CN114425081A - 钙调蛋白作为新靶点在防治糖尿病下肢外周血管疾病中的应用 - Google Patents
钙调蛋白作为新靶点在防治糖尿病下肢外周血管疾病中的应用 Download PDFInfo
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Abstract
本发明公开了一种糖尿病血管并发症防治的新靶点及其在糖尿病下肢外周血管疾病中的应用。本发明发现一种新的糖尿病血管并发症防治靶点，即钙调蛋白(calmodulin,CaM)，其在血管内皮表达水平的下调是糖尿病血管内皮损伤的重要原因。腺相关病毒携带CALM1的基因疗法能逆转糖尿病血管内皮CaM的表达，增强一氧化氮合酶的活性及一氧化氮和血管内皮生长因子的生成，促进血管新生，防治糖尿病血管并发症。本发明也明确了低分子量褐藻多糖硫酸酯(low molecular weight fucoidan，LMWF)治疗糖尿病血管并发症的分子机制，即LMWF通过减少CaM的泛素‑蛋白酶体降解，缓解糖尿病所致CaM表达的下调。本发明进一步揭示了糖尿病血管并发症防治的分子机制，对新治疗方案研究和应用具有重要的意义。
Description
技术领域
本技术发明涉及一种糖尿病血管合并症治疗的新靶点和新的有效治疗手段，具体地说是涉及一种采用基因疗法来增加糖尿病血管内皮中靶蛋白-钙调蛋白(calmodulin,CaM)的表达在防治糖尿病血管合并症中的应用。本发明属于基因治疗技术领域。
背景技术
糖尿病(diabetes mellitus,DM)是一种由胰岛素分泌异常所引起、以高血糖为主要临床表现的慢性代谢性疾病，严重危害人类的健康和生活质量。糖尿病患者血管由于受多种有害刺激，如高血糖、高血脂、氧化应激和炎症反应，使血管内皮功能紊乱和多种血管病变，进而容易诱发糖尿病血管并发症(Paneni F, et al.Diabetes and vasculardisease:pathophysiology,clinical consequences,and medical therapy:partI.European Heart Journal.2013；34:2436-2443.)。其中，内皮功能障碍所致的内皮修复功能障碍和微小血管生成能力降低，是糖尿病血管并发症发病的一个重要原因(Sena CM,et al.Endothelial dysfunction-a major mediator of diabetic vasculardisease.Biochim Biophys Acta.2013；1832:2216-31.)。外周动脉疾病(peripheralarterial disease, PAD)，特别是下肢动脉疾病是临床常见的一种疾病，发病率高，分布人群广，病理过程比较复杂，涉及内皮细胞肿胀，血小板积聚，白细胞黏附及微血栓形成等，最终导致了肢体末梢微循环灌注障碍。糖尿病合并的PAD中，发生在下肢的外周血管病，也就是糖尿病足，比较常见，以缺血与/或合并感染较为常见，临床表现为跛行，溃疡，甚至坏疽，在最后阶段截肢可能是许多患者的唯一选择(Schaper N,et al.Peripheral vasculardisease and type 2diabetes mellitus.Diabetes Metab Res Rev.2000；16Suppl 1:S11-5.2000:S11-5)。
