SM22α调节GLUT4转位的机制及其在血管平滑肌细胞增殖中的意义

发布时间：2018-04-23 03:33

本文选题：葡萄糖转运蛋白4 + 肌动蛋白　；参考：《河北医科大学》2016年博士论文

【摘要】：目的:糖尿病患者血管成型术后再狭窄和动脉粥样硬化的发生率明显高于正常人。血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖需要消耗高水平的葡萄糖,增强的葡萄糖转运、分解代谢和线粒体生物氧化是增殖的细胞能量转换的重要特征。葡萄糖转运体4(GLUT4)介导的葡萄糖摄取是血管平滑肌细胞糖摄取的主要机制,GLUT4在损伤诱导的新生内膜中表达明显增加。PDGF可通过激活PI3K促进GLUT4转位进而促进葡萄糖摄取,而PI3K激活介导的皮层肌动蛋白(actin)重构是GLUT4转位所必须的。平滑肌(smooth muscle,SM)22α,是一种actin细胞骨架相关蛋白,其主要功能是与actin结合,参与血管平滑肌细胞微丝聚合和骨架重构。在血管平滑肌细胞增殖相关的疾病如动脉粥样硬化斑块、腹主动脉瘤以及肿瘤组织中表达下调。我们先前实验证明,高表达SM22α可通过阻断PDGF-BB刺激所激活的Ras-ERK1/2信号通路,进而抑制血管平滑肌细胞增殖,并且,敲低SM22α可引起类似PDGF-BB刺激所产生的actin细胞骨架的重构。但是,SM22α是否参与PDGF-BB诱导的GLUT4转位尚不清楚。本研究旨在探讨SM22α在PDGF-BB诱导的GLUT4转位中的作用及其与细胞增殖活性的关系,揭示其作用的分子机制。方法:利用免疫荧光、Western blot技术检测PDGF-BB诱导的血管平滑肌细胞GLUT4转位及与细胞骨架重构的关系;利用荧光葡萄糖2-NBDG检测血管平滑肌细胞及动脉葡萄糖摄取;通过敲低SM22α,确定SM22α参与PDGF-BB诱导的GLUT4转位和葡萄糖摄取;通过细胞计数和Brd U实验检测GLUT4介导的葡萄糖摄取与血管平滑肌细胞增殖的相关性;利用Sm22α-/-小鼠建立颈总动脉损伤模型,用高效液相色谱法检测组织葡萄糖含量,结合膜蛋白提取和Western blot技术,探讨缺失SM22α在GLUT4转位介导的葡萄糖摄取与损伤诱导的新生内膜形成之间的关系。用免疫荧光技术检测平滑肌细胞微管的聚合情况;通过敲低或敲除或用腺病毒过表达SM22α,观察SM22α与微管稳定性的关系;利用Western blot技术检测α-Tubulin乙酰化水平;用JC-1法检测线粒体膜电位。结果:1 SM22α通过调节微丝骨架重构参与PDGF-BB诱导的GLUT4转位和糖摄取1.1 PDGF-BB诱导血管平滑肌细胞GLUT4转位和葡萄糖摄取PDGF-BB刺激血管平滑肌细胞0、5、10、15、20、30 min,提取膜蛋白,Western blot方法检测膜上GLUT4的表达量。结果显示,PDGF-BB刺激可诱导GLUT4向膜上转位,膜组分中GLUT4含量于15 min达到峰值,随后下降。荧光葡萄糖2-NBDG摄取实验显示,用PDGF-BB处理的细胞其2-NBDG的摄取也在15 min达到峰值,变化趋势与GLUT4膜转位一致,提示细胞对葡萄糖的摄取增加。1.2肌动蛋白重构调节PDGF-BB诱导的GLUT4转位细胞免疫荧光技术检测细胞膜上GLUT4的表达和细胞骨架的变化。结果显示,PDGF-BB刺激15 min,随着细胞骨架重构,皮层F-actin聚合增加,GLUT4从胞质转位至细胞边缘,与细胞皮层聚合的F-actin发生共定位。而用细胞骨架稳定剂JPK预处理细胞后,PDGF-BB诱导的细胞骨架解聚和皮层F-actin聚合受抑制,同时GLUT4膜转位明显减少。2-NBDG摄取分析也显示用JPK预处理后,PDGF-BB刺激引起的血管平滑肌细胞葡萄糖摄取被抑制。以上结果表明,PDGF-BB诱导的GLUT4膜转位和葡萄糖摄取依赖于细胞骨架重构。1.3 SM22α抑制GLUT4膜转位用SM22α特异性小干扰RNA(si SM22α)敲低血管平滑肌细胞内源性SM22α,鬼笔环肽荧光染色观察PDGF-BB刺激前后细胞骨架的变化。结果显示,相对于si Con组,敲低SM22α后,血管平滑肌细胞基础状态下应力纤维密度明显减少,而PDGF-BB刺激引起的皮层F-actin聚合则更加明显。同时,细胞免疫荧光结果显示,敲低SM22α后PDGF-BB诱导的GLUT4转位也明显增加。Western blot方法检测膜蛋白提取物中GLUT4的含量也得到相似结果。进一步用Sm22α-/-小鼠的细胞进行验证,与野生型(WT)相比较,PDGF-BB诱导的Sm22α-/-小鼠的细胞皮层F-actin聚合和GLUT4转位均明显增加。以上结果证实了我们的假设,下调SM22α通过增强actin动力学和皮层F-actin聚合从而促进PDGF-BB诱导的GLUT4转位。1.4下调SM22α表达可促进PDGF-BB诱导的葡萄糖摄取和代谢利用分别检测敲低SM22α的大鼠VSMC及Sm22α-/-小鼠细胞PDGF-BB诱导的荧光葡萄糖2-NBDG的摄取情况。结果显示,敲低或敲除SM22α可促进PDGF-BB诱导的2-NBDG摄取,与相同条件下GLUT4转位增强相一致。而用GLUT4活性抑制剂Indinavir预孵育后,PDGF-BB诱导的2-NBDG摄取增加受到抑制,说明GLUT4是糖摄取的主要载体。相对于si Con组,用小干扰RNA敲低SM22α表达,可增强PDGF-BB诱导的己糖激酶和乳酸脱氢酶活性。以上结果表明,PDGF-BB可加速细胞糖代谢,SM22α缺失能够促进这一过程。2敲除SM22α通过加速葡萄糖摄取而促进血管平滑肌细胞增殖2.1 GLUT4及其介导的葡萄糖摄取参与PDGF-BB诱导的细胞增殖用C57BL/6J野生型小鼠复制左侧颈总动脉结扎模型。HE染色结果显示,与假手术组相比,结扎的血管新生内膜明显增厚;组织免疫荧光染色结果也表明,新生内膜处GLUT4表达显著增加。体外研究显示,PDGF-BB诱导的GLUT4表达,呈时间依赖性;同时伴随细胞核增殖性抗原PCNA表达的增加。在相同刺激条件下,细胞培养基(低糖DMEM)中葡萄糖消耗也明显增加。细胞计数和Brd U分析结果显示,抑制GLUT4活性的细胞其增殖活性显著降低。以上结果表明,在PDGF-BB刺激下,GLUT4表达及其介导的葡萄糖摄取参与了细胞增殖。2.2 Sm22α-/-小鼠主动脉GLUT4介导的2-NBDG摄取增加取野生型(WT)及Sm22α-/-小鼠颈总动脉,加入PDGF-BB和2-NBDG共同孵育15 min,制备动脉冰冻切片,观察动脉组织摄取2-NBDG的荧光强度。结果显示,Sm22α-/-小鼠颈总动脉在PDGF-BB刺激下,2-NBDG摄取显著增加,荧光强度明显高于野生型小鼠的主动脉,这一效应可被GLUT4特异抑制剂indinavir解除。2.3 GLUT4参与Sm22α-/-小鼠损伤后新生内膜的形成体外研究已证实GLUT4介导的葡萄糖摄取参与细胞增殖,SM22α缺失可促进这一过程。为了在体内验证这一发现,选雄性WT及Sm22α-/-小鼠复制左侧颈总动脉结扎模型。于结扎术后28天取颈总动脉制备切片。HE染色结果表明,与WT对照组相比,Sm22α-/-小鼠在结扎的第28天血管新生内膜明显增厚。取WT及Sm22α-/-小鼠结扎侧颈总动脉进行总蛋白及膜蛋白提取,检测GLTU4表达。结果表明,相对于WT组,Sm22α-/-小鼠损伤动脉总蛋白和膜组分中GLTU4表达均高于WT小鼠,膜组分与总蛋白GLTU4比值也有所增加。用高效液相色谱法检测动脉组织中葡萄糖含量,结果发现,Sm22α-/-小鼠损伤动脉组织葡萄糖含量也明显高于野生型小鼠,与内膜增生程度相一致。以上结果表明,SM22α缺失可促进体内GLUT4转位和葡萄糖摄取,加重内膜增生程度。3 SM22α增强血管平滑肌细胞微管稳定性3.1 SM22a增强血管平滑肌细胞微管的稳定性为检测SM22α在微管稳定性中的作用,用SM22α特异性小干扰RNA(si SM22α)敲低血管平滑肌细胞内源性SM22α,α-tubulin抗体进行免疫荧光染色,结果显示,si Con组微管从核周向细胞边缘呈放射状分布;敲低SM22α后,微管解聚并向核周聚集。