SIRT1延缓小鼠下肢缺血后血流恢复的机制研究

发布时间：2017-12-31 07:11

本文关键词：SIRT1延缓小鼠下肢缺血后血流恢复的机制研究　出处：《河北医科大学》2017年博士论文　论文类型：学位论文

【摘要】：正常组织功能的维持依赖于血管供氧和营养物质。组织缺血后血流恢复速度取决于血管新生和动脉生成过程。沉默信息调节因子1(silent information regulator 1,SIRT1)是一类依赖于NAD的去乙酰化酶,广泛参与调节细胞生理活动和病理过程。有报道发现SIRT1通过抑制肿瘤血管新生而降低肿瘤生长,然而其对动脉生成的作用还不十分清楚,本研究旨在探寻SIRT1对动脉生成的调节及机制。目的:本研究以Sirt1转基因(Sirt1-transgenic,Sirt1-Tg)小鼠和血管平滑肌细胞作为研究对象,通过建立下肢缺血模型和低氧诱导模型,试图从体内外证实SIRT1在动脉生成中的作用,并探讨其作用机制,以期为揭示缺血及缺血相关心血管疾病的发生机制提供研究证据。方法:利用Sirt1-Tg小鼠建立股动脉结扎下肢缺血模型,评价SIRT1与组织缺血后血流恢复的关系;应用SIRT1激动剂和抑制剂进一步验证血流恢复与SIRT1活性的关系;利用免疫荧光染色分析缺血下肢腓肠肌毛细血管和小动脉密度;利用内皮细胞增殖、迁移、成管等体外实验和基质胶塞、伤口愈合、视网膜铺片等体内实验,确定SIRT1活性和血管新生的关系;利用蛋白质免疫印迹、免疫共沉淀和免疫沉淀等技术,从蛋白质相互作用、蛋白质修饰的角度探讨SIRT1对动脉生成的关键因子血小板衍生生长因子B(platelet-derived growth factor B,PDGFB)表达的调节及其分子机制。结果:1 SIRT1激活抑制小鼠下肢缺血后血流恢复1.1 Sirt1-Tg小鼠下肢缺血后血流恢复延迟为了探究SIRT1对组织缺血后血液灌注恢复的影响,小鼠进行股动脉结扎建立下肢缺血模型,采用激光散斑对比分析技术监测下肢血流恢复情况。与WT(wild-type,WT)小鼠比较,Sirt1-Tg小鼠血流恢复明显延迟,SIRT1抑制剂EX527腹腔注射使转基因小鼠血流恢复明显改善。1.2 SIRT1激活抑制小鼠下肢缺血后血流恢复复制小鼠下肢缺血模型,于不同时间点腹腔注射SIRT1激动剂白藜芦醇(resveratrol,RSV)。结果显示,延长用药时间可加重血流恢复的延迟;反之,SIRT1抑制剂EX527可加快小鼠缺血下肢的血流恢复速度。结果表明,SIRT1激活抑制小鼠下肢缺血后血流恢复。1.3 Sirt1-Tg小鼠下肢缺血后腓肠肌动脉生成能力降低为了识别毛细血管和小动脉,在小鼠股动脉结扎后14天,使用CD31(内皮细胞标志物)和平滑肌(smooth muscle,SM)α-actin(平滑肌细胞标志物)抗体对腓肠肌组织切片进行免疫荧光染色。结果显示,Sirt1-Tg小鼠下肢缺血侧腓肠肌组织毛细血管密度(CD31阳性区)和SMα-actin阳性的小动脉密度明显低于WT小鼠,壁细胞(NG2阳性)的比例也显著下降。结果表明,SIRT1激活抑制小鼠下肢缺血后动脉生成。2 SIRT1抑制血管新生2.1 SIRT1激活抑制内皮细胞增殖和成管功能为了探究SIRT1激活对内皮细胞功能的影响,低氧处理RSV或EX527预孵育的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)以及小鼠内皮细胞,结果显示,SIRT1激活抑制两种内皮细胞的增殖活性;SIRT1过表达抑制小鼠内皮细胞的迁移。为了进一步验证SIRT1在血管新生中的作用,采用基质胶进行内皮细胞体外成管实验,RSV预孵育的HUVEC成管能力降低。结果表明,SIRT1激活可抑制内皮细胞增殖、迁移和成管能力。2.2体内SIRT1过表达抑制血管新生为了探究体内SIRT1活性对血管新生的影响,将含有血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)的基质胶植入WT和Sirt1-Tg小鼠皮下,发现Sirt1-Tg小鼠的基质胶塞中的血管扩张和壁细胞(NG2阳性)覆盖降低。并且Sirt1-Tg小鼠的伤口愈合延迟。为进一步验证SIRT1对血管新生的抑制作用,分析出生5天小鼠视网膜毛细血管,发现Sirt1-Tg小鼠视网膜毛细血管分支点的数目及血管长度明显低于WT小鼠;用SIRT1激活剂RSV腹腔注射的新生WT小鼠,其视网膜血管新生受到明显抑制,而应用SIRT1抑制剂EX527处理的小鼠其视网膜血管新生与未处理的对照组比较增强。结果显示,激活SIRT1可抑制血管新生过程。2.3 SIRT1抑制内皮细胞Wnt/β-catenin信号通路和VEGFA表达VEGFA是Wnt/β-catenin信号通路的靶基因之一,在血管新生中发挥重要作用。小鼠股动脉结扎,Western blot检测双下肢腓肠肌,结果显示,Sirt1-Tg小鼠VEGFA表达缺血侧与对照侧的比值明显低于WT小鼠,β-catenin及其上游的DVL3表达水平降低,与VEGFA一致。RSV预孵育的HUVEC,低氧诱导后,VEGFA、β-catenin和DVL3的表达(低氧与常氧的比值)显著低于对照组;反之,EX527预孵育以抑制SIRT1活性,三种蛋白的表达增加。结果表明,SIRT1通过抑制Wnt/β-catenin信号通路继而下调VEGFA的表达。3 SIRT1通过抑制低氧诱导的血管平滑肌细胞PDGFB表达阻碍动脉生成3.1 SIRT1抑制缺血诱导的PDGFB表达为了探究Sirt1-Tg小鼠下肢缺血后动脉生成障碍的分子机制,检测PDGFB表达和ERK活化。Western blot分析显示,Sirt1-Tg小鼠腓肠肌PDGFB表达缺血侧/对照侧比值明显低于WT小鼠,同时p-ERK的比值也显著降低;定量RT-PCR显示,Sirt1-Tg小鼠腓肠肌PDGFB m RNA表达的比值(缺血侧/对照侧)较WT小鼠显著下降。然而,Sirt1-Tg小鼠腹腔注射EX527则可逆转这一现象。说明抑制SIRT1活性可促进缺血诱导的PDGFB表达和信号转导活性。3.2 SIRT1抑制低氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)介导的PDGFB基因转录HIF-1α参与动脉生成。为探究SIRT1抑制PDGFB表达的机制,低氧处理WT小鼠VSMC。结果显示,HIF-1α表达与PDGFB蛋白和m RNA水平成正相关。为了进一步确定HIF-1α参与缺血诱导的PDGFB表达调节,基于PDGFB基因中含有HIF-1α结合位点进行Ch IP分析。