CN116785411A - Glp-1(32-36)在治疗下肢动脉闭塞中的应用 - Google Patents
Glp-1(32-36)在治疗下肢动脉闭塞中的应用 Download PDFInfo
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Abstract
一种GLP‑1(32‑36)在制备治疗下肢动脉闭塞药物中的应用，尤其是对EPCs的高糖损伤起到保护作用，改善其促血管生成功能，尤其是改善糖尿病下肢动脉闭塞后组织血流灌注，为PAD的治疗提供新方案。
Description
技术领域
本发明涉及一种多肽的医药第二用途，尤其涉及一种GLP-1(32-36)作为药物而促进缺血下肢新生血管形成的应用。
背景技术
外周动脉疾病(PeripherialArterial Disease，PAD)是与糖尿病相关的最严重的慢性并发症之一，是一种进行性动脉粥样硬化性疾病，长期高糖应激引起的血管内皮损伤、随后的动脉粥样硬化和血栓形成是PAD的发病机制。其特点是供应下肢的动脉狭窄和闭塞(Stem cells translational medicine.2014；3(9):1090-9)，通常表现为跛行,但可能发展为严重肢体缺血症导致截肢(Trends Cardiovasc Med.2016；26(6):495-512)。
PAD的病变范围主要集中于膝以下末端小血管，究其原因可能与晚期糖基化终产物、氧化应激动脉硬化、血栓形成异常、低度感染、内皮功能障碍及微血管功能障碍相关。其中内皮细胞功能受损是其启动和关键的因素。内皮细胞在血管壁和管腔中分泌多种旁分泌因子。在病理情况下，内皮功能障碍引起结构性、血液动力学和功能性血管异常，改变血管反应性和舒张性，并形成动脉粥样硬化。
PAD的治疗包括纠正不良生活方式，如：戒烟、控制体重，严格控制血糖、血压、血脂，抗血小板及抗凝治疗，扩血管药物治疗，综合干预后仍不能改善症状和溃疡愈合时再考虑给予血管重建。但总体来说，治疗效果远不及预期。有部分研究也报道了干细胞移植有可能改善糖尿病下肢缺血的症状和体征，但是由于干细胞获取和使用涉及到的伦理问题及外周血动员后可能造成的凝血功能障碍，并未在临床广泛使用。PAD当前的治疗方案主要为调脂、抗血小板聚集、扩血管等支持治疗，尚无特效药，亟需研发能改善糖尿病下肢缺血后促进血管新生的药物。
内皮祖细胞(Endothelial progenitor cells，EPCs)在触发血管生成以及维持内皮稳态方面起着关键作用。大量研究证实EPCs在血管修复和血管生成中起关键作用。EPCs通过直接整合到缺血部位形成新血管(Autophagy,2018,14(10):1677-1692；Biol RevCamb Philos Soc,2015,90(3):927-963)或分泌促血管生成因子(Diabetes,1993,42(6):801-813)来促进血管生成。局部或全身施用来自骨髓(FEBS Lett,2003,546(1):154-158)、脐带血(Proc Natl Acad Sci U S A,2001,98(12):6611-6616)或外周血(Int J Cardiol,2014,174(2):230-242)的EPCs可以增强缺血部位新血管形成，改善患有后肢或心肌缺血动物缺血组织功能。然而，在1型(Cardiovasc Diabetol,2005,4:9)和2型(CurrPharmBiotechnol,2011,12(3):386-391)糖尿病中，EPCs的数量和功能发生了改变，循环EPCs的数量均减少且功能受损，导致缺血部位的血管生成困难(preclinical andclinical evidence.2020,17(9):585-607)，血管生成和修复能力减弱。血管再生疗法是预防截肢的基石,并在PAD患者的治疗中扮演着关键角色。
如果能促进缺血部位的血供重建，改善组织缺氧，将从根本上改善PAD的预后(Journal of diabetes.2017；9(2):133-40)。因此，增强EPCs抗氧化损伤，改善EPCs促血管生成功能，是PAD药物开发的策略之一。然而,很少有科学数据可用来确定最优血管再生策略。