过表达SM22α的细胞,其微管呈束状存在。与WT相比较,Sm22α-/-小鼠细胞微管解聚并向核周聚集;补救SM22α表达后,微管结构恢复至WT状态。结果表明,SM22α表达增强微管的稳定性。3.2 SM22α增强a-tubulin 40位赖氨酸残基乙酰化α-tubulin第40位赖氨酸残基乙酰化修饰是公认的是微管稳定性标志。细胞免疫荧光和Westhern blot结果均表明,敲低SM22α可降低α-tubulin乙酰化水平,而过表达SM22α则增强α-tubulin的乙酰化水平,与SM22α对微管存在形式的影响相一致。3.3敲除SM22α降低生长接触抑制,促进线粒体分裂和膜电位增加细胞计数结果显示,WT组的细胞在融合状态下,继续培养2,3,4天时,细胞密度无明显差异;而Sm22α-/-细胞在2,3,4天后仍保持增殖活性,数量明显增加,在第4天时,细胞密度达到WT组的1.68倍。JC-1染色结果表明,WT组细胞线粒体多呈长杆状、线状并紧密连接的立体网络的融合状态;而Sm22α-/-细胞线粒体则呈颗粒状、点状散在分布的分裂状态,而且线粒体膜电位也明显高于野生型细胞。结论:1 PDGF-BB诱导的GLUT4转位和糖摄取参与血管平滑肌细胞增殖。2缺失SM22α可诱导皮层细胞骨架聚合,增强PDGF-BB诱导的GLUT4膜转位和糖摄取及代谢活性。3 SM22α是一种新的增殖相关糖代谢调节因子。4 SM22α增强血管平滑肌细胞微管的稳定性,介导生长接触抑制效应。
[Abstract]:Objective: the incidence of restenosis and atherosclerosis in diabetic patients after angioplasty is significantly higher than that of normal people. The proliferation of vascular smooth muscle cell (VSMC) needs to consume high levels of glucose, enhanced glucose transport, and the importance of catabolism and linear granular biological oxidation. Glucose transporter 4 (GLUT4) - mediated glucose uptake is the main mechanism of glucose uptake in vascular smooth muscle cells. The expression of GLUT4 in injured neointima increases significantly by activating PI3K to promote GLUT4 translocation by activating PI3K and promoting glucose uptake, while PI3K activated cortical actin (actin) reconfiguration is GLUT4 transposition The necessary. Smooth muscle (SM) 22 alpha is a actin cytoskeleton related protein whose main function is to combine with actin to participate in microfilament and skeleton remodeling in vascular smooth muscle cells. The expression of vascular smooth muscle cell proliferation related diseases such as atherosclerotic plaques, abdominal aortic aneurysm and tumor tissue are down regulated. Previous experiments showed that high expression of SM22 alpha could inhibit the proliferation of vascular smooth muscle cells by blocking the Ras-ERK1/2 signaling activated by PDGF-BB stimulation, and knocking low SM22 alpha could cause the remodeling of the actin cytoskeleton similar to PDGF-BB stimulation. However, it is not clear whether SM22 alpha participates in PDGF-BB induced GLUT4 transposition. The purpose of this study is to explore the role of SM22 alpha in the PDGF-BB induced GLUT4 transposition and the relationship with cell proliferation activity, and to reveal the molecular mechanism of its role. Methods: using immunofluorescence, Western blot technique to detect the GLUT4 transposition of vascular smooth muscle cells induced by PDGF-BB and the relationship with cytoskeleton reconstruction, and the use of fluorescent glucose 2-NBDG to detect blood. Tube smooth muscle cells and arterial glucose uptake; SM22 alpha involved in PDGF-BB induced GLUT4 transposition and glucose uptake by knocking low SM22 alpha; the correlation between GLUT4 mediated glucose uptake and vascular smooth muscle cell proliferation through the cell count and Brd U test; the use of Sm22 A / - mice to establish a carotid artery injury model with high efficiency. The glucose content in tissue was detected by liquid chromatography, and the relationship between the glucose uptake mediated by GLUT4 transposition and the formation of the neointima induced by damage was investigated with membrane protein extraction and Western blot technology. The polymerization of microtubules in smooth muscle