VEGF介导的骨骼肌类型转变对血管新生的促进作用及其机制

发布时间：2018-01-07 10:09

本文关键词：VEGF介导的骨骼肌类型转变对血管新生的促进作用及其机制　出处：《河北医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：糖尿病足(diabetic foot,DF)是最严重的糖尿病并发症之一,致残致死率极高,且花费巨大。糖尿病患者下肢截肢多是由足溃疡导致的,外周动脉疾病(peripheral arterial disease,PAD)是影响糖尿病足溃疡最重要的因素,也是唯一独立的影响截肢预后的因素。糖尿病高糖环境下血管内皮细胞功能受损是PAD的主要发病机制。目前针对血管内皮细胞的研究很多,但很少有研究关注骨骼肌与血管新生的关系。骨骼肌是主要的糖代谢器官,同时又是血管内皮细胞生长的外环境,骨骼肌纤维存在两种类型,无氧呼吸型(糖化型)、有氧呼吸型(氧化型),两者可相互转化。研究表明,氧化肌纤维的代谢水平较高,摄糖能力更强。我们推测骨骼肌纤维类型的转变将有助于改善糖尿病环境下血管内皮细胞生长的微环境,增强血管内皮细胞的活性,促进下肢的血管新生。我们发现,运动可使VEGF增多,VEGF可显著增加小鼠毛细血管密度,且骨骼肌纤维也从无氧型变为有氧型。本课题旨在通过VEGF研究是否可通过调控肌纤维类型的转变,增多氧化肌纤维的含量,提高骨骼肌的摄糖能力,改善糖尿病下周围血管内皮细胞的微环境,增强血管内皮细胞的功能,促进下肢血管新生,改善糖尿病下肢缺血,为今后临床应用基因治疗奠定理论和实验基础,解决糖尿病足患者制动情况下糖代谢障碍及下肢缺血的难题。第一部分自主运动情况下,小鼠骨骼肌纤维类型和毛细血管密度的变化目的:明确在自主运动后,小鼠骨骼肌VEGF表达的变化,毛细血管密度的改变,骨骼肌纤维类型的转变和氧化代谢的变化。方法:将C57BL/6小鼠分为运动组和安静组,安静组置于普通笼中,运动组置于装备自主跑轮计数系统的笼中,分别在1w,2w,4w取骨骼肌组织,通过酶联免疫吸附试验(ELISA)检测VEGF水平,测定柠檬酸合酶的活性表示氧化代谢水平,免疫荧光方法检测毛细血管密度(CD31)和骨骼肌纤维类型(MHCs)。结果:1运动后,小鼠骨骼肌组织中VEGF表达增加相比安静组(205.962±9.712 pg/ml),运动1w组,运动2w组,运动4w组VEGF含量(449.164±15.280,556.818±22.659,549.366±14.410pg/ml)明显增多(P0.05)。2运动后,骨骼肌纤维类型和毛细血管密度的变化共聚焦荧光显微镜下可观察到,相比安静组,运动组小鼠骨骼肌氧化肌纤维(红色,MHCIIa)比例增加,毛细血管密度(绿色,CD31)增加。3骨骼肌柠檬酸合成酶活性的变化用试剂盒测定安静组和运动组骨骼肌柠檬酸合成酶活性,相比安静组(0.023±0.002μmol/min/ml),运动1w组,运动2w组,运动4w组骨骼肌柠檬酸合成酶活性(0.029±0.002,0.036±0.001,0.034±0.001μmol/min/ml),明显升高(P0.05)。第二部分建立糖尿病小鼠下肢缺血模型,明确VEGF对骨骼肌类型转变和血管新生的作用目的:明确VEGF对糖尿病情况下骨骼肌纤维类型的转化具有直接的调控作用,并能改善糖尿病下肢缺血小鼠的下肢血流灌注。方法:C57BL/6小鼠予以高脂喂养6周末禁食12h后,腹腔注射100mg/kg STZ,造模成功后行左侧下肢股动脉结扎,制作糖尿病小鼠下肢缺血模型,腓肠肌分别注射Ad-GFP或Ad-VEGF-GFP,荧光显微镜下观察腺病毒的转染效率,通过酶联免疫吸附试验(ELISA)检测VEGF水平,测定柠檬酸合酶的活性表示氧化代谢水平,免疫荧光方法检测骨骼肌纤维类型(MHCs),激光多普勒监测双下肢血流灌注。结果:1荧光显微镜下通过腺病毒的GFP标记观察VEGF的转染效率Ad-VEGF-GFP和Ad-GFP转染效率高,且持续到2周。2 ELISA方法检测骨骼肌VEGF表达的变化腺病毒注射后2周,Ad-VEGF-GFP转染组VEGF表达(653.373±55.348 pg/ml)明显高于Ad-GFP对照组(180.339±15.000 pg/ml)(P0.05)。3 VEGF使糖尿病小鼠缺血下肢氧化肌纤维增多腺病毒肌注后14天,荧光显微镜下观察到Ad-VEGF-GFP组小鼠骨骼肌氧化肌纤维(红色,MHCIIa)比例增加。4骨骼肌柠檬酸合成酶活性的变化术后14天,相比空载组(0.018±0.002μmol/min/ml),VEGF组(0.030±0.003μmol/min/ml)骨骼肌柠檬酸合成酶活性明显升高(P0.05)。5激光多普勒监测双下肢血流灌注Ad-VEGF-GFP转染组血流灌注恢复水平显著高于Ad-GFP空载组(P0.05),提示VEGF基因转染有促糖尿病下肢血管新生的作用。第三部分通过VEGF上调氧化肌纤维,并观察肌纤维类型转换后对血管内皮细胞活性的作用及机制。目的:明确VEGF可通过调节骨骼肌类型的转变促进血管新生,并探讨其机制。方法:培养小鼠成肌细胞(C2C12)并诱导分化成肌小管并通过半定量PCR验证肌小管上VEGF受体的表达,然后加入不同浓度(5,10,20ng/ml)的重组VEGF细胞因子(rh VEGF)刺激肌小管,用PBS+BSA作为阴性对照,在不同时间(6h,12h,24h)提取细胞RNA,用实时定量PCR方法确定氧化肌纤维MHCIIa(Myhc 2a)表达量最高的VEGF浓度和时间。通过Transwell小室将氧化肌纤维表达量最高时的肌小管与脐静脉血管内皮细胞(HUVEC)共培养,观察HUVEC活性的变化。葡萄糖氧化酶法测定肌小管转换为氧化肌纤维前后,葡萄糖消耗量的变化。Western检测肌小管转换为氧化肌纤维前后PGC-1α,GLUT4和COXIV表达的变化。结果:1肌小管VEGF受体的表达半定量PCR方法可检测到C2C12肌小管上有VEGFR1和VEGFR2m RNA的表达。2 VEGF促进骨骼肌类型转变rh VEGF处理后,Myhc 2a逐渐升高,在VEGF浓度为20ng/ml,干预12h时,表达量最高(P0.0001),随着时间的延长,表达量下降。免疫荧光检测氧化肌纤维(MHCIIa,红色)的表达,可见VEGF干预组氧化肌纤维比对照组明显增多。3 HUVEC活性的变化A组(空白对照组)单纯HUVEC迁移和管状形成能力作为对照,B组(正常肌小管对照组)C2C12肌小管与HUVEC共培养后,HUVEC迁移和管状形成能力无显著变化,可见正常的肌小管对HUVEC并无明显作用。D组C2C12肌小管给予20ng/ml VEGF干预12h,即氧化肌纤维比例最高的时候与HUVEC共培养,可见HUVEC迁移和管状形成能力明显增强,比C组(VEGF对照组)20ng/ml VEGF干预12h的HUVEC迁移和管状形成能力也增强,可见是氧化肌纤维促进了HUVEC的迁移和管状形成能力。24h时各组差异最明显。4葡萄糖消耗量与空白组(0.829±0.150mmol/L)相比,VEGF组(2.126±0.220mmol/L)肌小管葡萄糖消耗量明显增加(P0.05),但小于阳性对照胰岛素组(2.918±0.182 mmol/L)。5肌小管中PGC-1α,GLUT4和COXIV的表达情况肌小管加入20ng/ml VEGF,PBS+BSA作为阴性对照,干预12h后,氧化肌纤维增多,PGC-1α,GLUT4和COXIV表达显著增加。结论:1小鼠自主运动可以引起骨骼肌VEGF蛋白表达增加,氧化肌纤维增多,毛细血管密度增加,氧化代谢能力增强。2 VEGF可以增加糖尿病下肢缺血小鼠骨骼肌氧化肌纤维含量,增强氧化代谢水平,改善下肢血流灌注。3 VEGF可通过上调PGC-1α增加骨骼肌氧化肌纤维含量,增强线粒体生物合成,提高骨骼肌的摄糖能力,改善血管内皮细胞的微环境,增强血管内皮细胞的活性。4通过VEGF介导的骨骼肌类型转变可增强血管内皮细胞活性,促进血管新生,改善糖尿病下肢缺血,为今后临床应用基因治疗奠定了理论和实验基础,为解决糖尿病足患者制动情况下糖代谢障碍及下肢缺血的难题提供了新的思路。