目前临床上用于预防和治疗糖尿病PAD的方法包括：1)控制危险因素的一般治疗，如戒烟、控制血糖、血脂、血压及运动疗法等；2)以抗血小板聚集、扩张血管为主的药物治疗，如阿司匹林、氯吡格雷、西洛他唑等；3)血运重建：包括内科介入和血管外科手术治疗(Heikkila K,et al.Improving 1-Year Outcomes of Infrainguinal LimbRevascularization:Population-Based Cohort Study of 104 000Patients inEngland. Circulation,2018；137:1921-1933.)；4)尚处于临床试验阶段的生长因子基因治疗(
P,et al.Double VEGF/HGF Gene Therapy in Critical Limb IschemiaComplicated by Diabetes Mellitus.J Cardiovasc Transl Res. 2021；14:409-415.)(Belch J,Hiatt W,et al.Effect of fibroblast growth factor NV1FGF onamputation and death: a randomised placebo-controlled trial of gene therapyin critical limb ischaemia.Lancet.2011；377:1929-37.)和干细胞移植术(KawamotoA,et al.Intramuscular transplantation of G-CSF-mobilized CD34(+)cells inpatients with critical limb ischemia:a phase I/IIa,multicenter,single-blinded,dose-escalation clinical trial.Stem Cells. 2009；27:2857-64.)。但是，糖尿病下肢PAD患者早期不易被发现而错过最佳控制和治疗期，进展为严重的下肢缺血(Critical leg ischemia，CLI)，只能进行血运重建，对于不能进行血运重建患者，唯一的治疗方法是口服抗血小板药物、他汀类药物、糖尿病治疗和戒烟等，但预后特别差，1年截肢率为40％，死亡率高达20％(Freisinger E,et al.Impact of diabetes on outcome incritical limb ischemia with tissue loss:a large-scaled routine dataanalysis.Cardiovasc Diabetol.2017；16:41.)。近年来，血管生成的生长因子基因治疗和干细胞移植术被认为是一种很有前景的治疗方式，但是干细胞疗法的安全性受到种类来源、制备质量和副作用等影响且目前的生长因子疗法大多只针对单一生物标志物，如血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和肝细胞生长因子(HGF)等(Madonna R,et al.Insights into gene therapy for critical limb ischemia:thedevil is in the details.Vascul Pharmacol.2012；57:10-4.)。由于糖尿病PAD发生发展所涉及的生化网络的复杂性，使得这些试验结果多变不一，目前只有HGF作为候选通过了二期临床。因此，进一步深入了解DM血管并发症发生的相关分子机制，开发具有多种靶点或作用机制的药物有望成为糖尿病血管并发症的未来治疗方法。