发明内容
本发明的一个目的在于提供一种GLP-1(32-36)在制备对EPC的高糖损伤起到保护作用的药物中的应用，改善EPCs促血管生成功能。
本发明的另一个目的在于提供一种GLP-1(32-36)在制备治疗血管闭塞疾病的药物中的应用，为血管闭塞提供治疗新方案。
本发明的再一个目的在于提供一种GLP-1(32-36)在制备治疗下肢动脉闭塞的药物中的应用，改善糖尿病下肢动脉闭塞，为DPAD的治疗提供新方案。
GLP-1(32-36)是GLP-1蛋白水解的主要最终产物(Regulatory peptides.1995；58(3):149-56)。通常认为，GLP-1(7-36)经GLP-1受体作用进入胞内，而被酶切成若干的短肽，GLP-1(32-36)即为其中之一。若直接通过GLP-1(7-36)的方式给药，其是否能够进入胞内则尚不清楚，其能产生何种作用亦没有相关的公开信息。经验证，在体内和体外的高血糖模型中，GLP-1(32-36)对于EPCs和人脐静脉内皮细胞(HUVECs)具有促进血管生成的作用，尤其是增强血液灌注和缺血组织的血管生成，在STZ(链脲佐菌素)诱导的后肢缺血模型(HLI)1型糖尿病小鼠，这些效果依赖功能跨膜GLP-1受体(GLP-1R)。GLP-1(32-36)可改善高糖诱导的线粒体分裂异常和功能障碍，并通过PFKFB3介导的糖酵解促进线粒体代谢。通过靶向的代谢组学来检测代谢物的变化验证，GLP-1(32-36)增强了内皮细胞的糖酵解。在GLP-1R-/-小鼠中，GLP-1(32-36)促进下肢血流灌注的作用也被阻断。
因此，GLP-1(32-36)通过激活eNOS-NO-cGMP通路改善GLP-1R介导的代谢紊乱来保护内皮细胞免受高血糖的影响，其单独或与其它药物共同使用对EPCs的高糖损伤起到保护作用，用于治疗下肢动脉闭塞。
将GLP-1(32-36)作为活性成分用于对EPCs的高糖损伤起到保护作用的药物，以改善受高糖损伤的动脉血管功能，改善患者的下肢血管闭塞临床症状，尤其是改善糖尿病外周动脉病变，尤其是下肢动脉闭塞，为PAD的治疗提供新方案。
GLP-1(32-36)在输送至患处时，通常需要借助于辅料或载体，比如：与蛋白或聚合物相结合，或者由聚合物包裹于其中等方式。
通过靶向的代谢组学来检测代谢物的变化，表明GLP-1(32-36)增强内皮细胞糖酵解。在GLP-1R-/-小鼠中，GLP-1(32-36)促进下肢血流灌注的作用被阻断。总之，研究表明GLP-1(32-36)通过激活PFKFB3介导的糖酵解通路改善GLP-1R介导的代谢紊乱来保护内皮细胞免受高血糖的影响。
在本发明的方案中，还包括对GLP-1(32-36)的一个或几个氨基酸进行替换，或对一个或几个氨基酸进行化学修饰，以改善多肽的稳定性。
将GLP-1(32-36)与其它辅料相混合，制成药物以治疗血管闭塞，尤其是下肢动脉闭塞。
这些药用辅料既可以是各种制剂中常规使用的，如：但不仅限于等渗剂、缓冲液、矫味剂、赋形剂、填充剂、粘合剂、崩解剂和润滑剂等；也可以是为了与所述物质相适应而选择使用的，如：乳化剂、增溶剂、抑菌剂、止痛剂和抗氧剂等，这类辅料能有效提高组合物所含化合物的稳定性和溶解性或改变化合物的释放速率和吸收速率等，从而改善各种化合物在生物体内的代谢，进而增强组合物的给药效果。
在水溶液注射剂中，辅料一般包括等渗剂和缓冲液，以及必要的乳化剂(如：Tweeen-80、Pluronic和Poloxamer等)、增溶剂和抑菌剂等。此外，还包括含有药学上可接受的其它药用辅料，如：抗氧剂、pH调节剂和止痛剂等。
用于制取口服液体制剂的辅料一般包括溶剂，以及必要的矫味剂、抑菌剂、乳化剂和着色剂等。
