本文關(guān)鍵詞：VEGF介導(dǎo)的骨骼肌類型轉(zhuǎn)變對(duì)血管新生的促進(jìn)作用及其機(jī)制　出處：《河北醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血管內(nèi)皮生長(zhǎng)因子 血管新生 骨骼肌類型 氧化代謝 糖尿病周圍血管病

【摘要】：糖尿病足(diabetic foot,DF)是最嚴(yán)重的糖尿病并發(fā)癥之一,致殘致死率極高,且花費(fèi)巨大。糖尿病患者下肢截肢多是由足潰瘍導(dǎo)致的,外周動(dòng)脈疾病(peripheral arterial disease,PAD)是影響糖尿病足潰瘍最重要的因素,也是唯一獨(dú)立的影響截肢預(yù)后的因素。糖尿病高糖環(huán)境下血管內(nèi)皮細(xì)胞功能受損是PAD的主要發(fā)病機(jī)制。目前針對(duì)血管內(nèi)皮細(xì)胞的研究很多,但很少有研究關(guān)注骨骼肌與血管新生的關(guān)系。骨骼肌是主要的糖代謝器官,同時(shí)又是血管內(nèi)皮細(xì)胞生長(zhǎng)的外環(huán)境,骨骼肌纖維存在兩種類型,無氧呼吸型(糖化型)、有氧呼吸型(氧化型),兩者可相互轉(zhuǎn)化。研究表明,氧化肌纖維的代謝水平較高,攝糖能力更強(qiáng)。我們推測(cè)骨骼肌纖維類型的轉(zhuǎn)變將有助于改善糖尿病環(huán)境下血管內(nèi)皮細(xì)胞生長(zhǎng)的微環(huán)境,增強(qiáng)血管內(nèi)皮細(xì)胞的活性,促進(jìn)下肢的血管新生。我們發(fā)現(xiàn),運(yùn)動(dòng)可使VEGF增多,VEGF可顯著增加小鼠毛細(xì)血管密度,且骨骼肌纖維也從無氧型變?yōu)橛醒跣�。本課題旨在通過VEGF研究是否可通過調(diào)控肌纖維類型的轉(zhuǎn)變,增多氧化肌纖維的含量,提高骨骼肌的攝糖能力,改善糖尿病下周圍血管內(nèi)皮細(xì)胞的微環(huán)境,增強(qiáng)血管內(nèi)皮細(xì)胞的功能,促進(jìn)下肢血管新生,改善糖尿病下肢缺血,為今后臨床應(yīng)用基因治療奠定理論和實(shí)驗(yàn)基礎(chǔ),解決糖尿病足患者制動(dòng)情況下糖代謝障礙及下肢缺血的難題。第一部分自主運(yùn)動(dòng)情況下,小鼠骨骼肌纖維類型和毛細(xì)血管密度的變化目的:明確在自主運(yùn)動(dòng)后,小鼠骨骼肌VEGF表達(dá)的變化,毛細(xì)血管密度的改變,骨骼肌纖維類型的轉(zhuǎn)變和氧化代謝的變化。方法:將C57BL/6小鼠分為運(yùn)動(dòng)組和安靜組,安靜組置于普通籠中,運(yùn)動(dòng)組置于裝備自主跑輪計(jì)數(shù)系統(tǒng)的籠中,分別在1w,2w,4w取骨骼肌組織,通過酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)VEGF水平,測(cè)定檸檬酸合酶的活性表示氧化代謝水平,免疫熒光方法檢測(cè)毛細(xì)血管密度(CD31)和骨骼肌纖維類型(MHCs)。結(jié)果:1運(yùn)動(dòng)后,小鼠骨骼肌組織中VEGF表達(dá)增加相比安靜組(205.962±9.712 pg/ml),運(yùn)動(dòng)1w組,運(yùn)動(dòng)2w組,運(yùn)動(dòng)4w組VEGF含量(449.164±15.280,556.818±22.659,549.366±14.410pg/ml)明顯增多(P0.05)。2運(yùn)動(dòng)后,骨骼肌纖維類型和毛細(xì)血管密度的變化共聚焦熒光顯微鏡下可觀察到,相比安靜組,運(yùn)動(dòng)組小鼠骨骼肌氧化肌纖維(紅色,MHCIIa)比例增加,毛細(xì)血管密度(綠色,CD31)增加。