昆布多糖又称低分子量褐藻多糖硫酸酯(low molecular weight fucoidan，LMWF)是从海带以及昆布中提取出来的褐藻多糖硫酸酯，对其进行进一步降解和硫化得到的一种分子量为7～9kDa的高硫酸化的多糖聚合物。LMWF具有口服可吸收，副作用少，广泛的生物活性，如抗氧化应激、抗炎、抗血小板聚集、扩张血管等优点，尤其对糖尿病血管疾病疗效明显(Liu T,et al.Low molecular-weight fucoidan protects against hindlimbischemic injury in type 2diabetic mice through enhancing endothelial nitricoxide synthase phosphorylation.J Diabetes.2018；10:820-834.)，并已于2010年获得专利(CN 101912408)。为进一步探寻糖尿病血管内皮损伤和LMWF治疗糖尿病足的分子机制，本发明研究了高糖处理的血管内皮细胞和糖尿病动物的血管和内皮祖细胞，发现糖尿病及高糖刺激降低内皮细胞血管新生能力的同时，伴随着钙调蛋白 (Calmodulin，CaM)表达水平的下调，而LMWF改善糖尿病或高糖条件下内皮细胞血管新生能力的同时，上调CaM的蛋白表达水平，即CaM可能是LMWF发挥糖尿病血管保护作用的重要靶标，针对该靶标进行基因治疗发现糖尿病足的症状减轻和病理性损伤得到明显的改善。CaM是一类重要的小分子钙离子信号传感蛋白，具有高度保守的基因序列，是多种组织中效应物传递信号的重要蛋白质，它可通过细胞内钙离子的增加而被激活。CaM可与100多种靶蛋白结合，调节靶蛋白的活性，参与了许多不同的钙依赖性信号转导通路，在细胞运动、代谢、运输、分泌、受精、增殖、程序性细胞死亡、自噬过程和肿瘤发生等方面起着调节作用，同时也参与结构完整性和细胞间通讯。CaM在细胞内主要经泛素-蛋白酶体(UPS)进行降解。CaM的半衰期受钙离子浓度及氧化应激的影响，氧化引起CaM构象的改变，使其被识别和降解，从而影响了CaM丰度的改变(Tarcsa E,et al.Ca2+-free Calmodulin and Calmodulin Damaged by in VitroAging Are Selectively Degraded by 26S Proteasomes withoutUbiquitination.Journal of Biological Chemistry. 2000；275:20295-20301.)。同时，CaM是内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)激活的上游调节器，而eNOS对调节内皮功能至关重要，CaM可以通过增强eNOS的活性，产生NO，调节血管舒张功能(Rochette L,et al.Nitric oxide synthase inhibition and oxidative stressin cardiovascular diseases: possible therapeutic targets.Pharmacol Ther.2013；140:239-57.)。目前公认糖尿病血管eNOS功能失调：包括的信号转导脱偶联、磷酸化水平降低及NO合成障碍、以及参与硝基过氧化物的生成，可引发血管内皮和平滑肌功能紊乱，最终导致糖尿病血管内皮功能障碍和血管并发症的发生。
因此，本发明指出CaM的蛋白表达水平降低是糖尿病相关内皮功能障碍和血管生成受损的重要原因，同时开发了携带编码人CALM1基因的腺相关病毒(CaM-AAV)的基因药物对糖尿病小鼠的PAD进行了治疗，并阐述了LMWF治疗糖尿病足具体的分子机制。
发明内容
本发明的目的是提供一种糖尿病血管合并症治疗的新靶点，具体地说是涉及一种采用基因疗法来增加靶蛋白CaM的表达在预防和/或治疗糖尿病血管合并症中的应用。
本发明所提供的CaM作为治疗新靶点使其在制备预防和/或治疗糖尿病血管疾病产品中的应用。所述产品包括药物和保健品。