用于制取片剂的辅料一般包括填充剂(如：淀粉、糖粉、糊精、乳糖、可压性淀粉、微晶纤维素、硫酸钙、磷酸氢钙和甘露醇等)、粘合剂(如：乙醇、淀粉浆、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素、乙基纤维素、羟丙基甲基纤维素、明胶溶液、蔗糖溶液和聚乙烯吡咯烷酮的水溶液或醇溶液等)、崩解剂(如：干淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮和交联羧甲基纤维素钠)和润滑剂(如：硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇4,000、聚乙二醇6,000和月桂醇硫酸镁等)等。
用于制取乳剂的辅料一般为水、油(如：脂肪酸)、乳化剂，以及必要的防腐剂和矫味剂等。
用于制取颗粒剂的辅料与片剂类似，但造粒过程不同。根据需要，将制得的颗粒剂与助流剂混合后装入胶囊即得胶囊剂。
各种辅料与化合物制成有利于给药(drug delivery)的剂型，如：但不仅限于水溶液注射剂、粉针剂、丸剂、散剂、片剂、贴剂、栓剂、乳剂、霜剂、凝胶剂、颗粒剂、胶囊剂、气雾剂、喷雾剂、粉雾剂、缓释剂和控释剂等。此外，还可以为实现特定的给药目的或方式，如：缓释给药、控释给药和脉冲给药等，而使用的辅料，如：但不仅限于明胶、白蛋白、壳聚糖、聚醚和聚酯类高分子材料，如：但不仅限于，聚乙二醇、聚氨酯、聚碳酸酯及其共聚物等。所称的“有利于给药”的主要表现有：但不仅限于提高治疗效果、提高生物利用度、降低毒副作用和提高患者顺应性等。
将本发明所示的化合物与其它辅料相结合，比如：化学偶联，以进一步改善化合物的药效，降低毒作用和延长给药周期等。这些辅料通常是聚合物，比如：聚酯、聚醚和聚酰胺等。
将药物和医疗器械相结合制成的含药医疗器械也已经较为常见，比如：含止血剂的敷料。GLP-1(32-36)还作为活性成分装载或涂覆于支架材料上，用于制成治疗血管闭塞，尤其是下肢动脉闭塞的医疗器械。常见的支架材料如：PLA、PLGA和PET等。
附图说明
图1为各实验组相比于甘露醇组总血管生成长度的统计结果图；
图2为各实验组相比于甘露醇组划痕迁移统计结果图；
图3为各实验组相比于甘露醇组每个细胞的NO荧光强度统计结果图；
图4为各实验组相比于甘露醇组p-eNOS/eNOS比值统计结果图；
图5为各实验组相比于甘露醇组VEGFR2荧光强度平均值统计结果图；
图6为各实验组相比于对照组小鼠下肢血流灌注统计结果图；
图7为各实验组相比于对照组小鼠下肢腓肠肌CD31染色荧光强度统计结果图；
图8为各实验组相比于对照组小鼠下肢腓肠肌CD31/GAPDH比值统计结果图；
图9为各实验组相比于甘露醇组细胞划痕迁移统计结果图；
图10为各实验组中细胞Sca-1+/Flk-1+比值统计结果图；
图11为透射电镜观察各实验组的线粒体形态图；
图12为测得各实验组动物的线粒体氧化物统计图；
图13为测得各实验组动物的线粒体膜电位统计图；
图14为测得各实验组动物的mtDNA统计图；
图15为测得各实验组动物的基础耗氧率统计图；
图16为测得各实验组动物的最大耗氧率统计图。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围中。
本发明以下实施例所用的各项试验方法具体说明如下：
1)细胞培养
HUVECs(人脐静脉内皮细胞)是在ATCC(Manassas,VA)购买的，储存于实验室。细胞从3到6代使用和培养在37℃和5％的二氧化碳在DMEM低葡萄糖(葡萄糖5.56mM)和含10％胎牛血清(GIBCO)1％Lglutamine，1％的非必需氨基酸，100u/ml青霉素G、100μg/ml链霉素(100×GIBCO)。
BM-EPC(骨髓源性EPC)和UCB-EPC(脐带血源性EPC)在添加了10％胎牛血清(FBS)的内皮生长因子培养基(EGM-2)中生长。细胞培养在37℃，5％CO2在潮湿的空气中。
2)体外高血糖模型
HUVECs和UCB-EPCs挑战高葡萄糖(33mM)在DMEM和EGM-2然后孵化37℃和5％的二氧化碳的存在各种GLP-1肽(100nM)1h。四个分组进一步的研究在细胞保护机制模型：
i)高葡萄糖(HG)；
ii)HG+GLP-1(7-36)(100nM)；
