VEGF121和VEGF165对血管内皮细胞通透性影响的研究

发布时间：2018-03-25 10:02

本文选题：VEGF121　切入点：VEGF165　出处：《河北医科大学》2011年硕士论文

【摘要】：目的:血管内皮生长因子(vascular endothelial growth factor ,VEGF)发现于1989年,是一种能特异的作用于血管内皮细胞的生长因子,具有促血管生成等活性。血管内皮细胞生长因子(VEGF)家族成员包括: VEGF-A、VEGF-B、胎盘生长因( PIGF)、VEGF-C、VEGF-D、VEGF-E、VEGF-F和它们的受体VEGFR-1、2、3及Neuropilins,VEGF受体特异性地分布于血管内皮等细胞,VEGF通过与其受体的结合而发挥促进血管新生和增强血管通透性的生理功能。在人类,VEGF-A占主导作用,其共有8种亚型,包括:VEGF121, VEGF145, VEGF148,VEGF165, VEGF165b,VEGF 183,VEGF 189和VEGF206,其中VEGF 121和VEGF 165是主要的分泌亚型。一氧化氮(Nitric oxide,NO)广泛分布于动物和植物的体内,具有多种的生理病理学功能,是作用最强的舒血管物质之一。NO作为信号传导因子与鸟苷酸环化酶结合,使之活化,cGMP含量升高,cGMP可降低血管平滑肌中的Ca~(2+)的浓度,引起血管平滑肌舒张,血管扩张、血管通透新增加。血管新生(Angiogenesis)及通透性增加在肿瘤、风湿性关节炎、心血管疾病、糖尿病并发症等疾病发生、发展中起着重要作用,而血管新生与VEGF有着密切的关系。近年研究发现,在诸多皮肤病如特应性皮炎、银屑病、尖锐湿疣等的皮损和血清中VEGF水平均高于正常,所以认为其与这些皮肤病的发生发展密切相关,因此,研究血管内皮生长因子在皮肤病的表达及其在病理过程中所发挥的作用,不仅有助于阐明诸多皮肤病的发病机制,而且可通过对其作用靶点的研究,将为今后进一步探讨其治疗措施提供新的思路。 VEGF121和VEGF165是皮肤组织主要的表达亚型,两者参与各种病理生理过程,以往的各种研究主要着眼于VEGF-A的研究,具体到亚型的很少,所以,本研究旨在通过研究两种不同亚型的VEGF对血管内皮细胞通透性影响,以及不同亚型VEGF刺激血管内皮细胞后NO释放量的测定,明确引起血管通透性增加的主要VEGF亚型,从而为研发VEGF阻断剂,靶向治疗存在VEGF异常表达的皮肤病提供可靠的理论依据,同时可将毒副作用降到最低。方法: 1人类脐静脉内皮细胞(HUVEC)的体外培养EGM-2完全培养基37℃、5% CO_2培养箱静置培养,2-3天更换一次培养基。 2双腔通透性测定试剂盒(In Vitro Vascular Permeability Assay kit;ECM640)建立通透性检测模型,浓度为25ng/ml的VEGF121或VEGF165刺激人类脐静脉内皮细胞。 3荧光酶标仪(Exmission: 490 nm, Emission: 520 nm)测定底层培养基中不同时间FITC-dextran量的变化。 4人类脐静脉内皮细胞的体外培养,浓度为25ng/ml的VEGF121或VEGF165刺激人类脐静脉内皮细胞,Griess法测定不同时间培养基中NO含量。 5制图,运用统计学软件SPSS13.0对实验数据进行统计分析。结果: 1经VEGF165或VEGF121刺激人类脐静脉内皮细胞,通透性物质(FITC-dextran)测定 1.1荧光酶标仪测定不同刺激时相的通透性物质(FITC-dextran)的量,应用重复测量的方差分析,在不同刺激物之间培养基中FITC-dextran的量有明显的差异,p0.05,即经VEGF121处理的血管内皮细胞,渗透到细胞层外的FITC-dextran显著多于VEGF165和空白对照组。说明VEGF121对细胞通透性影响较VEGF165显著。 1.2在不同刺激时相之间通透性物质有明显的差异,p0.05,即随着刺激时间的增加,渗透到细胞层外的通透性物质(FITC-dextran)也明显增加。 1.3刺激时间和不同刺激物之间存在交互作用,p0.05。 2经VEGF165或VEGF121刺激人类脐静脉内皮细胞,培养基中NO产量的测定 2.1 Griess试剂盒测定培养基中NO含量,应用重复测量的方差分析,在不同刺激物之间NO产量有明显的差异,p0.05,即经VEGF121处理的血管内皮细胞,分泌的NO显著多于VEGF165和空白对照组。VEGF121对细胞NO产量的影响较VEGF165显著。 2.2在不同刺激时相之间分泌的NO的量有明显的差异,p0.05,即随着刺激时间的增加,分泌的NO也明显增加。 2.3刺激时间和不同刺激物之间存在交互作用,p0.05。结论: VEGF121和VEGF165均能够引起血管内皮细胞通透性增加,刺激内皮细胞产生NO。但是,VEGF121对通透性的影响远高于VEGF165,VEGF121刺激血管内皮细胞所产生的NO含量高于VEGF165,VEGF121可能是导致血管通透性增加的主要血管内皮生长因子亚型。
[Abstract]:Objective: vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) was found in 1989, is a growth factor specific effects on vascular endothelial cells and angiogenic activity. Vascular endothelial growth factor (VEGF) family members include: VEGF-A, VEGF-B, placental growth due to VEGF-C (PIGF). VEGF-D, VEGF-E, VEGF-F, VEGFR-1,2,3 and Neuropilins and their receptors in vascular endothelial cells, such as the distribution of VEGF receptors, VEGF with its receptors play in promoting angiogenesis and vascular permeability enhancing physiological function. In humans, VEGF-A is dominant, the total of 8 subtypes, including: VEGF121, VEGF145, VEGF148, VEGF165, VEGF165b, VEGF 183, VEGF 189 and VEGF206 VEGF 121 and VEGF 165, which is the main secretory subtypes.
Nitric oxide (Nitric oxide NO) is widely distributed in animal and plant body, with a variety of physiological and pathological functions, is one of the strongest vasodilator.NO as signal transduction factor and guanylate cyclase with the activation, increasing the content of cGMP, cGMP can decrease in vascular smooth muscle Ca~ (2+) concentration, caused by vascular smooth muscle relaxation and vasodilation, increased vascular permeability.
Neovascularization (Angiogenesis) and the permeability increase in cancer, rheumatoid arthritis, cardiovascular disease, diabetes complications, plays an important role in the development of neovascularization has a close relationship with the VEGF. Recent studies have found that in many skin diseases such as atopic dermatitis, psoriasis, skin lesions and serum level of VEGF the condyloma acuminatum were higher than normal, so it is closely related with the occurrence and development of skin disease so on expression of vascular endothelial growth factor in skin disease and its role in the pathological process of the role, not only help to elucidate the pathogenesis of many skin diseases, but also through the study on its function the target, will provide new ideas for the further study of the treatment measures.