本发明利用分子生物学、动物学、细胞学及生物化学等几大研究平台的技术手段进行证明，首次将CaM 作为一种治疗靶点应用于糖尿病血管并发症治疗领域。基于在糖尿病PAD发生发展过程中，CaM通过调控血管内皮细胞新生并利用病理学技术，观察其对糖尿病PAD的治疗作用。此外，探究LMWF是否是通过作用于该靶蛋白来发挥其治疗糖尿病血管并发症的作用。
使用本发明具有以下有益效果：
1、本发明明确了CaM的蛋白表达与糖尿病及糖尿病血管内皮损伤和血管并发症的关系，提供了一种新的糖尿病血管并发症防治靶点。本发明通过糖尿病骨髓内皮祖细胞(BM-EPCs)及高糖培养的人脐静脉内皮细胞(HUVECs)的研究发现，CaM蛋白表达的恢复能改善糖尿病情况下内皮细胞功能障碍从而提出其可能是防治糖尿病血管并发症的靶点，同时两细胞可作为糖尿病血管并发症药物的发现与筛选的有效工具。
2、同时，本发明的成果为糖尿病内皮损伤提出新的病理生理学机制，在新的治疗方案、药物研发及疾病诊断过程中具有十分重要的指导意义。
3、LMWF改善糖尿病血管内皮损伤的分子作用机制以及在糖尿病并发症中治疗作用已获4项国家发明专利，但具体的药物作用靶点不清。在本专利中已明确，LMWF通过减少CaM的泛素-蛋白酶体途径的降解，从而逆转由于糖尿病导致CaM蛋白表达的下调，而恢复eNOS/NO/VEGF信号通路的功能，促进血管新生，改善由于血管内膜受损导致糖尿病血流障碍和合并症的发生和发展。
附图说明
下面结合附图和实施例对本发明作进一步说明：
图1为本发明的人脐静脉血管内皮细胞进行高糖处理后(模拟糖尿病)，eNOS和CaM表达的改变图；
图2为本发明用质粒过表达CaM对db/db小鼠骨髓内皮祖细胞(BM-EPCs)蛋白表达及功能的影响图；
图3为本发明db/db小鼠骨骼肌中eNOS与CaM存在相互作用减弱图；
图4为本发明用腺相关病毒携带CALM1基因(CaM-AAV)对db/db小鼠下肢缺血模型足底血流恢复的治疗作用图；
图5为本发明用CALM1基因(CaM-AAV)对db/db小鼠下肢缺血模型腓肠肌病理改变的治疗作用图；
图6为本发明用CALM1基因(CaM-AAV)对db/db小鼠下肢缺血模型腓肠肌进行CD34、VEGF染色评价对血管新生密度的治疗作用图；
图7为本发明LMWF能逆转高糖下HUVECs细胞CaM表达水平下调作用图；
图8为本发明LMWF能增加高糖下内皮eNOS与CaM存在相互作用图；
图9为本发明LMWF通过降低CaM的泛素-蛋白酶体代谢而上调CaM的表达作用图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所用的昆布多糖为低分子量褐藻多糖硫酸酯(LMWF)，其制备方法及应用于糖尿病血管疾病治疗及下肢动脉闭塞的外周血管病大鼠治疗分别于2010年(CN101912408)和2016年(CN 105663154) 获得专利。
实施例1、钙调蛋白作为新靶点在防治糖尿病下肢缺血模型中的应用
材料与方法
1、实验动物的分组、给药与造模：
8～10周龄雄性db/db小鼠和周龄性别匹配的C57BL/6小鼠适应性饲养至12～13周，随机分组：Non- DM Sham组(左侧股动脉分离，但不结扎和去除)；Non-DM Model组(左侧股动脉结扎和去除)；DM Sham 组(左侧股动脉分离，但不结扎和去除)；DM AAV-Vector组(左侧股动脉结扎和去除之前1周，左侧腓肠肌注射AAV-Vector 4×1010gc/4个注射点)；DMAAV-CaM组(左侧股动脉结扎和去除前1周，左侧腓肠肌注射AAV-CaM 4×1010gc/4个注射点)。
2、外周血管病(PAD)手术造模：
左侧股动脉结扎和去除造成C57BL/6和db/db糖尿病小鼠下肢缺血模型。手术全程用异氟烷气麻装置麻醉小鼠，麻醉后，把小鼠仰卧于固定板上，在显微镜下完成操作手术，左侧腹股沟中点与膝盖处连线，为手术部位，用棉球沾取碘伏，反复擦拭三次以上，再用不锈钢钢双面刀片备皮，弯镊配合组织剪开皮肤，组织，暴露股动脉，动静脉和神经分离，游离股动脉，2把弯镊配合充分剔除股动脉上残余的结缔组织。股动脉远近段结扎和去除，用丝线缝合伤口。碘伏消毒后，腹腔注射青霉素1ml预防感染，共注射青霉素 7天。
