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ANG-1在AS髖關(guān)節(jié)韌帶中表達(dá)增加并通過Notch信號(hào)通路調(diào)節(jié)血管再生

發(fā)布時(shí)間:2018-04-28 11:07

  本文選題:強(qiáng)直性脊柱炎 + 全髖關(guān)節(jié)置換 ; 參考:《第二軍醫(yī)大學(xué)》2017年博士論文


【摘要】:第一部分:強(qiáng)直性脊柱炎髖關(guān)節(jié)韌帶基質(zhì)降解和血管生成增加研究背景:強(qiáng)直性脊柱炎(Ankylosing Spondylitis,AS)是一種自身炎癥性疾病,炎癥常常累及中軸關(guān)節(jié)及外周大關(guān)節(jié)的肌腱韌帶附著點(diǎn),晚期韌帶肌腱附著點(diǎn)部位易形成韌帶骨贅,而炎癥和新骨形成常伴發(fā)有血管再生。髖關(guān)節(jié)作為AS中最常受累的外周大關(guān)節(jié),目前國內(nèi)外的研究中,還沒有關(guān)于AS髖關(guān)節(jié)韌帶中血管再生方面的研究。研究目的:本部分實(shí)驗(yàn)中,我們在手術(shù)的過程中獲取AS和股骨頸骨折(Femur Neck Fracture,FNA)髖關(guān)節(jié)周圍韌帶分別作為實(shí)驗(yàn)組和對照組,研究AS髖關(guān)節(jié)周圍韌帶是否伴發(fā)有血管再生增加。實(shí)驗(yàn)方法:血管周圍基質(zhì)降解是血管再生的第一步。我們通過RT-PCR檢測纖連蛋白(fibronectin),層黏連蛋白(Laminin),1型膠原(Collagen 1),4型膠原(Collagen 4),基質(zhì)金屬蛋白酶(matrix metalloproteinases,MMP1,MMP2,MMP9),基質(zhì)金屬蛋白酶抑制劑(inhibitors of matrix metalloproteinases,TIMP1,TIMP2,TIMP3)表達(dá)水平的變化來證明血管周圍基質(zhì)降解情況。CD31是血管內(nèi)皮細(xì)胞的表面標(biāo)志物,Fibronectin和Laminin是能反應(yīng)血管周圍基質(zhì)降解情況,我們通過CD31與Fibronectin,CD31與Laminin雙標(biāo)免疫熒光的方法來證明AS髖關(guān)節(jié)周圍基質(zhì)降解,血管密度及其形態(tài)的變化。實(shí)驗(yàn)結(jié)果:RT-PCR結(jié)果提示,與FNF髖關(guān)節(jié)韌帶對比,AS患者髖關(guān)節(jié)韌帶組織中MMP1,MMP2,MMP9表達(dá)分別增加7.21倍,2.17倍和2.19倍,而Fibronectin,Laminin,Collagen1,Collagen4,TIMP1,TIMP2,TIMP3表達(dá)分別降低1.92倍,2.44倍,2.04倍,2.63倍,2.33倍,10.73倍,2.78倍。雙標(biāo)免疫熒光結(jié)果提示,與FNF相比,AS患者髖關(guān)節(jié)韌帶組織血管密度增加,Fibronectin,Laminin表達(dá)水平明顯降低,這些結(jié)果證明在AS髖關(guān)節(jié)韌帶組織中血管周圍基質(zhì)降解增加,血管再生增加。結(jié)論:與FNF髖關(guān)節(jié)韌帶組織相比,AS髖關(guān)節(jié)韌帶組織中基質(zhì)降解增加,血管再生增加。第二部分:強(qiáng)直性脊柱炎髖關(guān)節(jié)韌帶組織及成纖維細(xì)胞中Ang1表達(dá)增加,TNF-α刺激AS成纖維細(xì)胞后Ang1及Tie2表達(dá)增加研究背景:血管再生過程中,許多細(xì)胞因子參與其中,包括血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF),成纖維細(xì)胞生長因子,血管生成素家族(Angiopoietins,Angs)及其受體等。本課題組之前的基因芯片結(jié)果已經(jīng)證實(shí)Angs家族中的Ang1在AS髖關(guān)節(jié)韌帶組織中表達(dá)上調(diào)。研究目的:本部分實(shí)驗(yàn)中,我們將進(jìn)一步證明Ang1、Ang2、Tie2受體及CD31在AS髖關(guān)節(jié)韌帶組織中表達(dá)水平的變化,Ang1,Ang2在成纖維細(xì)胞中表達(dá)水平的變化。TNF-α刺激成纖維細(xì)胞,探索引起AS中Ang1表達(dá)上調(diào)的原因。實(shí)驗(yàn)方法:首先,我們通過RT-PCR在基因水平檢測AS髖關(guān)節(jié)組織中Ang1,Ang2,Tie2表達(dá)水平的變化,通過Western-Blot在蛋白水平檢測AS測髖關(guān)節(jié)組織中Ang1,Ang2,Tie2表達(dá)水平的變化。通過免疫組化對Ang1,Ti2及CD31行半定量和定位檢測,通過CD31與Tie2雙標(biāo)免疫熒光法定位Tie2表達(dá)部位。其次,獲取韌帶組織后,膠原酶消化法獲取成纖維細(xì)胞,通過CD90和CD29鑒定成纖維細(xì)胞。通過RT-PCR和Western-Blot方法分別檢測AS成纖維細(xì)胞中Ang1,Ang2及Tie2在基因水平和蛋白水平的變化。通過Ang1與CD90雙標(biāo)免疫熒光方法檢測AS成纖維細(xì)胞中Ang1表達(dá)變化,并對Ang1合成部位進(jìn)行定位分析。通過ELISA的方法檢測成纖維細(xì)胞培養(yǎng)上清液中Ang1,Ang2濃度。最后,為了進(jìn)一步探討引起AS成纖維細(xì)胞中Ang1表達(dá)上調(diào)的原因,我們用TNF-α刺激成纖維細(xì)胞后檢測Ang1配體和Tie2受體的表達(dá)。實(shí)驗(yàn)結(jié)果:RT-PCR結(jié)果提示,與FNF的髖關(guān)節(jié)韌帶組織相對比,AS的髖關(guān)節(jié)韌帶組織中Ang1,Ang2,Tie2表達(dá)分別上調(diào)5.32倍,3.56倍和2.13倍。