cells was detected by immunofluorescence technique; by knocking down or knocking down or using adenovirus. SM22 alpha was expressed and the relationship between SM22 alpha and microtubule stability was observed; Western blot technique was used to detect the level of alpha -Tubulin acetylation; the mitochondrial membrane potential was detected by JC-1. Results: 1 SM22 alpha was involved in PDGF-BB induced GLUT4 transposition and glucose uptake by 1.1 PDGF-BB to induce the GLUT4 transposition of vascular smooth muscle cells and glucose uptake by the regulation of microfilament framework remodeling. 0,5,10,15,20,30 min of vascular smooth muscle cells was stimulated by PDGF-BB, and membrane protein was extracted and Western blot was used to detect the expression of GLUT4 on the membrane. The results showed that PDGF-BB stimulation could induce the transposition of GLUT4 to the membrane. The GLUT4 content in the membrane components reached the peak of the 15 min and then decreased. The uptake of 2-NBDG was also at the peak of 15 min, the change trend was consistent with the GLUT4 membrane transposition, suggesting that the uptake of glucose increased by.1.2 actin reconfiguration and regulation of PDGF-BB induced GLUT4 transposition cell immunofluorescence technique to detect the expression of GLUT4 on the cell membrane and the change of cytoskeleton. The results showed that PDGF-BB stimulation was 15 min, with finer. Cytoskeleton reconstruction, cortical F-actin polymerization increased, GLUT4 translocated from cytoplasm to cell edge, and Co located with the F-actin of cell cortical aggregation. After the cells were pretreated with cytoskeleton stabilizer JPK, PDGF-BB induced cytoskeleton depolymerization and cortical F-actin polymerization were inhibited. GLUT4 membrane transposition significantly decreased.2-NBDG uptake analysis at the same time. The glucose uptake of vascular smooth muscle cells induced by PDGF-BB stimulation was inhibited after JPK pretreatment. The results showed that the GLUT4 membrane transposition and glucose uptake induced by PDGF-BB were dependent on the cytoskeleton remodeling of.1.3 SM22 a to inhibit the GLUT4 membrane translocation with SM22 a specific small interference RNA (Si SM22 alpha) knocking down the endogenous SM22 alpha in the vascular smooth muscle cells. The cytoskeleton changes before and after PDGF-BB stimulation were observed before and after the stimulation of the Si Con group. The results showed that the stress density of the vascular smooth muscle cells decreased significantly compared to the Si Con group, and the F-actin polymerization caused by PDGF-BB stimulation was more obvious. PDGF-BB induced GLUT4 transposition also significantly increased the.Western blot method to detect the content of GLUT4 in the membrane protein extract. Further use Sm22 alpha / - mouse cells to verify, and compared with the wild type (WT), PDGF-BB induced Sm22 alpha / - mouse cell cortical F-actin polymerization and GLUT4 transposition were significantly increased. The result confirms our hypothesis that down regulation of SM22 alpha by enhancing actin kinetics and cortical F-actin polymerization to promote PDGF-BB induced GLUT4 transposition.1.4 down regulation of SM22 alpha expression can promote PDGF-BB induced glucose uptake and metabolic utilization to detect VSMC and Sm22 alpha / - mouse cell PDGF-BB induced fluorescence glucose induced