3、激光多普勒散斑血流测定：
小鼠股动脉结扎手术后1d及每周使用激光多普勒散斑成像系统采集血流情况至实验结束。用异氟烷气麻装置麻醉小鼠，将其仰卧于黑色的背景板上，足部自然分开，充分暴露足底。测量之前固定仪器探头和动物双侧肢之间的最佳位置，点击“摄像头”图标，然后开始连续扫描血流成像图。对彩色数字图像进行分析，量化从膝关节到脚趾区域的血流。计算血流灌注平均值，计算每组每只动物缺血肢(左，L)与非缺血肢(右，R)的比值，比较不同组别的血流恢复程度。通过这种方式采集数据，尽量避免由不同时间、不同仪器、不同背景之间造成的系统差异。
4、苏木精-伊红染色和CD34、VEGF免疫组化染色
4.1苏木精-伊红(HE)染色：
取4％甲醛固定的腓肠肌组织48h，按常规石蜡切片过程进行梯度乙醇脱水、二甲苯透明、浸蜡、包埋，4μm厚度切片。石蜡切片用经二甲苯脱蜡；乙醇洗掉二甲苯：100％乙醇3min×3；95％乙醇3min× 3；80％乙醇3min×3；70％乙醇3min×3；去离子水洗3次洗掉乙醇；苏木精染细胞核5min；水洗苏木精 3次；1％盐酸-乙醇分化15s；水洗；伊红染细胞质1min；水洗伊红3次；分色，脱水：70％乙醇2min； 80％乙醇2min；95％乙醇2min×2；100％乙醇2min×2；二甲苯透明：10min×3；中性树胶封片：避免气泡，迅速，防止组织发黑；37℃烘箱烤干一周。显微镜下观察，细胞核被苏木精染成紫蓝色，多数细胞质及非细胞成分被伊红染成粉红色。病理扫描仪阅片，保存照片。
4.2免疫组化染色：
用于免疫组化的组织石蜡块切成4μm的厚度，组织切片脱蜡和水化后，用PBS(PH7.4)冲冼3次，每次3分钟，于玻璃染色缸内加50mL H2O2液(甲醇50mL+H2O2 1.5mL，浓度为3％)，室温12分钟，PBS 冲洗3次，洗去2个片子之间的H2O2液，于玻璃染色缸内加50mL EDTA液(50×浓缩，稀释50倍使用 1×EDTA)，92℃水浴锅加热10分钟，冷却至室温，加PBS冲洗3次，滴加正常非免疫动物血清20-50μL，于湿盒内，放于37℃烘箱孵育1小时之后，然后直接滴加25μL一抗，置于37℃烘箱孵育1小时，然后 4℃过夜，第二天，拿出湿盒室温复温1小时，水滴法加PBS冲洗3次，滴加30μL生物素标记的二抗， 37℃孵育1小时，水滴法加PBS冲洗3次，应用DAB溶液显色5-6分钟，蒸馏水洗3次，苏木素复染核1-2分钟，蒸馏水洗去苏木精染液，洗3次，梯度乙醇脱水，最后二甲苯透明2min×3次，中性树胶封片。 37℃烤箱，晾干一周。病理扫描仪阅片，每张切片高倍镜(×400倍)下随机选择5个不同的视野，使用 Image-Pro Plus 5.1.计量每个视野中阳性表达水平。
5、Western blot测定
将动物组织转移至预冷的含有合适剂量磷酸酶抑制剂的组织裂解液的EP管中，冰上超声破碎，4℃， 10000rpm离心10min，将上清转移到新的EP管中。细胞中蛋白提取，将处理好的细胞用PBS洗两遍后，加入含磷酸酶抑制剂的裂解液，冰上裂解并用细胞刮收集，裂解20min后，4℃，10000rpm离心10min，将上清转移到新的预冷EP管中，弃去沉淀。将样品进行蛋白定量，加入1/4体积的5x Loading Buffer(普利来公司)，混匀，金属浴98℃加热变性10min(TECHNE)，-80℃冻存，以备上样。热变性后的蛋白样品，震荡混匀，每孔加入等量40μg的蛋白样品，进行电泳。样品在恒压80V电泳1h后，恒压120V继续电泳，直到溴酚蓝前沿到达底部，停止电泳。电泳结束后，使用硝酸纤维素膜(NC膜)，冰水浴，150mA， 90min，进行转膜。将NC膜浸于含5％脱脂奶粉的TBST溶液中室温封闭1h。用TBST稀释一抗，4℃孵育摇床过夜。第二天，用TBST溶液清洗3次，每次8min。在室温，二抗孵育1h，之后用TBST溶液清洗3次，每次8min。配制ECL发光液，室温混匀静置2分钟，覆盖PVDF膜蛋白结合面，化学发光凝胶成像仪曝光并保存图像。用ImageJ软件对Western blot结果进行半定量分析，反映目的蛋白的相对表达水平。
