; AngII can promote the expression of EPCs Flk-1 mRNA. Angiotensin II type 1 receptor (angiotensin II type 1 receptor, AT1R) antagonist valsartan protein kinase C (protein, kinase, C, PKC) inhibitors were able to antagonize this effect (P0.05).
3, compared with the blank control group, different concentration of AngII can cause apoptosis under serum free EPCs decreased (P0.05); different concentrations of AngII can make the EPCs secretion of NO increased (P0.05); different concentrations of AngII can make the number of adherent cells in the short term the increase of EPCs (P0.05) are. In a concentration dependent manner, the effect of valsartan can inhibit AngII (P0.05).
conclusion
The biological characteristics of a certain concentration of AngII can improve the performance of BM-EPCs, in order to enhance the activity of cell proliferation, decreased apoptosis rate, NO secretion increased, enhanced adhesion ability, and the effect of AngII is mediated by AT1R. The results suggest that AngII may promote angiogenesis effect is produced by the effect on EPCs.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【引证文献】

相关硕士学位论文 前1条

1 杜瑞;FoxO4在血管紧张素Ⅱ诱导的内皮祖细胞凋亡中的机制研究[D];山西医科大学;2010年

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