本文選題：VEGF121　切入點：VEGF165　出處：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:血管內(nèi)皮生長因子(vascular endothelial growth factor ,VEGF)發(fā)現(xiàn)于1989年,是一種能特異的作用于血管內(nèi)皮細(xì)胞的生長因子,具有促血管生成等活性。血管內(nèi)皮細(xì)胞生長因子(VEGF)家族成員包括: VEGF-A、VEGF-B、胎盤生長因( PIGF)、VEGF-C、VEGF-D、VEGF-E、VEGF-F和它們的受體VEGFR-1、2、3及Neuropilins,VEGF受體特異性地分布于血管內(nèi)皮等細(xì)胞,VEGF通過與其受體的結(jié)合而發(fā)揮促進(jìn)血管新生和增強血管通透性的生理功能。在人類,VEGF-A占主導(dǎo)作用,其共有8種亞型,包括:VEGF121, VEGF145, VEGF148,VEGF165, VEGF165b,VEGF 183,VEGF 189和VEGF206,其中VEGF 121和VEGF 165是主要的分泌亞型。 一氧化氮(Nitric oxide,NO)廣泛分布于動物和植物的體內(nèi),具有多種的生理病理學(xué)功能,是作用最強的舒血管物質(zhì)之一。NO作為信號傳導(dǎo)因子與鳥苷酸環(huán)化酶結(jié)合,使之活化,cGMP含量升高,cGMP可降低血管平滑肌中的Ca~(2+)的濃度,引起血管平滑肌舒張,血管擴張、血管通透新增加。 血管新生(Angiogenesis)及通透性增加在腫瘤、風(fēng)濕性關(guān)節(jié)炎、心血管疾病、糖尿病并發(fā)癥等疾病發(fā)生、發(fā)展中起著重要作用,而血管新生與VEGF有著密切的關(guān)系。近年研究發(fā)現(xiàn),在諸多皮膚病如特應(yīng)性皮炎、銀屑病、尖銳濕疣等的皮損和血清中VEGF水平均高于正常,所以認(rèn)為其與這些皮膚病的發(fā)生發(fā)展密切相關(guān),因此,研究血管內(nèi)皮生長因子在皮膚病的表達(dá)及其在病理過程中所發(fā)揮的作用,不僅有助于闡明諸多皮膚病的發(fā)病機制,而且可通過對其作用靶點的研究,將為今后進(jìn)一步探討其治療措施提供新的思路。 VEGF121和VEGF165是皮膚組織主要的表達(dá)亞型,兩者參與各種病理生理過程,以往的各種研究主要著眼于VEGF-A的研究,具體到亞型的很少,所以,本研究旨在通過研究兩種不同亞型的VEGF對血管內(nèi)皮細(xì)胞通透性影響,以及不同亞型VEGF刺激血管內(nèi)皮細(xì)胞后NO釋放量的測定,明確引起血管通透性增加的主要VEGF亞型,從而為研發(fā)VEGF阻斷劑,靶向治療存在VEGF異常表達(dá)的皮膚病提供可靠的理論依據(jù),同時可將毒副作用降到最低。 方法: 1人類臍靜脈內(nèi)皮細(xì)胞(HUVEC)的體外培養(yǎng)EGM-2完全培養(yǎng)基37℃、5% CO_2培養(yǎng)箱靜置培養(yǎng),2-3天更換一次培養(yǎng)基。 2雙腔通透性測定試劑盒(In Vitro Vascular Permeability Assay kit;ECM640)建立通透性檢測模型,濃度為25ng/ml的VEGF121或VEGF165刺激人類臍靜脈內(nèi)皮細(xì)胞。 3熒光酶標(biāo)儀(Exmission: 490 nm, Emission: 520 nm)測定底層培養(yǎng)基中不同時間FITC-dextran量的變化。 4人類臍靜脈內(nèi)皮細(xì)胞的體外培養(yǎng),濃度為25ng/ml的VEGF121或VEGF165刺激人類臍靜脈內(nèi)皮細(xì)胞,Griess法測定不同時間培養(yǎng)基中NO含量。 5制圖,運用統(tǒng)計學(xué)軟件SPSS13.0對實驗數(shù)據(jù)進(jìn)行統(tǒng)計分析。 結(jié)果: 1經(jīng)VEGF165或VEGF121刺激人類臍靜脈內(nèi)皮細(xì)胞,通透性物質(zhì)(FITC-dextran)測定 1.1熒光酶標(biāo)儀測定不同刺激時相的通透性物質(zhì)(FITC-dextran)的量,應(yīng)用重復(fù)測量的方差分析,在不同刺激物之間培養(yǎng)基中FITC-dextran的量有明顯的差異,p0.05,即經(jīng)VEGF121處理的血管內(nèi)皮細(xì)胞,滲透到細(xì)胞層外的FITC-dextran顯著多于VEGF165和空白對照組。說明VEGF121對細(xì)胞通透性影響較VEGF165顯著。 1.2在不同刺激時相之間通透性物質(zhì)有明顯的差異,p0.05,即隨著刺激時間的增加,滲透到細(xì)胞層外的通透性物質(zhì)(FITC-dextran)也明顯增加。 1.3刺激時間和不同刺激物之間存在交互作用,p0.05。 2經(jīng)VEGF165或VEGF121刺激人類臍靜脈內(nèi)皮細(xì)胞,培養(yǎng)基中NO產(chǎn)量的測定 2.1 Griess試劑盒測定培養(yǎng)基中NO含量,應(yīng)用重復(fù)測量的方差分析,在不同刺激物之間NO產(chǎn)量有明顯的差異,p0.05,即經(jīng)VEGF121處理的血管內(nèi)皮細(xì)胞,分泌的NO顯著多于VEGF165和空白對照組。VEGF121對細(xì)胞NO產(chǎn)量的影響較VEGF165顯著。 2.2在不同刺激時相之間分泌的NO的量有明顯的差異,p0.05,即隨著刺激時間的增加,分泌的NO也明顯增加。 2.3刺激時間和不同刺激物之間存在交互作用,p0.05。 結(jié)論: VEGF121和VEGF165均能夠引起血管內(nèi)皮細(xì)胞通透性增加,刺激內(nèi)皮細(xì)胞產(chǎn)生NO。但是,VEGF121對通透性的影響遠(yuǎn)高于VEGF165,VEGF121刺激血管內(nèi)皮細(xì)胞所產(chǎn)生的NO含量高于VEGF165,VEGF121可能是導(dǎo)致血管通透性增加的主要血管內(nèi)皮生長因子亞型。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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