Western-Blot結(jié)果提示,AS髖關(guān)節(jié)韌帶組織中Ang1,Ang2,Tie2的表達(dá)增加,對其定量分析提示,AS髖關(guān)節(jié)韌帶組織中Ang1(P0.001),Ang2(P0.001),Tie2(P0.001)蛋白的灰度值明顯高于FNF,而Ang1灰度值/Ang2灰度值的比值沒有差異。免疫組化的結(jié)果提示,AS髖關(guān)節(jié)韌帶組織中Ang1,CD31,Tie2蛋白表達(dá)均上調(diào),Ang1蛋白在血管周圍及其遠(yuǎn)離血管的部位均有表達(dá),Tie2主要在血管周圍表達(dá)。CD31與Tie2雙標(biāo)免疫熒光結(jié)果證實(shí)Tie2在主要在內(nèi)皮細(xì)胞上表達(dá)。細(xì)胞鑒定結(jié)果提示,膠原酶消化法獲得的細(xì)胞81.1%為成纖維細(xì)胞。成纖維細(xì)胞RT-PCR與Western-Blot的結(jié)果提示,AS成纖維細(xì)胞中Ang1在基因水平及蛋白水平表達(dá)均上調(diào)。CD90與Ang1雙標(biāo)免疫熒光結(jié)果提示Ang1在AS成纖維細(xì)胞胞漿中的表達(dá)明顯高于FNF,ELISA結(jié)果提示,AS成纖維細(xì)胞上清培養(yǎng)液中Ang1的濃度明顯高于FNF(36.3±5.08 ng/ml VS 17.06±1.54 ng/ml,p0.001)。當(dāng)用TNF-α刺激AS成纖維細(xì)胞后,Ang1以劑量依賴的方式表達(dá)上調(diào),并且Ang1的受體Tie2也以劑量依賴的方式表達(dá)上調(diào),FNF成纖維細(xì)胞中未發(fā)現(xiàn)這種情況。結(jié)論:與FNF相比,Ang1,Ang2,Tie2無論基因水平還是蛋白水平在AS髖關(guān)節(jié)韌帶組織中均表達(dá)增加。Tie2受體在AS髖關(guān)節(jié)韌帶組織中表達(dá)增加,并且主要在的內(nèi)皮細(xì)胞中表達(dá)。AS成纖維細(xì)胞中,Ang1在基因和蛋白水平均表達(dá)增加,由胞漿合成分泌至細(xì)胞外,通過旁分泌的方式作用在韌帶組織中的內(nèi)皮細(xì)胞上。TNF-α能刺激AS髖關(guān)節(jié)韌帶成纖維細(xì)胞,Ang1配體及Tie2受體表達(dá)上調(diào),可能與AS本身的炎癥性病理變化有關(guān)。第三部分:AS成纖維細(xì)胞上清液及COMP-Ang1促進(jìn)了內(nèi)皮細(xì)胞的遷移和小管形成研究背景:血管再生由血管周圍環(huán)境所決定,當(dāng)周圍環(huán)境不能為組織提供足夠的營養(yǎng)物質(zhì)和氧氣時(shí),低氧組織就是釋放促血管再生因子,促使內(nèi)皮細(xì)胞從已有血管的上出芽形成新的血管。血管再生的過程中,血管周圍基質(zhì)的降解是第一步,第一部分實(shí)驗(yàn)已經(jīng)證實(shí)。隨后內(nèi)皮細(xì)胞從原有血管的上通過出芽,形成分枝,每一個(gè)新的出芽都會(huì)通過尖端細(xì)胞與周圍的出芽連接起來,最終形成連續(xù)的管腔。血管再生是多基因共同作用的結(jié)果,那么,COMP-Ang1與成纖維細(xì)胞上清液能否促進(jìn)內(nèi)皮細(xì)胞的遷移和小管形成呢?研究目的:本部分實(shí)驗(yàn)的目的是檢測AS和FNF成纖維細(xì)胞細(xì)胞上清液中Ang1/Tie2信號(hào)系統(tǒng)是否能促進(jìn)內(nèi)皮細(xì)胞的遷移和小管形成,同時(shí)用Ang1的重組蛋白COMP-Ang1檢測其是否促進(jìn)內(nèi)皮細(xì)胞的遷移和小管形成,以驗(yàn)證Ang1促進(jìn)血管再生的作用。實(shí)驗(yàn)方法:通過劃痕實(shí)驗(yàn)證明Ang1/Tie2信號(hào)系統(tǒng)能促進(jìn)內(nèi)皮細(xì)胞的遷移,通過體外小管形成實(shí)驗(yàn)證明Ang1/Tie2信號(hào)系統(tǒng)能促進(jìn)內(nèi)皮細(xì)胞的血管管腔形成。實(shí)驗(yàn)結(jié)果:實(shí)驗(yàn)結(jié)果提示,AS與FNF成纖維細(xì)胞條件培養(yǎng)基均能促進(jìn)內(nèi)皮細(xì)胞的遷移和小管形成,AS成纖維細(xì)胞條件培養(yǎng)基的作用更加明顯。COMP-Ang1以劑量依賴方式促進(jìn)內(nèi)皮細(xì)胞的遷移和小管形成,當(dāng)在內(nèi)皮細(xì)胞培養(yǎng)液中加入Tie2抑制劑后,內(nèi)皮細(xì)胞的遷移和小管形成明顯被抑制。說明Ang1/Tie2信號(hào)系統(tǒng)能促進(jìn)內(nèi)皮細(xì)胞遷移和小管形成。結(jié)論:Ang1/Tie2系統(tǒng)通過促進(jìn)內(nèi)皮細(xì)胞遷移和管腔形成而促進(jìn)血管再生。第四部分:Ang1通過Notch-1信號(hào)通路調(diào)節(jié)血管再生研究背景:我們的實(shí)驗(yàn)研究已經(jīng)證明Ang1/Tie2在AS髖關(guān)節(jié)韌帶及成纖維細(xì)胞中高表達(dá),并且能促進(jìn)內(nèi)皮細(xì)胞的遷移和小管形成。然而,目前Ang1/Tie2通過何種信號(hào)通路促進(jìn)血管再生的機(jī)制還不完全明確,研究表明在類風(fēng)濕性關(guān)節(jié)炎的滑膜中,VEGF/Ang2能通過Notch-1信號(hào)通路共同促進(jìn)血管形成[1],也有研究表明,酒精以Notch-1信號(hào)通路和Ang1依賴的方式促進(jìn)內(nèi)皮細(xì)胞的成血管活性[2]。然而,Ang1/Tie2信號(hào)系統(tǒng)與Notch-1信號(hào)通路在調(diào)節(jié)血管再生之間的相互關(guān)系并沒有研究。研究目的:本部分實(shí)驗(yàn)旨在研究Ang1/Tie2信號(hào)系統(tǒng)與Notch-1信號(hào)通路之間的關(guān)系,證明Ang1/Tie2信號(hào)系統(tǒng)通過Notch-1信號(hào)通路調(diào)節(jié)內(nèi)皮細(xì)胞的遷移和小管形成。