结果显示,HIF-1α与PDGFB增强子序列(HBS1)结合活性在低氧诱导后明显增加;构建包含HIF-1α结合位点的的荧光素酶报告基因质粒,转染293T细胞,结果显示,低氧诱导的荧光素酶表达活性升高。过表达SIRT1,则抑制HIF-1α与PDGFB基因HBS1增强子的结合;激活SIRT1(RSV预孵育),同样抑制低氧诱导的荧光素酶报告基因活性;反之,抑制SIRT1活性(EX527预孵育)则促进低氧诱导的荧光素酶表达。结果表明,SIRT1抑制低氧诱导的HIF1α激活及其介导的PDGFB基因转录。3.3 SIRT1介导HIF1α的去乙酰化失活为了探究SIRT1抑制HIF-1α介导的PDGFB表达的分子机制,免疫沉淀分析HIF-1α乙酰化水平。结果显示,低氧可诱导VSMC中HIF-1α乙酰化;过表达或激活SIRT1可降低低氧诱导的HIF-1α乙酰化,同时PDGFB蛋白表达降低;反之用EX527抑制SIRT1活性则可逆转HIF-1α的去乙酰化和PDGFB表达的降低。为了进一步验证SIRT1介导HIF-1α的去乙酰化失活,进行免疫共沉淀检测SIRT1与HIF-1α的相互作用。结果显示,低氧时SIRT1与HIF-1α的相互作用减弱;过表达或激活SIRT1,二者的相互作用增强,而抑制SIRT1活性二者的相互作用减弱。结果表明,SIRT1介导的HIF-1α去乙酰化失活可抑制缺氧诱导的PDGFB基因表达。结论:1 SIRT1激活抑制小鼠下肢缺血后动脉生成,导致下肢血流恢复延迟。2 SIRT1通过抑制Wnt/β-catenin信号通路而下调VEGFA表达,进而抑制内皮细胞增殖和血管新生。3 SIRT1介导的HIF-1α去乙酰化失活可抑制缺氧诱导的PDGFB表达和动脉生成。
[Abstract]:To maintain normal tissue function depends on the blood oxygen and nutrition after ischemia. Blood flow recovery rate depends on angiogenesis and arteriogenesis. Silent information regulator 1 (silent information 1 regulator, SIRT1) is a class of histone deacetylase NAD dependent, widely involved in the regulation of cell physiological activities and pathological processes. SIRT1 through the inhibition of tumor angiogenesis and reduce tumor growth reported, however the artery formation is not clear, the purpose of this study was to explore the effect of SIRT1 on arterial and mechanism of the formation. Objective: in this study, Sirt1 transgenic (Sirt1-transgenic, Sirt1-Tg) and vascular smooth muscle cells of mice as the research object, and through the establishment of model hypoxia induced lower limb ischemia model, trying to confirm the role of SIRT1 in the artery in the generation from outside the body, and to explore its mechanism, in order to reveal the ischemia and lack of Study on the mechanism of blood provide evidence of cardiovascular diseases. Methods: the Sirt1-Tg mice femoral artery ligation model of lower limb ischemia, SIRT1 after ischemia and evaluate the relationship between blood flow recovery; application of SIRT1 agonists and antagonists to further verify the recovery of blood flow relationship with SIRT1 activity; dyeing analysis of ischemic gastrocnemius muscle capillary and arteriolar density by immunofluorescence use; endothelial cell proliferation, migration, tube formation in vitro assay and Matrigel plugs, wound healing, the retinas of mice, to determine the relationship between SIRT1 activity and angiogenesis; using Western blotting, immunoprecipitation and immunoprecipitation, from protein interaction, protein modification of key factor platelet the formation of arterial SIRT1 derived growth factor B (platelet-derived growth factor B, PDGFB) expression and its molecular regulation The mechanism of activation of SIRT1. Results: 1 the recovery of blood flow of mice 1.1 Sirt1-Tg after ischemia of lower limb blood flow recovery delay in order