3骨骼肌檸檬酸合成酶活性的變化用試劑盒測(cè)定安靜組和運(yùn)動(dòng)組骨骼肌檸檬酸合成酶活性,相比安靜組(0.023±0.002μmol/min/ml),運(yùn)動(dòng)1w組,運(yùn)動(dòng)2w組,運(yùn)動(dòng)4w組骨骼肌檸檬酸合成酶活性(0.029±0.002,0.036±0.001,0.034±0.001μmol/min/ml),明顯升高(P0.05)。第二部分建立糖尿病小鼠下肢缺血模型,明確VEGF對(duì)骨骼肌類型轉(zhuǎn)變和血管新生的作用目的:明確VEGF對(duì)糖尿病情況下骨骼肌纖維類型的轉(zhuǎn)化具有直接的調(diào)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小鼠予以高脂喂養(yǎng)6周末禁食12h后,腹腔注射100mg/kg STZ,造模成功后行左側(cè)下肢股動(dòng)脈結(jié)扎,制作糖尿病小鼠下肢缺血模型,腓腸肌分別注射Ad-GFP或Ad-VEGF-GFP,熒光顯微鏡下觀察腺病毒的轉(zhuǎn)染效率,通過酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)VEGF水平,測(cè)定檸檬酸合酶的活性表示氧化代謝水平,免疫熒光方法檢測(cè)骨骼肌纖維類型(MHCs),激光多普勒監(jiān)測(cè)雙下肢血流灌注。結(jié)果:1熒光顯微鏡下通過腺病毒的GFP標(biāo)記觀察VEGF的轉(zhuǎn)染效率Ad-VEGF-GFP和Ad-GFP轉(zhuǎn)染效率高,且持續(xù)到2周。2 ELISA方法檢測(cè)骨骼肌VEGF表達(dá)的變化腺病毒注射后2周,Ad-VEGF-GFP轉(zhuǎn)染組VEGF表達(dá)(653.373±55.348 pg/ml)明顯高于Ad-GFP對(duì)照組(180.339±15.000 pg/ml)(P0.05)。3 VEGF使糖尿病小鼠缺血下肢氧化肌纖維增多腺病毒肌注后14天,熒光顯微鏡下觀察到Ad-VEGF-GFP組小鼠骨骼肌氧化肌纖維(紅色,MHCIIa)比例增加。4骨骼肌檸檬酸合成酶活性的變化術(shù)后14天,相比空載組(0.018±0.002μmol/min/ml),VEGF組(0.030±0.003μmol/min/ml)骨骼肌檸檬酸合成酶活性明顯升高(P0.05)。5激光多普勒監(jiān)測(cè)雙下肢血流灌注Ad-VEGF-GFP轉(zhuǎn)染組血流灌注恢復(fù)水平顯著高于Ad-GFP空載組(P0.05),提示VEGF基因轉(zhuǎn)染有促糖尿病下肢血管新生的作用。第三部分通過VEGF上調(diào)氧化肌纖維,并觀察肌纖維類型轉(zhuǎn)換后對(duì)血管內(nèi)皮細(xì)胞活性的作用及機(jī)制。目的:明確VEGF可通過調(diào)節(jié)骨骼肌類型的轉(zhuǎn)變促進(jìn)血管新生,并探討其機(jī)制。方法:培養(yǎng)小鼠成肌細(xì)胞(C2C12)并誘導(dǎo)分化成肌小管并通過半定量PCR驗(yàn)證肌小管上VEGF受體的表達(dá),然后加入不同濃度(5,10,20ng/ml)的重組VEGF細(xì)胞因子(rh VEGF)刺激肌小管,用PBS+BSA作為陰性對(duì)照,在不同時(shí)間(6h,12h,24h)提取細(xì)胞RNA,用實(shí)時(shí)定量PCR方法確定氧化肌纖維MHCIIa(Myhc 2a)表達(dá)量最高的VEGF濃度和時(shí)間。通過Transwell小室將氧化肌纖維表達(dá)量最高時(shí)的肌小管與臍靜脈血管內(nèi)皮細(xì)胞(HUVEC)共培養(yǎng),觀察HUVEC活性的變化。