實(shí)驗(yàn)方法:首先,驗(yàn)證COMP-Ang1是否激活Notch1信號(hào)通路。用不同濃度的COMP-Ang1刺激內(nèi)皮細(xì)胞,通過RT-PCR從基因水平檢測Notch-1及其下游靶基因Hey1,Hey2,Hes5的變化,通過Western-Blot從蛋白水平檢測Notch-1受體及其配體DLL-4表達(dá)的變化。隨后,COMP-Ang1刺激內(nèi)皮細(xì)胞的同時(shí)加入Tie2受體抑制劑抑制Ang1/Tie2信號(hào)系統(tǒng),同樣通過RT-PCR從基因水平檢測Notch-1及其下游靶基因Hey1,Hey2,Hes5的變化,通過Western-Blot從蛋白水平檢測Notch-1受體及其配體DLL-4表達(dá)的變化。其次,驗(yàn)證Ang1/Tie2信號(hào)系統(tǒng)通過Notch-1信號(hào)通路調(diào)節(jié)血管再生,以不同的COMP-Ang1濃度刺激內(nèi)皮細(xì)胞,檢測其對內(nèi)皮細(xì)胞的遷移和小管形成的影響,隨后在加入COMP-Ang1刺激內(nèi)皮細(xì)胞的同時(shí)加入γ分泌酶抑制劑DAPT抑制Notch-1信號(hào)通路,檢測其對內(nèi)皮細(xì)胞的遷移和小管形成實(shí)驗(yàn)的影響。實(shí)驗(yàn)結(jié)果:不同濃度的COMP-Ang1刺激內(nèi)皮細(xì)胞后,Notch-1受體及其DLL-4配體在內(nèi)皮細(xì)胞中以劑量依賴的方式表達(dá)上調(diào),其下游的靶基因Hey1,Hey2,Hes5表達(dá)也上調(diào)。同樣不同濃度的COMP-Ang1刺激內(nèi)皮細(xì)胞,同時(shí)抑制Tie2受體后,與無COMP-Ang1刺激組內(nèi)皮細(xì)胞相對比,Notch-1受體及DLL-4配體表達(dá)無變化,同時(shí)其下游的靶基因Hey1,Hey2,Hes5表達(dá)也無明顯變化。當(dāng)以不同濃度的COMP-Ang1刺激內(nèi)皮細(xì)胞后,內(nèi)皮細(xì)胞的遷移和小管形成增加。不同濃度的COMP-Ang1刺激內(nèi)皮細(xì)胞的同時(shí)抑制Notch-1信號(hào)通路后,與無COMP-Ang1刺激組內(nèi)皮細(xì)胞相對比,內(nèi)皮細(xì)胞的遷移和小管形成無明顯變化。結(jié)論:Ang-1/Tie2信號(hào)系統(tǒng)可能通過Notch-1信號(hào)通路調(diào)節(jié)血管再生。
[Abstract]:The first part: the research background of the matrix degradation and angiogenesis of the hip ligament in ankylosing spondylitis: Ankylosing Spondylitis (AS) is a kind of self inflammatory disease. Inflammation often involves the attachment point of the tendon and ligament of the middle and peripheral joints, and the ligamentous ligament is easily formed at the point of the late ligament attachment. Inflammation and new bone formation are often accompanied by vascular regeneration. The hip joint is the most frequently involved large joint in AS. There is no study on vascular regeneration in the AS hip ligament at home and abroad. Objective: in this part of the study, we obtained the AS and the femoral neck fracture (FNA) during the operation. The periarticular ligaments were used as the experimental group and the control group to investigate whether the AS periarticular ligaments were associated with vascular regeneration. Experimental methods: the degradation of the perivascular matrix was the first step of vascular regeneration. We detected fibronectin (fibronectin), laminin (Laminin), type 1 collagen (Collagen 1), and type 4 collagen (Collage (Collage)). N 4), matrix metalloproteinases (matrix metalloproteinases, MMP1, MMP2, MMP9), the changes in the expression level of matrix metalloproteinase inhibitors (inhibitors of matrix metalloproteinases, TIMP1, TIMP2, and TIMP2) to prove that the degradation of the perivascular matrix is the surface marker of the endothelial cells of the blood tube, and it is capable of reacting the blood. CD31 and Fibronectin, CD31 and Laminin double standard immunofluorescence methods were used to demonstrate the degradation of matrix around the hip joint, blood vessel density and its morphological changes. Experimental results: RT-PCR results suggested that the MMP1, MMP2, MMP9 expression in the hip ligament tissue of AS patients increased by 7., respectively, compared with the FNF hip ligament. 