VEGF121 and VEGF165 are the main subtypes expressed in skin tissue, both of which are involved in various pathophysiological processes of all previous studies mainly focus on the VEGF-A, specific to subtype rarely, therefore, this study aims to study two different subtypes of VEGF on vascular endothelial cell permeability, and determine the release of NO different subtypes of vascular endothelial cells after VEGF stimulation, to clear the cause of increased vascular permeability of VEGF subtype, and for the development of VEGF blocking agent, target to provide a reliable theoretical basis for the treatment of abnormal expression of VEGF skin disease, and can be toxic and side effects to a minimum.
Method:
1 in vitro culture of human umbilical vein endothelial cells (HUVEC) in vitro, EGM-2 completely culture medium 37 C, 5% CO_2 incubator statically culture, 2-3 days to replace the culture medium.
2 dual chamber permeability assay kit (In Vitro Vascular Permeability Assay kit; ECM640) to establish a permeability detection model, 25ng/ml VEGF121 or VEGF165 to stimulate human umbilical vein endothelial cells.
The 3 fluorescence enzyme labeling instrument (Exmission: 490 nm, Emission: 520 nm) was used to determine the changes of FITC-dextran in the substrate at different time.
4 human umbilical vein endothelial cells were cultured in vitro, and the concentration of 25ng/ml VEGF121 or VEGF165 stimulated human umbilical vein endothelial cells. Griess method was used to determine NO content in different time medium.
5 the statistical analysis of the experimental data was carried out by using the statistical software SPSS13.0.
Result:
1 the human umbilical vein endothelial cells stimulated by VEGF165 or VEGF121, and the determination of the permeability substance (FITC-dextran)
1.1 fluorescence eliasa permeability material phase determination of different stimulation (FITC-dextran) the amount of variance analysis of repeated measurement applications, the amount of FITC-dextran in the culture medium had obvious differences between different stimuli P0.05, namely VEGF121 treated endothelial cells penetrate into the cell layer was more than VEGF165 and FITC-dextran the blank control group. VEGF121 of cell permeability influence VEGF165 significantly.
1.2, there was a significant difference in permeability between different stimuli. P0.05, with the increase of stimulation time, FITC-dextran increased significantly.
There was a interaction between 1.3 stimulation time and different stimulants, p0.05.
2 stimulation of human umbilical vein endothelial cells by VEGF165 or VEGF121, and the determination of NO production in culture medium
2.1 Griess kit for determination of NO content in culture medium, analysis of variance of repeated measurement applications, between different stimuli was significantly different, the yield of NO P0.05, namely VEGF121 treated endothelial cells, the secretion of NO was significantly higher than VEGF165 and.VEGF121 on NO cells group blank effect yield control than VEGF165 significantly.
2.2 there is a significant difference in the amount of NO secreted between different stimuli, P0.05, that is, as the time of stimulation increases, the secretion of NO is also significantly increased.
There was a interaction between 2.3 stimulation time and different stimulants, p0.05.
Conclusion:
VEGF121 and VEGF165 could increase the permeability of vascular endothelial cells, stimulate endothelial cells to produce NO. but the effect of VEGF121 on permeability is much higher than that of VEGF165, NO in VEGF121 stimulated by vascular endothelial cells is higher than that of VEGF165 and VEGF121 may lead to increased vascular permeability of vascular endothelial growth factor isoforms.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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