ANG-1在AS髋关节韧带中表达增加并通过Notch信号通路调节血管再生

发布时间：2018-04-28 11:07

本文选题：强直性脊柱炎 + 全髋关节置换　；参考：《第二军医大学》2017年博士论文

【摘要】：第一部分:强直性脊柱炎髋关节韧带基质降解和血管生成增加研究背景:强直性脊柱炎(Ankylosing Spondylitis,AS)是一种自身炎症性疾病,炎症常常累及中轴关节及外周大关节的肌腱韧带附着点,晚期韧带肌腱附着点部位易形成韧带骨赘,而炎症和新骨形成常伴发有血管再生。髋关节作为AS中最常受累的外周大关节,目前国内外的研究中,还没有关于AS髋关节韧带中血管再生方面的研究。研究目的:本部分实验中,我们在手术的过程中获取AS和股骨颈骨折(Femur Neck Fracture,FNA)髋关节周围韧带分别作为实验组和对照组,研究AS髋关节周围韧带是否伴发有血管再生增加。实验方法:血管周围基质降解是血管再生的第一步。我们通过RT-PCR检测纤连蛋白(fibronectin),层黏连蛋白(Laminin),1型胶原(Collagen 1),4型胶原(Collagen 4),基质金属蛋白酶(matrix metalloproteinases,MMP1,MMP2,MMP9),基质金属蛋白酶抑制剂(inhibitors of matrix metalloproteinases,TIMP1,TIMP2,TIMP3)表达水平的变化来证明血管周围基质降解情况。CD31是血管内皮细胞的表面标志物,Fibronectin和Laminin是能反应血管周围基质降解情况,我们通过CD31与Fibronectin,CD31与Laminin双标免疫荧光的方法来证明AS髋关节周围基质降解,血管密度及其形态的变化。实验结果:RT-PCR结果提示,与FNF髋关节韧带对比,AS患者髋关节韧带组织中MMP1,MMP2,MMP9表达分别增加7.21倍,2.17倍和2.19倍,而Fibronectin,Laminin,Collagen1,Collagen4,TIMP1,TIMP2,TIMP3表达分别降低1.92倍,2.44倍,2.04倍,2.63倍,2.33倍,10.73倍,2.78倍。双标免疫荧光结果提示,与FNF相比,AS患者髋关节韧带组织血管密度增加,Fibronectin,Laminin表达水平明显降低,这些结果证明在AS髋关节韧带组织中血管周围基质降解增加,血管再生增加。结论:与FNF髋关节韧带组织相比,AS髋关节韧带组织中基质降解增加,血管再生增加。第二部分:强直性脊柱炎髋关节韧带组织及成纤维细胞中Ang1表达增加,TNF-α刺激AS成纤维细胞后Ang1及Tie2表达增加研究背景:血管再生过程中,许多细胞因子参与其中,包括血管内皮生长因子(vascular endothelial growth factor,VEGF),成纤维细胞生长因子,血管生成素家族(Angiopoietins,Angs)及其受体等。本课题组之前的基因芯片结果已经证实Angs家族中的Ang1在AS髋关节韧带组织中表达上调。研究目的:本部分实验中,我们将进一步证明Ang1、Ang2、Tie2受体及CD31在AS髋关节韧带组织中表达水平的变化,Ang1,Ang2在成纤维细胞中表达水平的变化。TNF-α刺激成纤维细胞,探索引起AS中Ang1表达上调的原因。实验方法:首先,我们通过RT-PCR在基因水平检测AS髋关节组织中Ang1,Ang2,Tie2表达水平的变化,通过Western-Blot在蛋白水平检测AS测髋关节组织中Ang1,Ang2,Tie2表达水平的变化。通过免疫组化对Ang1,Ti2及CD31行半定量和定位检测,通过CD31与Tie2双标免疫荧光法定位Tie2表达部位。