[Abstract]:Diabetic foot (diabetic foot DF) is one of the most serious complications of diabetes mellitus, rate of disability is high and costly. In diabetic patients with lower extremity amputation is caused by foot ulcer, peripheral arterial disease (peripheral arterial, disease, PAD) is one of the most important factors of diabetic foot ulcer, and is the only independent effect of amputation prognostic factors. Vascular endothelial cell function in high glucose environment damage is the main pathogenesis of PAD. At present a lot of research on vascular endothelial cells, but few studies have focused on the relationship between skeletal muscle and angiogenesis. Skeletal muscle glucose metabolism is the main organ of the external environment, is also the growth of vascular endothelial cells, there are two types of skeletal muscle fibers, anaerobic respiration type (saccharifying), aerobic respiration type (oxidized), which can be transformed into each other. The study shows that the oxidation of muscle fibers of high metabolism, intake of sugar Stronger. We speculate that the microenvironment changes of skeletal muscle fiber types will be helpful to improve the environment of diabetic vascular endothelial cell growth, enhance endothelial cell activity, promote the lower limb angiogenesis. We found that the movement may cause the increase of VEGF, VEGF significantly increased capillary density in mice, and skeletal muscle fibers from anaerobic type into aerobic type. The aims of the study is VEGF by changing the regulation of muscle fiber types, and increase the content of oxidative muscle fibers, improve skeletal muscle glucose uptake, improve the microenvironment of skin cells in diabetic peripheral vascular, enhance the function of endothelial cells, promote vasculogenesis, improve diabetes lower limb ischemia, lay a theoretical and experimental basis for future clinical application of gene therapy in patients with diabetic foot, solve the brake case of glycometabolism disorder and lower limb ischemia first problem. The independent movement, changes of skeletal muscle fiber types and capillary density in mice Objective: to determine the independent movement after the change of the expression of VEGF in skeletal muscle of mice, capillary density changes, changes and oxidative metabolism of skeletal muscle fiber type. Methods: C57BL/6 mice were divided into exercise group and control group, control group the cage, in the exercise group equipment independent running wheel counting system of the cage, respectively in 1W, 2W, 4W of skeletal muscle tissue by enzyme linked immunosorbent assay (ELISA) detection of VEGF level determination of citrate synthase activity of oxidative metabolism, the capillary density of fluorescence detection (CD31) and skeletal muscle fiber types (MHCs). Results: 1 after exercise, skeletal muscle tissue in mice increased VEGF expression compared to the quiet group (205.962 + 9.712 pg/ml), 1W group, 2W group, 4W group exercise, exercise the content of VEGF (449.164 + 15.280,5 56.818 + 22.659549.366 + 14.410pg/ml (P0.05).2) increased significantly after exercise, changes of skeletal muscle fiber and capillary density of confocal fluorescence microscope can be observed, compared to control group, exercise group mice skeletal muscle oxidative muscle fibers (red, MHCIIa) ratio increases, the capillary density (green, CD31) to increase the change of acid.3 synthase activity in skeletal muscle of citric acid synthase activity determination of the quiet group and