by PDGF-BB The results of BDG uptake showed that knocking down or knocking SM22 alpha could promote the 2-NBDG uptake induced by PDGF-BB, which was consistent with the GLUT4 transposition enhancement under the same conditions. The increase of 2-NBDG uptake induced by PDGF-BB was inhibited after Indinavir preincubation with the GLUT4 active inhibitor, indicating that GLUT4 was the main carrier of sugar uptake. RNA knocks low SM22 alpha expression and enhances PDGF-BB induced hexokinase and lactate dehydrogenase activity. The above results show that PDGF-BB can accelerate cell sugar metabolism, and SM22 alpha deletion can promote the process of.2 knockout SM22 alpha by accelerating glucose uptake and promoting vascular smooth muscle cells to increase 2.1 GLUT4 and its mediated glucose uptake to participate PDG The proliferation of F-BB induced cell proliferation in the left cervical artery ligation model in C57BL/6J wild type mice showed that the neointima of ligated vascular thickening was obviously thickened compared with the sham group, and the results of tissue immunofluorescence staining showed that the expression of GLUT4 in the neointima was significantly increased. In vitro studies showed that PDGF-BB induced GLUT4 expression, It was time dependent and accompanied by an increase in the expression of nuclear proliferative antigen PCNA. The glucose consumption in the cell culture medium (low sugar DMEM) was also significantly increased under the same stimulation conditions. Cell count and Brd U analysis showed that the proliferation activity of cells inhibiting GLUT4 activity was significantly reduced. The above results showed that GLUT4 was stimulated by PDGF-BB, GLUT4 The expression and its mediated glucose uptake participated in the increase of 2-NBDG uptake mediated by GLUT4 in the proliferation of.2.2 Sm22 alpha - / - mouse aorta and the increasing of the common carotid artery of the wild type (WT) and Sm22 alpha / - mice. The 15 min was incubated with PDGF-BB and 2-NBDG to prepare the frozen section of the arteries and to observe the fluorescence intensity of the 2-NBDG in the arterial tissue. The results showed that Sm22 alpha - / - / - / - Under the stimulation of PDGF-BB, the uptake of 2-NBDG in the common carotid artery of mice increased significantly, and the fluorescence intensity was significantly higher than that of the aorta in the wild type mice. This effect could be relieved by.2.3 GLUT4, a specific inhibitor of GLUT4, to participate in the formation of neointima after Sm22 alpha / - mice injury, and that GLUT4 mediated glucose uptake was involved in cell proliferation, S. M22 alpha deletion could promote this process. In order to verify this discovery in the body, male WT and Sm22 alpha / - mice were selected to copy the left carotid artery ligation model. The.HE staining of the common carotid artery in the 28 day after ligation showed that the Sm22 alpha / - mice were thickened in the neointima of vascular neovascularization at the twenty-eighth day of ligation compared with the WT control group. WT and Sm were taken. The total protein and membrane protein of the 22 alpha - / - mice were ligated in 22 alpha - / - mice to detect the expression of the total protein and membrane protein. The results showed that the expression of GLTU4 in the total and membrane components of Sm22 A / - mice was higher than that of the WT group. The ratio of the