其次,获取韧带组织后,胶原酶消化法获取成纤维细胞,通过CD90和CD29鉴定成纤维细胞。通过RT-PCR和Western-Blot方法分别检测AS成纤维细胞中Ang1,Ang2及Tie2在基因水平和蛋白水平的变化。通过Ang1与CD90双标免疫荧光方法检测AS成纤维细胞中Ang1表达变化,并对Ang1合成部位进行定位分析。通过ELISA的方法检测成纤维细胞培养上清液中Ang1,Ang2浓度。最后,为了进一步探讨引起AS成纤维细胞中Ang1表达上调的原因,我们用TNF-α刺激成纤维细胞后检测Ang1配体和Tie2受体的表达。实验结果:RT-PCR结果提示,与FNF的髋关节韧带组织相对比,AS的髋关节韧带组织中Ang1,Ang2,Tie2表达分别上调5.32倍,3.56倍和2.13倍。Western-Blot结果提示,AS髋关节韧带组织中Ang1,Ang2,Tie2的表达增加,对其定量分析提示,AS髋关节韧带组织中Ang1(P0.001),Ang2(P0.001),Tie2(P0.001)蛋白的灰度值明显高于FNF,而Ang1灰度值/Ang2灰度值的比值没有差异。免疫组化的结果提示,AS髋关节韧带组织中Ang1,CD31,Tie2蛋白表达均上调,Ang1蛋白在血管周围及其远离血管的部位均有表达,Tie2主要在血管周围表达。CD31与Tie2双标免疫荧光结果证实Tie2在主要在内皮细胞上表达。细胞鉴定结果提示,胶原酶消化法获得的细胞81.1%为成纤维细胞。成纤维细胞RT-PCR与Western-Blot的结果提示,AS成纤维细胞中Ang1在基因水平及蛋白水平表达均上调。CD90与Ang1双标免疫荧光结果提示Ang1在AS成纤维细胞胞浆中的表达明显高于FNF,ELISA结果提示,AS成纤维细胞上清培养液中Ang1的浓度明显高于FNF(36.3±5.08 ng/ml VS 17.06±1.54 ng/ml,p0.001)。当用TNF-α刺激AS成纤维细胞后,Ang1以剂量依赖的方式表达上调,并且Ang1的受体Tie2也以剂量依赖的方式表达上调,FNF成纤维细胞中未发现这种情况。结论:与FNF相比,Ang1,Ang2,Tie2无论基因水平还是蛋白水平在AS髋关节韧带组织中均表达增加。Tie2受体在AS髋关节韧带组织中表达增加,并且主要在的内皮细胞中表达。AS成纤维细胞中,Ang1在基因和蛋白水平均表达增加,由胞浆合成分泌至细胞外,通过旁分泌的方式作用在韧带组织中的内皮细胞上。TNF-α能刺激AS髋关节韧带成纤维细胞,Ang1配体及Tie2受体表达上调,可能与AS本身的炎症性病理变化有关。第三部分:AS成纤维细胞上清液及COMP-Ang1促进了内皮细胞的迁移和小管形成研究背景:血管再生由血管周围环境所决定,当周围环境不能为组织提供足够的营养物质和氧气时,低氧组织就是释放促血管再生因子,促使内皮细胞从已有血管的上出芽形成新的血管。血管再生的过程中,血管周围基质的降解是第一步,第一部分实验已经证实。随后内皮细胞从原有血管的上通过出芽,形成分枝,每一个新的出芽都会通过尖端细胞与周围的出芽连接起来,最终形成连续的管腔。血管再生是多基因共同作用的结果,那么,COMP-Ang1与成纤维细胞上清液能否促进内皮细胞的迁移和小管形成呢?研究目的:本部分实验的目的是检测AS和FNF成纤维细胞细胞上清液中Ang1/Tie2信号系统是否能促进内皮细胞的迁移和小管形成,同时用Ang1的重组蛋白COMP-Ang1检测其是否促进内皮细胞的迁移和小管形成,以验证Ang1促进血管再生的作用。实验方法:通过划痕实验证明Ang1/Tie2信号系统能促进内皮细胞的迁移,通过体外小管形成实验证明Ang1/Tie2信号系统能促进内皮细胞的血管管腔形成。