to explore the effects of SIRT1 on blood perfusion recovery after ischemia inhibited limb ischemia in mice, mice model of lower limb ischemia femoral artery ligation, using laser speckle contrast analysis technology to monitor the recovery of lower limb blood flow (wild-type, and WT. WT) mice, Sirt1-Tg mice blood recovery was significantly delayed, intraperitoneal injection of EX527 SIRT1 inhibitor restored to significantly improve.1.2 SIRT1 inhibiting activation of lower extremity ischemia mice after restoration of blood flow replication of lower limb ischemia mouse model of transgenic mice blood flow at different time points by intraperitoneal injection of SIRT1 agonist resveratrol (resveratrol, RSV). The results showed that prolonged treatment delay time can increase the recovery of blood flow; on the other hand, the SIRT1 inhibitor EX527 can accelerate the recovery of blood flow velocity of lower limb ischemia in mice. Results That SIRT1 inhibiting activation of lower extremity ischemia mice after restoration of blood flow.1.3 Sirt1-Tg mice gastrocnemius artery ischemia of lower extremity after generation capacity is reduced in order to identify the capillaries and small arteries, femoral artery ligation in mice after 14 days, the use of CD31 (endothelial cell marker) and smooth muscle (smooth muscle, SM) -actin (alpha smooth muscle cell markers) antibody immunofluorescence staining of tissue sections of gastrocnemius. The results showed that the capillary density in Sirt1-Tg mouse hindlimb ischemia side gastrocnemius muscle (positive CD31) and SM alpha -actin positive arteriolar density was significantly lower than that in WT mice, parietal cells (NG2 positive) proportion decreased significantly. The results showed that the activation of SIRT1 inhibited mice after ischemia of lower extremity artery formation.2 SIRT1 inhibits angiogenesis 2.1 activation of SIRT1 inhibited endothelial cell proliferation and tube function in order to explore the SIRT1 on endothelial cell activation function the influence of hypoxia treatment RSV Or EX527 preincubation of human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC) and the results showed that SIRT1 of endothelial cells in mice, inhibit the activation of two kinds of endothelial cell proliferation activity; overexpression of SIRT1 inhibits the migration of endothelial cells in mice. In order to further verify the role of SIRT1 in angiogenesis, using matrix glue in vitro endothelial cell tube formation, RSV preincubation of HUVEC tube into reduced ability. The results showed that SIRT1 activation can inhibit endothelial cell proliferation, migration and tube formation of.2.2 in vivo overexpression of SIRT1 inhibits angiogenesis in vivo in order to explore the effect of SIRT1 activity on angiogenesis, including vascular endothelial