6、免疫共沉淀Co-IP
CaM/Ubi相互作用实验：HUVECs在1x106细胞/mL的初始密度下在10cm的培养皿培养。第二天，在更换细胞培养液的同时加入10μM MG132，孵育1h。随后，加入LMWF 30μg/mL继续培养12h。将培养皿中的细胞培养液吸净，然后加入400μL Co-IP细胞裂解液。用细胞刮刀将培养皿中的细胞刮下，收集在提前准备好的1.5mL的离心管内，14000rcf/min低温离心15min，提取蛋白上清液。用BCA蛋白定量试剂盒，将蛋白定量为3mg。每个蛋白样品中加入20μL A-Sepharose，4℃摇床孵育2h。随后，200 rcf/min，离心2min，提取蛋白上清液。用10μLanti-calmodulin antibody抗体4℃孵育一夜。第二天，在蛋白上清液中加A-Sepharose 25μL，4℃摇床，孵育4h。在冰冷Co-IP裂解缓冲液中洗涤三次。然后用 1×蛋白质样品缓冲液25μL处理样品。95℃变性5min后，用手掌离心机离心2-3次，取上清，进行蛋白质免疫印迹。
eNOS/CaM相互作用实验：将小鼠腓肠肌组织剪碎后加Co-IP组织裂解液1mL，4℃冰箱摇床裂解1 h，随后4℃，14000rcf/min离心15min，将上清液转移至新的1.5mL EP管中。随后过程与CaM/Ubi相互作用实验类似。
7、细胞培养
人脐静脉内皮细胞(HUVECs)购于美国菌种保藏中心(ATCC)。人脐静脉内皮细胞培养在Dulbecco 改良的Eagle培养基(DMEM)(Gibco)含10％胎牛血清(Gibco)和5.5mM葡萄糖，培养于37℃，5％二氧化碳培养箱中48小时。从第2代至8代的细胞用于所有实验。对于高糖刺激，细胞用33mM葡萄糖处理48小时，同时加入药物等干预。
8、分离和培养小鼠骨髓内皮祖细胞
C57BL/6和db/db小鼠脱颈或麻醉处死，用75％酒精浸泡鼠双腿。无菌条件下取鼠股骨和胫骨，分离干净肌肉组织，用无菌PBS洗2次，置于6cm培养皿中。以1mL注射器吸取5mL纯DMEM培养基反复冲洗骨髓腔，至冲洗液清亮，骨干发白。将冲洗液充分吹打，混匀，获得细胞悬液。将淋巴细胞分离液 (Ficoll-paque 1.084)约5mL加入15mL离心管中，细胞悬液以1:1比例缓慢加入其中，保持两液体间清晰界面。室温下400g，离心30min(降低升降速度均为0)，轻轻吸取离心管第二层(中间)乳白色云雾状单个核细胞层到新的15mL离心管中，加入8ml PBS洗涤2次(室温离心1000rpm，5min)。弃上清，保留下层细胞，混匀于3mL含10％FBS，EGM-2完全培养基中，吹打悬液。吸出各1mL悬液加于3个含有3mL培养基的6cm培养皿水平混匀。37℃，5％CO2细胞培养箱培养至第3-4天，吸走未贴壁的细胞和碎片，换液。然后每隔2天换一次培养基，细胞培养至12-14天进行实验。
小鼠骨髓内皮祖细胞随机分为四组：(1)非糖尿病组(Ctrl)；(2)糖尿病组(db/db)；(3)糖尿病+空载体组(Vector)；(4)糖尿病+CaM过表达质粒组(CaM-wt)。
9、过表达质粒转染