iii)HG+GLP-1(32-36)(100nM)；
iv)甘露醇组(Man)不含HG和GLP-1(7-36)。
利用shRNA慢病毒技术在UCB-EPCs中过表达GLP-1R。包含特定的细胞被感染了慢病毒成分对GLP-1R(shGLP-1R)(目标序列)或shRNA序列-(NC)对照组(GenePharma)12h。两组细胞进行预处理与GLP-1(7-36)或GLP-1(32-36)(100nM)1h，然后孵育高葡萄糖(33mM)正常HUVECs，不含高糖和/或利拉鲁肽进行比较。所有样本在孵育结束时采集。
3)实验动物
实验装置和动物护理得到了浙江大学动物政策和福利委员会的许可(批件号：(2021年度)第(172)号)小鼠被关在一个温度控制的房间里，按12小时的明暗周期进行，并接受标准实验室饮食。选用8-10周龄的C57BL/6J小鼠(GemPharmateh，南京，中国)，通过腹腔注射链霉菌素(STZ)(100mg/kg体重，每天1次，连续2天)建立1型糖尿病(T1DM)模型。用血糖仪测定血糖，验证糖尿病高血糖。注射STZ 5天后，夜间空腹血糖水平11.1mM或更高的小鼠被分为体重和GLP-1(32-36)处理组，或对照组每天腹腔注射1次，连续21天。所有动物实验协议均遵守美国国立卫生研究院发布的《实验室动物护理和使用指南》。所有实验均在HLI后28天处死成年雄性小鼠。
4)鼠后肢缺血模型
启动血管新生的过程在腓肠肌的后肢，用异氟烷麻醉鼠，然后从右股动脉和股静脉(从下方深股动脉腘动脉和静脉)的结扎，而左后肢作为假手术对照组(Natureprotocols.2009；4(12):1737-46)。后肢缺血后，每天给予生理盐水或药物治疗，持续28天。研究结束时，对小鼠实施安乐死，并收集肌肉组织，用4％的多聚甲醛固定或液氮冷冻，以供进一步的实验。
5)骨髓源性内皮祖细胞的分离与鉴定
通过percoll1-1083(Sigma,USA)密度梯度离心，从WT(WT-EPCs)、STZ诱导的T1DM和GLP-1R-/-(GLP-1R-/-EPCs)小鼠骨髓中分离EPCs，并在内皮细胞基础培养基2(EBM-2，瑞士)中添加2％胎牛血清(胎牛血清，FBS，美国)，根据以前建立的方法进行少量修改(Journal ofcellular and molecular medicine，2011；15(6):1299-309)。简单地说，用PBS洗去未贴壁的细胞，然后在培养3天后使用新的培养基。细胞集落在分离后第7天出现，被定义为EPCs，并在含20％胎牛血清的EBM-2中维持。分离的EPCs用于第2～3代的体外研究。培养24天后，收集细胞进行实验。
内皮祖细胞的荧光化学检测,在第七天,细胞首先孵化Dil-acetylated低密度脂蛋白(DiI-acLDL)37℃为4小时，然后用4％多聚甲醛固定后10分钟。与PBS洗两次,细胞反应与ulex europaeus agglutinin-1(UEA-1σ)1小时。用共聚焦显微镜(Leica,Wetzlar，德国)观察样品。双阳性染色的细胞被鉴定为分化的EPCs(Proc NatlAcad Sci U S A.2000；97(7):3422-7)
6)人脐血来源EPCs的分离与鉴定
在CPD溶液中采集人脐带血20-40mL。第二附属医院的机构审查委员会批准浙江大学所有的协议，并获得了知情同意((2021)伦审研第(1108)号)。人类脐血EPCs根据之前发表的方案进行了少量修改。简单地说，脐带血(50-100ml)用Hanks平衡盐溶液(HBSS；Invitrogen公司，NY,USA)，并小心地覆盖在等量的Histopaque 1077(Sigma,MO,USA)上，在室温下400g离心30分钟。收集白大衣，HBSS洗涤2次。随后，用含2％胎牛血清(Sigma)的EGM-2(Lonza,Basel,Switzerland)重悬细胞，然后在预涂有人类纤维连接蛋白(2μg/cm2,BDBiosciences,MA,USA)的6孔板的一个孔中培养。将平板置于37℃加5％CO2的潮湿环境中培养。24h后，用EGM-2培养基洗涤，去除未附着的细胞和碎片。每日更换培养基，连续7天，之后隔日更换。克隆期为14-21天，28天左右达到80％融合。