21 times, 2.17 times and 2.19 times, while Fibronectin, Laminin, Collagen1, Collagen4, TIMP1, TIMP2, TIMP3, respectively, decreased 1.92 times, 2.44 times, 2.04 times, 2.63 times, 2.33 times, 10.73 times, 2.78 times. The double standard immunofluorescence results showed that the density of hip toughened tissue and vascular density in AS patients was increased compared with FNF, and Fibronectin, Laminin expression levels decreased significantly. Some results showed that the degradation of the perivascular matrix in the AS hip ligament increased and the vascular regeneration increased. Conclusion: compared with the FNF hip ligament tissue, the matrix degradation in the AS hip ligament tissue increased and the vascular regeneration increased. The second part: the increased expression of Ang1 in the hip ligament tissue and fibroblasts of ankylosing spondylitis, TNF- alpha spines. The expression of Ang1 and Tie2 increased after stimulated AS fibroblasts. In the process of vascular regeneration, many cytokines were involved, including vascular endothelial growth factor (vascular endothelial growth factor, VEGF), fibroblast growth factor, angiopoietin family (Angiopoietins, Angs) and their receptors. The results have proved that Ang1 in the Angs family is up to up in the AS hip ligament tissue. Objective: in this part of the study, we will further demonstrate the changes in the expression level of Ang1, Ang2, Tie2 receptor and CD31 in the ligament tissue of AS hip joint, Ang1, Ang2 in the fibroblast cell expression level,.TNF- alpha stimulates fibroblasts. To explore the reasons for the up-regulated expression of Ang1 in AS. First, we detected the changes in the expression level of Ang1, Ang2, Tie2 in the hips of AS by RT-PCR at the gene level. We detected the changes in Ang1, Ang2, Tie2 expression level in the hip tissue by Western-Blot at the protein level. Quantitative and localization detection was used to locate Tie2 expression site by CD31 and Tie2 double standard immunofluorescence. Secondly, after obtaining ligaments, fibroblasts were obtained by collagenase digestion, and fibroblasts were identified by CD90 and CD29. RT-PCR and Western-Blot methods were used to detect Ang1, Ang2 and Tie2 in the gene level and protein water in AS fibroblasts. Ang1 and CD90 double standard immunofluorescence methods were used to detect the changes of Ang1 expression in AS