membrane components to the total protein GLTU4 was also increased. The glucose content in the arterial tissue was detected by high performance liquid chromatography. The results showed that the glucose content in the injured arterial tissue of Sm22 - / - / - mice was also significantly higher than that in the wild type mice, which was consistent with the degree of intimal hyperplasia. The above results showed that the deletion of SM22 alpha could promote the translocation of GLUT4 and the uptake of glucose in the body, and increased the degree of intimal hyperplasia by.3 SM22 a to enhance the microtubule stability of the smooth muscle cells of the smooth muscle cells by 3.1 SM22a enhanced blood vessels. The stability of smooth muscle cell microtubules was used to detect the role of SM22 alpha in microtubule stability. SM22 alpha specific small interference RNA (Si SM22 alpha) knocks endogenous SM22 A and alpha -tubulin antibody for immunofluorescence staining. The results showed that the microtubules of Si Con group were distributed radially from the periphery to the edge of the cell; after knocking down SM22 alpha, microtubules were knocked down. The microtubules in the cells expressed SM22 alpha were in a fascicular structure. Compared with WT, the microtubules of Sm22 alpha / - mouse cells were depolymerization and aggregated to the pericarp. After remedial SM22 alpha expression, the microtubule structure was restored to WT state. The results showed that SM22 alpha expression enhanced the stability of microtubules by.3.2 SM22 A and enhanced the a-tubulin 40 lysine residue acetylation The acetylation modification of alpha -tubulin fortieth lysine residues is recognized as a marker of microtubule stability. Both cell immunofluorescence and Westhern blot results show that knocking low SM22 alpha can reduce the level of alpha -tubulin acetylation, while overexpression of SM22 a enhances the level of acetylation of alpha -tubulin, and is consistent with the existence of SM22 a in the presence of microtubules in.3.3 knockout SM2. 2 alpha decreased growth contact inhibition, promoting mitochondrial division and increasing cell count by membrane potential showed that cells in WT group had no significant difference in cell density when they continued to cultivate 2,3,4 days in fusion state, while Sm22 alpha / - cells still maintained proliferation activity after 2,3,4 days, and the cell density reached 1.68 times.J in WT group at the time of fourth days. The results of C-1 staining showed that the mitochondria in the WT group were mostly rod like, linear and tightly connected stereoscopic network, while Sm22 alpha / - cell mitochondria were granular, punctate scattered in the distribution of split state, and the mitochondrial membrane potential was obviously higher than that of wild type cells. Conclusion: 1 PDGF-BB induced GLUT4 transposition and sugar uptake participate in blood Vascular smooth muscle cell proliferation,.2 deletion, SM22 a can induce cortical cytoskeleton polymerization, enhanced PDGF-BB induced GLUT4 membrane transposition, and glucose uptake and metabolic activity.3 SM22 alpha, a new proliferation related glycometabolic regulator.4 SM22 a to enhance the stability of vascular smooth muscle cells microtubules and mediate growth contact inhibition.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R54

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