继代培养后，EPCs的特征是乙酰化低密度脂蛋白(ac-LDL)摄取和凝集素结合实验，以及细胞表面标记物染色。在ac-LDL摄取和凝集素结合实验中，细胞与Dil-acLDL(10μg/ml)在37℃孵育4h，然后与3％多聚甲醛固定10min。PBS洗涤两次后，细胞与UlexEuropaeus凝集素-1(uea-1,10μg/ml,Sigma)反应1h。染色后用荧光显微镜(Olympus IX71,Olympus,Tokyo,Japan)拍照。双阳性染色细胞为分化的EPCs。流式细胞仪检测EPCs中CD34、CD133和VEGFR2的表达。将EPCs扩展到第四或第五代进行进一步分析。
7)免疫印迹
Western blotting检测不同处理下HUVECs、BM-EPCs和UCB-EPCs的蛋白表达及磷酸化水平。用磷酸盐缓冲液(pH7.2的PBS，Beyotime，杭州，中国)洗涤细胞三次，用含有蛋白酶抑制剂鸡尾酒的细胞裂解缓冲液(Beyotime)处理细胞，然后从培养板上提取。4℃，14,000×g离心10分钟。蛋白浓度测定采用Bradford assay kit(Beyotime)。用8-15％SDS-PAGE凝胶分离等量的蛋白(20μg蛋白/lane)。凝胶中的蛋白转移到聚亚氟乙烯膜(默克Millipore，达姆施泰特，德国)，然后用5％脱脂乳粉在含有0.05％吐温-20的PBS中在37℃下阻断1小时，并在4℃下与不同的一抗在1:1000稀释下孵育过夜。随后，将膜与适当的二抗按1:5000稀释孵育1小时。使用的抗β-actin、GAPDH、Fis1、Mfn2、OPA1、GLP-1R、total-Drp1、eNOS及其磷酸化形式(p-Drp1(Ser637和p-eNOS))的抗体来自Cell Signaling Technology(CST,Boston,USA)。与辣根过氧化物酶(HRP)结合的山羊抗兔或山羊抗鼠抗体购自KPL(Gaithersburg,MD,USA)。使用SuperSignal West Pico化学发光衬底(ThermoScientificTM，Waltham,USA)对免疫反应带进行可视化，并在化学发光成像系统(AmershamTMImageQuant 800)中捕捉图像。使用NIH ImageJ软件(TreeStar,San Carlos,CA,USA)进行密度分析定量条带。目的蛋白与参比蛋白的比值用于相对定量目的。
8)激光多普勒血流灌注成像(LDPI)
对小鼠进行麻醉，观察缺血后肢血液灌注恢复情况(Proc Natl Acad SciUSA.2006；103(29):11015-20)。在术前和术后0、3、7、14、21、28天使用LDPI系统(MoorLDLS,UK)对缺血肢体微血管灌注进行一系列无创评估。以左后肢为对照组。后肢灌注恢复以缺血肢体灌注(右)与健康肢体灌注(左)之比表示。
9)细胞疗法
术后尾静脉灌注1×106个慢病毒载体、携带GLP-1R、非特异性shRNA序列的慢病毒的EPCs。为了评估肢体灌注比[缺血肢体(右)/正常肢体(左)]，在缺血后0、3、7、14、21和28天，使用基于激光散斑对比分析技术的Pericam灌注散斑成像仪(PSI)进行实时微循环成像分析。
10)体外血管生成(试管形成)检测
采用基质管形成法检测HUVECs和EPCs的血管生成能力。评估GLP-1(32-36)(五肽)或其他化合物对血管新生的影响是一种简单的方法，方法是测量内皮细胞形成毛细血管样结构(管)的能力，内皮细胞以适当的细胞外基质支持以亚融合密度，以模拟血管新生的重组阶段。内皮祖细胞或HUVECs内胚层的增长保持medium-2(EGM2)处理五肽12h与过度或转染lentivirus-shGLP-1R或lentivirus-shNC 12h,然后播种Matrigel-GFR(生长因子)有或没有高葡萄糖(33mM)形成管。管被允许形成18-24小时，在37℃和5％的CO2加湿的培养箱。荧光图像由LeicaDMi8(Wetzlar，德国)捕捉，并使用NIH ImageJ软件进行分析。