fibroblasts and to locate the Ang1 synthesis site. The ELISA method was used to detect the Ang1, Ang2 concentration in the supernatant of fibroblast culture. Finally, the reasons for the up regulation of Ang1 expression in the AS fibroblasts were further discussed. The expression of Ang1 ligand and Tie2 receptor was detected with TNF- alpha stimulation of fibroblasts. Experimental results: RT-PCR results suggest that the expression of Ang1, Ang2, and Tie2 in the hip ligament tissue of AS is up to 5.32 times, 3.56 times and 2.13 times.Western-Blot, respectively, compared with FNF's hip ligament tissue. The quantitative analysis showed that the gray value of Ang1 (P0.001), Ang2 (P0.001) and Tie2 (P0.001) protein in the AS hip ligament was significantly higher than that of FNF, while the ratio of Ang1 gray value /Ang2 gray value was not different. Tie2 was expressed around the blood vessels, and the.CD31 and Tie2 double labeled immunofluorescence showed that Tie2 was mainly expressed on the endothelial cells. The results of cell identification suggested that 81.1% of the cells obtained by collagenase digestion were fibroblasts. The results of fibroblast cell RT-PCR and Western-Blot suggest that AS is a fibrous cell. The expression of Ang1 at the gene level and protein level in the vascular cells all up-regulated the.CD90 and Ang1 double standard immunofluorescence results, suggesting that the expression of Ang1 in the cytoplasm of AS fibroblasts was significantly higher than that of FNF. ELISA results suggested that the concentration of Ang1 in the culture medium of AS fibroblasts was significantly higher than that of FNF (36.3 + 5.08 ng/ml VS 17.06 + 1.54). After alpha stimulation of AS fibroblasts, Ang1 was up-regulated in a dose-dependent manner, and Ang1 receptor Tie2 was also up-regulated in a dose-dependent manner. No such situation was found in FNF fibroblasts. Conclusion: Ang1, Ang2, and Tie2, both in Ang1, Ang2, and protein levels, are increased in.Tie2 receptor in the AS hip ligament tissue compared with FNF. The expression of the body is increased in the AS hip ligament tissue, and the expression of the.AS fibroblasts in the endothelial cells is mainly expressed in the endothelial cells. The expression of Ang1 is increased at the level of gene and protein, and is secreted from the cytoplasm to the extracellular. The.TNF- alpha in the endothelial cells in the ligamentum tissue can stimulate the AS ligament fibroblasts by the paracrine way. The up-regulated expression of Ang1 ligand and Tie2 receptor may be associated with inflammatory pathological changes of AS itself. Third part: AS fibroblast supernatant and COMP-Ang1 promote endothelial cell migration and tubule formation research background: vascular