exercise group with lemon skeletal muscle kit, compared to the quiet group (0.023 + 0.002 mol/min/ml), 1W group, 2W group, 4W group exercise, exercise skeletal muscle citrate synthase activity (0.029 + 0.002,0.036 + 0.001,0.034 + 0.001 u mol/min/ml), obviously increased (P0.05). The second part is the establishment of diabetic mice model of lower limb ischemia, a clear effect of VEGF on the change of skeletal muscle type and angiogenesis Objective: to clear the VEGF of diabetes cases of skeletal muscle fiber types The transformation has a direct regulatory role, and can improve the blood perfusion of lower limbs of diabetic lower limb ischemia in mice. Methods: C57BL/6 mice were given high fat diet for 6 weeks after fasting 12h, intraperitoneal injection of 100mg/kg STZ, the successful model of the left lower limb after femoral artery ligation, making lower extremity ischemia in diabetic mice model, the gastrocnemius muscle were injected with Ad-GFP or Ad-VEGF-GFP the transfection efficiency of adenovirus, observed under fluorescence microscope by enzyme-linked immunosorbent assay (ELISA) detection of VEGF level determination of citrate synthase activity of oxidative metabolism, immunofluorescence to detect the skeletal muscle fiber type (MHCs), laser Doppler perfusion of lower extremities monitoring. Results: 1 fluorescence microscope GFP markers through the observation of VEGF adenovirus transfection efficiency of Ad-VEGF-GFP and Ad-GFP with high transfection efficiency, and continued to 2 weeks.2 ELISA method for detection of skeletal muscle VEGF expression changes of adenovirus 2 weeks after injection, the expression of Ad-VEGF-GFP VEGF transfection group (653.373 + 55.348 pg/ml) was higher than that of Ad-GFP group (180.339 + 15 pg/ml) (P0.05).3 VEGF to diabetic mice with ischemic limbs oxidative muscle fibers increased 14 days after intramuscular injection of adenovirus, fluorescence microscopy showed that Ad-VEGF-GFP mice skeletal muscle oxidative muscle fibers (red. MHCIIa) 14 days to increase the proportion of changes of acid synthase activity of.4 in skeletal muscle of lemon, compared with empty vector group (0.018 + 0.002 mol/min/ml), group VEGF (0.030 + 0.003 mol/min/ml) citric acid synthase activity in skeletal muscle increased significantly (P0.05).5 laser Doppler perfusion monitor limb perfusion recovery in Ad-VEGF-GFP transfection group no load group was significantly higher than that of Ad-GFP (P0.05), suggesting that VEGF gene transfection could promote angiogenesis of diabetic lower extremity function. The third part through the upregulation of VEGF oxidative muscle fibers, and observe the muscle fiber type conversion Effect and mechanism of vascular endothelial cell activity. Objective: to determine the VEGF by changing the regulation of skeletal muscle types and promote angiogenesis, and to explore its mechanism. Methods: cultured mouse myoblasts (C2C12) and induced to differentiate into myotubes and through VEGF receptor expression by semi quantitative PCR validation myotubule, then add different concentration (5,10,20ng/ml) of the recombinant VEGF cell factor (RH VEGF) to stimulate muscle tubules, with PBS+BSA as the negative control, at different time (6h, 12h, 24h) extraction of RNA cells to determine oxidative muscle fibers MHCIIa by real-time quantitative PCR method (Myhc 2a) expressed the highest VEGF concentration and time by Transwell chamber. The expression of oxidative muscle fibers was highest when myotubes