11)创面愈合情况分析
先前描述的伤口愈合划痕技术被用来评估细胞迁移(Cell.2019；176(4):944-5)。将HUVECs和EPCs镀在12孔板的孔中培养过夜，直到形成融合的单层，用200μL移液针尖划伤单层。在创伤后0h和24h，采用配备DFC295相机的DMIL显微镜系统，由LAS V4.0软件(Leica,Germany)控制，测量高糖(33mM)和GLP-1(32-36)对伤口愈合的影响。加入1μM丝裂霉素(Selleck Chemicals LLC,Houston,TX,USA)以排除细胞增殖的影响。
12)一氧化氮(NO)分泌量的测定
用二醋酸二氨基荧光素-fm(DAF-FM)二醋酸试剂盒测定NO含量。简单地说，将2mM的DAF-FM加入细胞中，在室温黑暗中孵育30分钟。将细胞洗去多余的未结合探针，然后在黑色透明底96孔板中孵育15分钟，使细胞内的二乙酸盐完全去酯化。NO产物在共聚焦荧光显微镜(IX81-FV1000,Olympus,Markham,Canada)上成像，并使用荧光激发/发射光谱仪在500/515nm波长进行测量，数据以相对荧光单位(RFU)表示。
13)mtDNA水平测定
采用AquaPure基因组DNA分离试剂盒(BioRad)提取总DNA。50ng的DNA与水稀释到4.5毫升,合并后10mM和0.5mL的正向和反向引物添加(mtDNA，向前引物：CTAGCCACCAAACCAAA，相反：CCAGCTATCACCAAGCTCGT,为nDNAmB2M1向前引物：ATGGGAAGCCGAACATACTG，相反：CAGTCTCAGTGGGGGTGAAT和5ml 2×RT2SYBR GreenqPCRMastermix(Qiagen)。反应在Eco qPCR体系(Illumina)上运行，设置为：50℃循环2min，95℃循环10分钟，95℃15s，60℃1min，循环40次。
14)透射电子显微镜
透射电子显微镜(TEM)评价线粒体形态。骨髓来源的EPCs在12孔板中孵育，制备TEM样本。样品处理程序参见：Viruses.2020；12(3)。将不同处理后的细胞样品在LEICAEMUC7超微组中切片。切片连续用乙酸铀酰和碱性柠檬酸铅染色5-10分钟，在日立H-7650TEM(东京，日本)中观察。
15)流式细胞仪
将HUVECs和EPCs置于24孔板中培养，测定线粒体活性氧(ROS)和线粒体膜电位(MMP)。经过不同处理后，去除培养基后用HBSS洗涤细胞。用5μM MitoSOX(ThermoFisherTM)探针线粒体ROS，用0.1μM JC-1(BD Biosciences)探针MMP，在37℃黑暗中探针30min(Molecular and cellular endocrinology.2022；545:111560)。
调查的影响GLP-1(32-36)五肽在EPC动员外周血循环(PB),以应对组织缺血,从小鼠骨髓中收集100μL PB，然后与异硫氰酸荧光素(FITC)anti-mouse抗体孵化，和APC anti-mouse Flk-1(VEGFR-2)抗体，因为来自单个核部分的双阳性Sca-1+/Flk-1+细胞被认为是循环的EPCs(Atherosclerosis.2010；212(2):426-35)。每次分析包括10万个细胞，并在HBSS中重悬，在BECKMAN COULTER CytoFLEX LX(BECKMAN COULTER,USA)上进行流式细胞分析。数据分析使用FlowJo软件版本10(Treestar)。
16)细胞生物能量分析