regeneration is determined by the peripheral environment, when the surrounding environment can not provide adequate nutrients and oxygen for tissue. In the process of regeneration of blood vessels, the degradation of the perivascular matrix is the first step, and the first part of the experiment has been confirmed. Then the endothelial cells form branches from the original blood vessels and form branching, each new bud. The results of COMP-Ang1 and fibroblast supernatants promote endothelial cell migration and tubule formation. Purpose: the purpose of this division is to detect AS and FNF fibroblast cells. Whether the Ang1/Tie2 signal system in the supernatant can promote the migration of endothelial cells and the formation of tubules. At the same time, using the recombinant protein COMP-Ang1 of Ang1 to detect the migration of endothelial cells and the formation of tubules, in order to verify the effect of Ang1 on promoting vascular regeneration. In vitro microtubule formation experiments show that Ang1/Tie2 signal system can promote the formation of vascular lumen in endothelial cells. Experimental results suggest that both AS and FNF fibroblast conditioned medium can promote endothelial cell migration and tubule formation, and the role of AS fibroblast conditioned medium is more.COMP-Ang1. The dose-dependent manner promotes endothelial cell migration and tubule formation. When Tie2 inhibitors are added to the endothelial cell culture, endothelial cell migration and tubule formation are obviously inhibited. The Ang1/Tie2 signal system can promote endothelial cell migration and tubule formation. Conclusion: the Ang1/ Tie2 system promotes endothelial cell migration and lumen by promoting endothelial cell migration and tubule formation. Formation and promoting vascular regeneration. Fourth part: Ang1 regulation of vascular regeneration through Notch-1 signaling: our experimental study has demonstrated that Ang1/Tie2 is highly expressed in the AS hip ligament and fibroblasts, and can promote endothelial cell migration and tubule formation. But what signal pathway is promoted by Ang1/Tie2 at present The mechanism of vascular regeneration is not completely clear. The study shows that in the synovial membrane of rheumatoid arthritis, VEGF/Ang2 can promote the formation of vascular [1] through the Notch-1 signaling pathway, and some studies suggest that alcohol promotes vascular activity [2]. in endothelial cells by Notch-1 signaling pathway and Ang1 dependence, however, Ang1/Tie2 signal system and Notch The relationship between the -1 signaling pathway and the regulation of vascular regeneration has not been studied. The purpose of this study is to study the relationship between the Ang1/Tie2 signal system and the