growth factor A (vascular endothelial growth factor A, VEGFA) of the Matrigel implantation of WT and Sirt1-Tg mice, found that vascular dilatation and wall plug stromal cells in Sirt1-Tg mice (NG2 positive) cover down Low. Sirt1-Tg mice and delayed wound healing. Inhibition of angiogenesis to validate the SIRT1 analysis, was born 5 days of retinal capillary in mice, found that the number and length of vessels of Sirt1-Tg mouse retinal capillary branch points was significantly lower than that of WT mice; activator RSV intraperitoneal injection with SIRT1 WT newborn mice, the retinal neovascularization is obvious comparison and application of SIRT1 enhanced inhibition, inhibitor EX527 in mice retinal neovascularization and the untreated control group. The results showed that the activation of SIRT1 can inhibit the angiogenesis of.2.3 SIRT1 inhibition of endothelial cell Wnt/ beta -catenin signaling pathway and the expression of VEGFA VEGFA is one of the target genes of Wnt/ beta -catenin signaling pathway play an important role in angiogenesis. In mice. Femoral artery ligation, Western blot detection of double gastrocnemius muscle, results showed that Sirt1-Tg VEGFA expression in ischemic mice The ratio of side and the control side was significantly lower than that in WT mice, the expression levels of beta -catenin and its upstream DVL3 decreased, consistent with the VEGFA.RSV preincubation of HUVEC induced by hypoxia, VEGFA, -catenin and DVL3 expression of beta (ratio of hypoxia and normoxia) was significantly lower than the control group; on the other hand, preincubation of EX527 to inhibit the activity of SIRT1, increase the expression of the three proteins. The results showed that the.3 of SIRT1 by PDGFB in vascular smooth muscle cells inhibits hypoxia induced expression of SIRT1 3.1 generation block arterial inhibition of ischemia induced PDGFB expression in order to explore the molecular mechanism of Sirt1-Tg mice after hindlimb ischemia artery dyspoietic SIRT1 expression by inhibiting Wnt/ beta -catenin signaling pathway and down-regulation of VEGFA. To detect the expression of PDGFB and activation of ERK.Western blot analysis showed that the expression of Sirt1-Tg in mouse gastrocnemius PDGFB ischemic / control side were significantly lower than those in WT mice, the ratio of p-ERK decreased significantly at the same time; Quantitative RT-PCR showed that the ratio of Sirt1-Tg mice gastrocnemius PDGFB m expression of RNA (ischemic side / control side) was significantly higher than that of WT mice decreased. However, Sirt1-Tg mice by intraperitoneal injection of EX527 can reverse this phenomenon. It inhibited the activity of SIRT1 can promote PDGFB expression and signal transduction of.3.2 activity of ischemia induced SIRT1 inhibition of hypoxia inducible factor 1 alpha (hypoxia-inducible alpha factor alpha 1, HIF-1) mediated PDGFB gene transcription in HIF-1 alpha generation. To explore the mechanism of SIRT1 artery to inhibit the expression of PDGFB, hypoxia treated WT mice VSMC. results showed that HIF-1 expression was positively correlated with PDGFB protein and m RNA level. In order to further determine the HIF-1 alpha is involved in ischemia induced PDGFB expression regulation of PDGFB gene containing HIF-1 alpha binding sites were Ch based on IP analysis. The results showed that HIF-1 alpha and PDGFB enhancer (HBS1) binding activity in hypoxia increased significantly after induction; construct HIF-1 alpha binding sites of luciferase reporter plasmid, the transfected 293T cells showed that hypoxia induced expression of luciferase activity increased. Overexpression of SIRT1 combined inhibition of HIF-1 alpha and PDGFB HBS1 gene enhancer; activated SIRT1 (RSV preincubation), also inhibited luciferase activity induced by hypoxia; conversely, inhibition the activity of SIRT1 (EX527 preincubation) promotes hypoxia induced luciferase expression. The results showed that SIRT1 inhibited hypoxia induced activation of HIF1 alpha and PDGFB mediated by.3.3 gene transcription mediated by SIRT1 HIF1 alpha to acetylation in order to explore the molecular mechanism of inactivation of SIRT1 expression inhibition of HIF-1 mediated by PDGFB, immunoprecipitation analysis of HIF-1 alpha acetylation level. The results showed that hypoxia VSMC HIF-1 acetylation induced by alpha; overexpression or activation of SIRT1 can reduce hypoxia induced HIF-1 alpha acetylation, and decreased expression of PDGFB protein; reverse The use of EX527 to inhibit the activity of SIRT1 decreased acetylation and PDGFB expression of reversed HIF-1 alpha. In order to verify the SIRT1 mediated deacetylation of HIF-1 alpha inactivation, interaction of immunoprecipitation to detect SIRT1 and HIF-1 alpha. The results showed that hypoxia interaction between SIRT1 and HIF-1 alpha had weakened; the expression or activation of SIRT1, enhance the interaction between the two, and inhibit the interaction of SIRT1 two activity decreased. The results showed that the expression of PDGFB gene SIRT1 mediated HIF-1 deacetylation alpha inactivation can inhibit hypoxia induced activation of SIRT1 1. Conclusion: inhibition of lower limb ischemia in mice after artery formation, resulting in lower limb blood flow recovery delay.2 SIRT1 Wnt/ by inhibiting the -catenin signaling pathway and down-regulation of VEGFA expression, PDGFB expression and inhibit arterial endothelial cell proliferation and angiogenesis of.3 SIRT1 mediated HIF-1 alpha deacetylation can inhibit hypoxia induced inactivation of students .

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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