为了测定线粒体的功能生物能能力，我们使用Seahorse XF96(AgilentTechnologies,USA)进行了耗氧率(OCR)和细胞外酸化率(ECAR)测试。简而言之，将HUVECs或EPCs以5000个细胞/孔的密度接种到海马XFp细胞培养微型板中，并按照上述“体外高血糖模型”方法进行相应处理。分光光度法由3种不同的注射剂组成。将细胞保存在培养基(25mM葡萄糖，1mM丙酮酸，2mM-谷氨酰胺，pH 7.4)中，然后第一次注射1μM寡霉素，寡霉素是一种负责阻断线粒体ATP生成的化学物质，第二次注射1.5μM FCCP(羰基氰基-4(三氟甲氧基)苯腙)，这是一种解偶联线粒体膜以评估最大细胞呼吸的化学物质，第三种是100μM鱼藤酮和1μM抗霉素a的组合，它们分别在电子传递链上阻断复合物I和III。每次注射后记录4个时间点，每次间隔约35min。OCR和ECAR由Seahorse XF-96软件自动记录和计算。每个实验组在每个分析时间点使用9个重复并进行分析。变化与基础率的百分比计算为变化值除以基线读数的平均值。每孔总蛋白的OCR和ECAR归一化，以pmol/min和mPH/min表达。
为了进行ATP检测，骨髓来源的EPCs以250000个/孔的浓度被镀在六孔板中并培养过夜。根据制造商说明，使用ATP测定试剂盒(Invitrogen，CA)对裂解液中的ATP浓度进行定量。PBS冲洗细胞，ATP释放缓冲液裂解细胞，将10μl裂解液和ATP标准品加入白色Nunc 96孔板中。然后，每孔加入荧光素酶缓冲液100μl，立即用InfiniteM200酶标仪(Tecan)测定发光。总ATP水平按蛋白量归一化，显示为与BM-EPCs水平的比值。
17)免疫荧光染色
HLI后28天的血管生成程度通过CD31和dystrophin(用于指示肌纤维)染色测量毛细血管密度来评估。简单地说，在第28天从缺血后肢解剖的缺血腓肠肌冰冻切片(6μm)用冷甲醇固定15min。PBS洗涤3次后，切片与封闭缓冲液(含5％山羊血清的PBS)孵育1小时。然后，切片与抗肌营养不良蛋白(Abcam)和CD31(Santa Cruz)一抗在4℃孵育过夜。PBS冲洗3次后，切片与相应的pe偶联二抗(Cell Signaling Technology)和fitc偶联二抗(BDBiosciences)在室温暗孵育1h。PBS冲洗3次后，用抗褪色试剂封片，共聚焦显微镜(OlympusIX71,Olympus)拍照。毛细血管密度以每根肌纤维CD31阳性毛细血管数目表示。
18)代谢组学分析
样品提取物采用液相色谱-esi-MS/MS系统3，由武汉美泰生物技术有限公司进行分析。分析条件为:UPLC:色谱柱Waters ACQUITY UPLC HSS T3C18(1.8μm,2.1mm*100mm)；列温度40℃；流速0.4mL/min；注入体积,2μL；溶剂体系，水(0.04％乙酸):乙腈(0.04％乙酸)；梯度程序，95:5V/V在0分钟，5:95V/V在11.0分钟，5:95V/V在12.0分钟，95:5V/V在12.1分钟，95:5V/V在14.0分钟。
19)统计
数据用三次独立实验的均值±SEM表示。图由GraphPadPrism 5(美国)绘制。统计学比较采用单因素方差分析(ANOVA)后进行LSD事后检验。所有统计分析均使用SPSS 24.0(美国)进行。在*P<0.05认为差异显著，**P<0.01和***P<0.001显著性更高或极显著，ns表示无统计学差异。
实施例1 GLP-1(32-36)对高糖(HG)作用下的HUVECs血管生成作用
采用体外高血糖模型，HUVEC和GLP-1(7-36)作为全长肽对照。血管形成和划痕恢复实验显示，高糖显著损害HUVECs的血管生成，GLP-1可明显恢复血管生成(图1和图2)。用western blotting检测eNOS的磷酸化水平，用DAF-FM二醋酸试剂盒检测NO的产生后，发现在HG-和GLP-1(32-36)处理的细胞中p-eNOS的表达(图3)和NO的荧光强度显著升高(图4)。进一步通过流式细胞术检测了VEGFR-2的表达，如图5所示，在五肽处理的细胞中，VEGFR-2显著增强。由此可见，GLP-1(32-36)对HUVEC血管生成的促进作用略好于GLP-1(7-36)，至少与GLP-1(7-36)具有相当的血管生成的促进作用。