葡萄糖氧化酶法測(cè)定肌小管轉(zhuǎn)換為氧化肌纖維前后,葡萄糖消耗量的變化。Western檢測(cè)肌小管轉(zhuǎn)換為氧化肌纖維前后PGC-1α,GLUT4和COXIV表達(dá)的變化。結(jié)果:1肌小管VEGF受體的表達(dá)半定量PCR方法可檢測(cè)到C2C12肌小管上有VEGFR1和VEGFR2m RNA的表達(dá)。2 VEGF促進(jìn)骨骼肌類型轉(zhuǎn)變r(jià)h VEGF處理后,Myhc 2a逐漸升高,在VEGF濃度為20ng/ml,干預(yù)12h時(shí),表達(dá)量最高(P0.0001),隨著時(shí)間的延長(zhǎng),表達(dá)量下降。免疫熒光檢測(cè)氧化肌纖維(MHCIIa,紅色)的表達(dá),可見VEGF干預(yù)組氧化肌纖維比對(duì)照組明顯增多。3 HUVEC活性的變化A組(空白對(duì)照組)單純HUVEC遷移和管狀形成能力作為對(duì)照,B組(正常肌小管對(duì)照組)C2C12肌小管與HUVEC共培養(yǎng)后,HUVEC遷移和管狀形成能力無顯著變化,可見正常的肌小管對(duì)HUVEC并無明顯作用。D組C2C12肌小管給予20ng/ml VEGF干預(yù)12h,即氧化肌纖維比例最高的時(shí)候與HUVEC共培養(yǎng),可見HUVEC遷移和管狀形成能力明顯增強(qiáng),比C組(VEGF對(duì)照組)20ng/ml VEGF干預(yù)12h的HUVEC遷移和管狀形成能力也增強(qiáng),可見是氧化肌纖維促進(jìn)了HUVEC的遷移和管狀形成能力。24h時(shí)各組差異最明顯。4葡萄糖消耗量與空白組(0.829±0.150mmol/L)相比,VEGF組(2.126±0.220mmol/L)肌小管葡萄糖消耗量明顯增加(P0.05),但小于陽性對(duì)照胰島素組(2.918±0.182 mmol/L)。5肌小管中PGC-1α,GLUT4和COXIV的表達(dá)情況肌小管加入20ng/ml VEGF,PBS+BSA作為陰性對(duì)照,干預(yù)12h后,氧化肌纖維增多,PGC-1α,GLUT4和COXIV表達(dá)顯著增加。結(jié)論:1小鼠自主運(yùn)動(dòng)可以引起骨骼肌VEGF蛋白表達(dá)增加,氧化肌纖維增多,毛細(xì)血管密度增加,氧化代謝能力增強(qiáng)。2 VEGF可以增加糖尿病下肢缺血小鼠骨骼肌氧化肌纖維含量,增強(qiáng)氧化代謝水平,改善下肢血流灌注。3 VEGF可通過上調(diào)PGC-1α增加骨骼肌氧化肌纖維含量,增強(qiáng)線粒體生物合成,提高骨骼肌的攝糖能力,改善血管內(nèi)皮細(xì)胞的微環(huán)境,增強(qiáng)血管內(nèi)皮細(xì)胞的活性。4通過VEGF介導(dǎo)的骨骼肌類型轉(zhuǎn)變可增強(qiáng)血管內(nèi)皮細(xì)胞活性,促進(jìn)血管新生,改善糖尿病下肢缺血,為今后臨床應(yīng)用基因治療奠定了理論和實(shí)驗(yàn)基礎(chǔ),為解決糖尿病足患者制動(dòng)情況下糖代謝障礙及下肢缺血的難題提供了新的思路。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 謝佳佳;楊慧;李瑜輝;賓建平;;下肢缺血性血管新生的血管內(nèi)皮生長(zhǎng)因子受體-2超聲分子成像研究[J];中華超聲影像學(xué)雜志;2014年12期

2 王彤,周國(guó)用,湯秀群,趙泳誼,馮自由,馬靜,凌文華;鏈脲佐菌素和高脂肪飼料誘導(dǎo)的實(shí)驗(yàn)性2型糖尿病小鼠模型[J];中國(guó)糖尿病雜志;2000年02期

，

本文編號(hào)：1392090