实验结果:实验结果提示,AS与FNF成纤维细胞条件培养基均能促进内皮细胞的迁移和小管形成,AS成纤维细胞条件培养基的作用更加明显。COMP-Ang1以剂量依赖方式促进内皮细胞的迁移和小管形成,当在内皮细胞培养液中加入Tie2抑制剂后,内皮细胞的迁移和小管形成明显被抑制。说明Ang1/Tie2信号系统能促进内皮细胞迁移和小管形成。结论:Ang1/Tie2系统通过促进内皮细胞迁移和管腔形成而促进血管再生。第四部分:Ang1通过Notch-1信号通路调节血管再生研究背景:我们的实验研究已经证明Ang1/Tie2在AS髋关节韧带及成纤维细胞中高表达,并且能促进内皮细胞的迁移和小管形成。然而,目前Ang1/Tie2通过何种信号通路促进血管再生的机制还不完全明确,研究表明在类风湿性关节炎的滑膜中,VEGF/Ang2能通过Notch-1信号通路共同促进血管形成[1],也有研究表明,酒精以Notch-1信号通路和Ang1依赖的方式促进内皮细胞的成血管活性[2]。然而,Ang1/Tie2信号系统与Notch-1信号通路在调节血管再生之间的相互关系并没有研究。研究目的:本部分实验旨在研究Ang1/Tie2信号系统与Notch-1信号通路之间的关系,证明Ang1/Tie2信号系统通过Notch-1信号通路调节内皮细胞的迁移和小管形成。实验方法:首先,验证COMP-Ang1是否激活Notch1信号通路。用不同浓度的COMP-Ang1刺激内皮细胞,通过RT-PCR从基因水平检测Notch-1及其下游靶基因Hey1,Hey2,Hes5的变化,通过Western-Blot从蛋白水平检测Notch-1受体及其配体DLL-4表达的变化。随后,COMP-Ang1刺激内皮细胞的同时加入Tie2受体抑制剂抑制Ang1/Tie2信号系统,同样通过RT-PCR从基因水平检测Notch-1及其下游靶基因Hey1,Hey2,Hes5的变化,通过Western-Blot从蛋白水平检测Notch-1受体及其配体DLL-4表达的变化。其次,验证Ang1/Tie2信号系统通过Notch-1信号通路调节血管再生,以不同的COMP-Ang1浓度刺激内皮细胞,检测其对内皮细胞的迁移和小管形成的影响,随后在加入COMP-Ang1刺激内皮细胞的同时加入γ分泌酶抑制剂DAPT抑制Notch-1信号通路,检测其对内皮细胞的迁移和小管形成实验的影响。实验结果:不同浓度的COMP-Ang1刺激内皮细胞后,Notch-1受体及其DLL-4配体在内皮细胞中以剂量依赖的方式表达上调,其下游的靶基因Hey1,Hey2,Hes5表达也上调。同样不同浓度的COMP-Ang1刺激内皮细胞,同时抑制Tie2受体后,与无COMP-Ang1刺激组内皮细胞相对比,Notch-1受体及DLL-4配体表达无变化,同时其下游的靶基因Hey1,Hey2,Hes5表达也无明显变化。当以不同浓度的COMP-Ang1刺激内皮细胞后,内皮细胞的迁移和小管形成增加。不同浓度的COMP-Ang1刺激内皮细胞的同时抑制Notch-1信号通路后,与无COMP-Ang1刺激组内皮细胞相对比,内皮细胞的迁移和小管形成无明显变化。结论:Ang-1/Tie2信号系统可能通过Notch-1信号通路调节血管再生。
[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.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.23

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