Notch-1 signaling pathway and to prove that the Ang1/Tie2 signal system regulates the migration of endothelial cells and the formation of the tubules through the Notch-1 signaling pathway. Whether G1 activates the Notch1 signaling pathway, stimulates the endothelial cells with different concentrations of COMP-Ang1, and detects the changes of Notch-1 and its downstream target gene Hey1, Hey2, Hes5 through RT-PCR at the gene level. The DLL-4 expression of the Notch-1 receptor and its ligand is detected by Western-Blot from the protein level. Subsequently, COMP-Ang1 stimulates endothelial cells to join simultaneously. The IE2 receptor inhibitor inhibits the Ang1/Tie2 signal system, and also detects the changes of Notch-1 and its downstream target gene Hey1, Hey2, Hes5 through RT-PCR. The changes in the expression of Notch-1 receptor and its ligand DLL-4 are detected by Western-Blot from the protein level. Secondly, the Ang1/Tie2 signal system is verified to regulate vascular regeneration through the Notch-1 signal pathway. The effects of the endothelial cells on endothelial cell migration and tubule formation were detected with different COMP-Ang1 concentrations, and the effects on the migration of endothelial cells and the tubule formation experiment were detected by adding the gamma secretase inhibitor DAPT to the endothelial cells with COMP-Ang1 stimulation, and the effects on the endothelial cell migration and tubule formation experiments were detected. After the COMP-Ang1 stimulation of the endothelial cells, the Notch-1 receptor and its DLL-4 ligand are up-regulated in a dose dependent manner in endothelial cells. The target genes downstream of the target gene, Hey1, Hey2, and Hes5 are also up-regulated. The COMP-Ang1 stimulates the endothelial cells of different concentrations and inhibits Tie2 receptors, and is compared with those without the COMP-Ang1 stimulation group, Notch. There was no change in the expression of -1 receptor and DLL-4 ligand, and there was no obvious change in the expression of target gene Hey1, Hey2 and Hes5 in the downstream. After stimulating endothelial cells with different concentrations of COMP-Ang1, the migration of endothelial cells and the formation of tubules were increased. The COMP-Ang1 stimulation of different concentrations of COMP-Ang1 inhibited Notch-1 signaling pathway, and without COMP-Ang1 spines. There was no obvious change in endothelial cell migration and tubule formation in stimulated group of endothelial cells. Conclusion: Ang-1/Tie2 signaling system may regulate vascular regeneration through Notch-1 signaling pathway.

【學(xué)位授予單位】:第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2017
【分類號(hào)】:R593.23

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