转染前一天细胞接种于6cm皿中，加血清不加双抗培养。每个皿用纯DMEM培养基洗2次，最后加纯DMEM培养基2mL。质粒转染2μg/6cm皿，质粒与lipo2000分别用opti-MEM培养基配好后，室温静置5min。含质粒的opti-MEM加入含lipo2000的opti-MEM中，混匀，室温静置15min。每个6cm皿加入质粒与lipo2000的混合液1mL。转染4小时之后，换不含双抗，含10％FBS的完全DMEM培养基。48小时后提蛋白，western-blot，测定蛋白表达变化。
10、CaM的半衰期测定实验
向每组细胞培养皿中铺入等量HUVECs细胞，待细胞贴壁并生长状态良好，生长密度约为80％。使用放线菌酮(20μg/mL)处理细胞，同时对各组细胞进行高糖及药物干预。收取0、4、8、12h的细胞，加入适量RIPA裂解液收集蛋白。western-blot，测定蛋白表达变化。
11、统计学方法
采用SPSS统计软件对所得数据进行处理。数据均以均数±标准差(X±SD)表示，两组数据进行独立样本t检验，P<0.05为差异具有统计学意义。
实验结果
1、在高糖处理的HUVECs(模拟糖尿病)中，eNOS活性降低及NO和VEGF生成障碍的同时，伴有CaM 表达的下调
检测高糖处理的HUVECs(模拟糖尿病)中eNOS蛋白表达和一氧化氮的含量，发现如图1显示，高糖条件下eNOS磷酸化(pS1177-eNOS,pS116-eNOS,pS633-eNOS)水平比等渗甘露醇组明显降低(P<0.01)，而eNOS抑制位点(pT495-eNOS)磷酸化水平比等渗组显著增加，差异具有统计学意义(P<0.01)，并且， NO含量和VEGF及VEGFR2的表达明显下降(P<0.01)。同时检测到内皮细胞中CaM的表达明显减少 (P<0.05)。
同时，提取糖尿病小鼠BM-EPCs培养后，进行功能及相关蛋白表达的检测发现如图2：1)与正常鼠相比，糖尿病小鼠的EPCs成管能力障碍；eNOS-S1177磷酸化下降，NO生成减少和VEGF等蛋白表达下降并伴有CaM表达下调。2)过表达CaM后，上述变化得到逆转。
以上结果首先说明糖尿病下的危险因素可使血管内皮细胞及BM-EPCs中CaM的表达下调，伴有血管及BM-EPCs的功能障碍和eNOS，NO水平的改变，而质粒过表达CaM可以保护糖尿病BM-EPCs的功能及eNOS，NO的水平，由此，我们得出CaM的病理性改变可能是糖尿病内皮损伤和血管并发症发生发展的重要机制之一。
2、糖尿病骨骼肌血管中eNOS与CaM的相互作用
应用免疫共沉淀的方法检测糖尿病db/db小鼠骨骼肌血管中eNOS与CaM是否存在相互作用及其强弱，结果如图3显示，与非糖尿病动物骨骼肌血管eNOS与CaM相互作用相比，糖尿病小鼠骨骼肌血管中eNOS与CaM的相互作用减弱。
上述结果表明，糖尿病血管内皮细胞功能障碍可能与eNOS与CaM的相互作用减弱有关，相互作用的降低减少了eNOS的激活，进而NO合成障碍，血管新生障碍。而CaM表达的下调可能是eNOS与CaM 的相互作用减弱的根本原因。CaM可以作为糖尿病血管并发症防治的一个关键靶点，故可以通过上调CaM 的水平，来增加eNOS/CaM相互作用，促进eNOS的激活，NO的产生，恢复血管和BM-EPCs功能，预防和治疗糖尿病血管并发症。
3、过表达CaM后对下肢缺血模型的db/db小鼠足底血流恢复的影响