and umbilical vein endothelial cells (HUVEC) were cultured to observe the changes of HUVEC activity. The determination method of glucose oxidase myotubule conversion of muscle fiber into oxygen before and after glucose elimination Conversion of tubular.Western to detect changes in muscle consumption and oxidative muscle fibers of PGC-1 alpha, the expression of GLUT4 and COXIV. Results: the expression of semi quantitative PCR method 1 myotubule VEGF receptor can be detected with VEGFR1 VEGFR2m and RNA C2C12 to promote muscle tubules on the expression of.2 VEGF in skeletal muscle of type change of RH after VEGF treatment Myhc 2A increased gradually when the concentration of VEGF was 20ng/ml, 12h interference (P0.0001), the highest expression level, with the extension of time, the expression amount decreased. Immunofluorescence oxidative muscle fibers (MHCIIa, red) expression changes observed in A group, VEGF intervention group and control group of oxidative muscle fiber viby.3 significantly increased the activity of HUVEC (control group) with HUVEC migration and tube formation ability as control, group B (normal control group myotubule) C2C12 co cultured myotubes with HUVEC, HUVEC migration and tube formation ability and no significant changes in normal myotubes visible on HUVEC There is no obvious effect of.D group C2C12 20ng/ml VEGF 12h to myotubule intervention, which is the highest proportion of oxidative muscle fibers when co cultured with HUVEC, the formation of visible HUVEC migration and tubular capacity significantly enhanced than that of C group (VEGF control group) 20ng/ml VEGF intervention 12h HUVEC migration and tube formation ability is also enhanced, visible oxidative muscle fibers to promote the migration and tubular HUVEC and blank group were the most obvious difference between.4 glucose consumption formation ability of.24h (0.829 + 0.150mmol/L), group VEGF (2.126 + 0.220mmol/L) myotubule glucose consumption increased significantly (P0.05), but less than the positive control group, insulin (2.918 + 0.182 mmol/L) PGC-1 alpha.5 myotubes in the GLUT4, and the expression of COXIV 20ng/ml VEGF PBS+BSA myotubule added, as a negative control, 12h after the intervention, oxidative muscle fibers increased, PGC-1 alpha, GLUT4 and COXIV expression increased significantly. Conclusion: 1 mice independent movement Can increase the expression of VEGF protein in skeletal muscle, oxidative muscle fiber increased, capillary density increased, oxidative capacity enhanced.2 VEGF can increase the diabetic lower limb ischemia in mouse skeletal muscle oxidative muscle fiber content, enhanced oxidative metabolism, improve blood perfusion of lower extremity.3 VEGF can increase skeletal muscle oxidative muscle fiber content through upregulation of PGC-1 alpha, enhanced mitochondrial biogenesis and improve the skeletal muscle glucose uptake, improve the microenvironment of vascular endothelial cells, skeletal muscle type transformation mediated by VEGF enhanced.4 activity of vascular endothelial cells can enhance vascular endothelial cell activity, promote angiogenesis, improve diabetic lower limb ischemia, laid the theoretical and experimental basis for future clinical application of gene therapy to provide. A new way to solve the problem of glycometabolism disorder and lower limb ischemia in patients with diabetic foot brake conditions.

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

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