实施例2 GLP-1(32-36)促进1型糖尿病小鼠下肢缺血模型的血液灌注和血管生成
stz诱导的小鼠糖尿病模型，并使用小鼠单侧后肢缺血模型测量治疗性血管生成，在HLI手术后第3、7、14、21、28天用LDPI评价血流恢复情况。如图6所示，28天后，与PBS对照组相比，GLP-1(32-36)和GLP-1(7-36)处理组的血流量恢复明显更高。
由于缺血腓肠肌血管以CD31表达为特征，图7所示，免疫荧光结果显示肽处理组比对照组有更多的CD31阳性毛细血管。图8所示，western blot结果也证实了这一点。可见，GLP-1(32-36)在各项试验中均略优于GLP-1(7-36)。这些结果表明，GLP-1(32-36)挽救了stz诱导的糖尿病小鼠缺血肢体的血管生成功能和血液灌注。
实施例3 GLP-1(32-36)介导的EPCs血管生成
使用传代3至6代的人类EPCs细胞，用过表达的慢病毒-shGLP-1R或随机对照慢病毒NC转染EPCs 12h后，分别用GLP-1(32-36)或(7-36)加或不加高糖(33mM)转染EPCs。如图9所示，GLP-1(32-36)或(7-36)增加了EPCs的迁移能力，提高EPCs的血管生成能力。
实施例4 GLP-1(32-36)拯救小鼠骨髓源性EPCs的动员
采用流式细胞术检测T1DM小鼠外周血单个核部分的双阳性Sca-1+/Flk-1+细胞数量。组织缺血可增强EPC的动员。如图10所示，在HLI后第3天，GLP-1(32-36)给药大大增强了T1DM小鼠组织缺血后EPC的动员，并在第7天达到峰值。
由于已有研究证明，PAD引起的糖尿病足溃疡修复延迟源于eNOS活性受损NO合成障碍和由此导致的EPCs从骨髓至外周循环动员障碍。Epc动员增强意味着促血管新生能力的增强。进一步研究证明，GLP-1(32-36)可改善高糖诱导的线粒体分裂异常和功能障碍，并通过eNOS-NO-cGMP途径促进线粒体代谢。
实施例5 GLP-1(32-36)对线粒体代谢的作用
从T1DM小鼠或注射了五肽的T1DM小鼠中分离并培养了原代BM-EPCs。透射电镜观察线粒体形态。在stz诱导的糖尿病小鼠中，线粒体超微结构显示，注射了五肽的细胞内细长的线粒体显著增加，而糖尿病小鼠的线粒体呈圆形和圆形(参见图11)。GLP-1(32-36)可有效降低线粒体氧化物的量(参见图12)，膜电位显著提高(参见图13)。注射GLP-1(32-36)可增加T1DM小鼠的mtDNA含量(参见图14)，还逆转了高糖对基础和最大耗氧率水平的影响，参见图15和图16。
这些结果表明，GLP-1(32-36)具有调控线粒体功能(线粒体动力学、氧化应激等表型)及线粒体代谢的作用，从而促进内皮细胞的血管新生能力。
Claims (10)
1.一种GLP-1(32-36)在制备对EPCs的高糖损伤起到保护作用的药物中的应用。
2.根据权利要求1所述的用途，其特征在于所述的GLP-1(32-36)来自于GLP-1(7-36)的降解或化学合成。
3.根据权利要求1所述的用途，其特征在于所述的GLP-1(32-36)还与聚合物、多肽或蛋白相结合。
4.一种GLP-1(32-36)在制备治疗糖尿病外周动脉病变的药物中的应用。
5.根据权利要求4所述的用途，其特征在于所述的GLP-1(32-36)来自于GLP-1(7-36)的降解或化学合成。
6.根据权利要求4所述的用途，其特征在于所述的GLP-1(32-36)还与聚合物、多肽或蛋白相结合。
7.一种GLP-1(32-36)在制备治疗下肢动脉闭塞药物中的应用。
8.根据权利要求1所述的用途，其特征在于所述的GLP-1(32-36)来自于GLP-1(7-36)的降解或化学合成。
9.根据权利要求1所述的用途，其特征在于所述的GLP-1(32-36)还与聚合物、多肽或蛋白相结合。
10.一种以GLP-1(32-36)为活性物质在制备用于治疗下肢动脉闭塞的药物或含药医疗器械中的应用。
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