进一步，我们应用编码人CALM1基因的腺相关病毒(CaM-AAV)对糖尿病小鼠的下肢缺血模型治疗，进行体内验证。对12-13w的小鼠左侧腓肠肌注射CaM-AAV(1×1010vg/注射点，4个注射位点)，一周后进行左后肢股动脉结扎和去除手术，造成下肢缺血模型，造模后1天、1w、2w、3w对血流恢复情况进行测定。如图4，A图为注射CaM-AAV、下肢缺血造模及血流测定的时间示意图。由B图，我们可知：1)在 C57BL/6小鼠中，与假手术组(Non-DM-Sham)相比，非糖尿病小鼠模型组(Non-DM-Model)下肢缺血 21天血流基本完全恢复；2)与非糖尿病手术组(Non-DM-Model)及与糖尿病假手术组(DM-Sham)相比，糖尿病Vector手术组(DM-Vector)血流恢复程度明显下降，且均有显著性差异(P<0.01，P<0.01)；而与DM-Vector组相比，CaM基因过表达治疗组(DM-CaM-AAV)较DM-Vector组呈现出更快的血流恢复，差异有统计学意义(P<0.01)。
因此，上述实验结果表明：1)首先糖尿病小鼠下肢缺血的血流恢复速度明显低于非糖尿病下肢缺血小鼠；2)其次，基因疗法过表达CaM能明显的促进db/db小鼠缺血下肢血流的恢复。
4、过表达CaM后减轻下肢缺血模型的db/db小鼠腓肠肌的病理改变
对过表达CaM后的db/db小鼠缺血腓肠肌进行HE染色，如图5显示发现：1)Non-DM-Sham组小鼠腓肠肌肌纤维呈多角形，肌纤维间存在一定的间隙。Non-DM-Model组小鼠腓肠肌出现萎缩，肌间隙增加，肌纤维显著肿胀、变圆，存在炎性细胞浸润；2)与DM-Sham组相比，DM-Vector缺血下肢腓肠肌肌纤维的边缘变钝变圆，肌间隙增大，肌纤维排列紊乱；3)与Non-DM-Model组相比，DM-Vector缺血下肢腓肠肌肌纤维形态、肌间隙、炎细胞浸润等进一步增加；4)与DM-Vector组相比，DM-CaM-AAV组小鼠，即基因疗法过表达CaM治疗的糖尿病小鼠能明显减少肌肉的间隙以及维持骨骼肌纤维结构的完整。
5、过表达CaM后促进外周血管病db/db小鼠缺血下肢血管新生
同样的对缺血腓肠肌进行免疫组织化学染色，如图6显示：1)与Non-DM-Sham组小鼠相比，正常 C57BL/6小鼠下肢缺血(Non-DM-Model)后会促进CD34和VEGF的表达(P<0.05)；2)与正常C57BL/6 小鼠组(Non-DM-Sham)比较，DM-Sham组腓肠肌组织中CD34及VEGF阳性染色面积比均明显减少(p <0.05)；3)且db/db小鼠下肢缺血(DM-Vector-AAV)后腓肠肌组织中CD34及VEGF阳性染色面积均未出现显著性差异(p>0.05)；4)经过CaM-AAV治疗(DM-CaM-AAV)后db/db小鼠腓肠肌组织中CD34 (p<0.01)及VEGF(p<0.05)阳性染色面积均明显增加，表明CaM-AAV可以明显促进db/db小鼠缺血下肢腓肠肌组织的血管新生。
6、昆布多糖可以逆转高糖下HUVECs细胞中CaM表达的下调
已知，LMWF具有口服可吸收，副作用少，广泛的生物活性，如抗氧化应激、抗炎、抗血小板聚集、扩张血管等优点，特别其对糖尿病足防治的药效明显。由此我们猜想LMWF是否是通过上调了CaM的表达而产生了对糖尿病足的治疗作用呢？
于是，对HUVECs应用33mM葡萄糖处理48小时来复制体外糖尿病模型进行了相关的研究。我们发现如图7所示，在HUVECs给与高糖刺激后，与糖尿病血管及BM-EPCs中的情况类似，CaM的表达明显下调，而LMWF的同时处理可逆转这一过程，上调CaM的表达水平。
7、昆布多糖可增强高糖下HUVECs中eNOS与CaM的相互作用
进而，我们探索了LMWF对eNOS与CaM相互作用的影响。我们使用免疫共沉淀的方法检测LMWF 对高糖下HUVECs中eNOS与CaM的相互作用的影响，结果如图8显示，与正常糖和等渗甘露醇对照组eNOS-CaM相互作用相比，高糖使得eNOS与CaM的相互作用减弱，而LMWF的同时处理显著增加了eNOS与CaM的相互作用。
由此，我们得出CaM是LMWF发挥治疗糖尿病足的作用靶点，具体的是，LMWF通过上调CaM的水平而发挥作用的。那么，LMWF提高内皮细胞CaM的蛋白水平的机制又是什么呢？
8、LMWF通过降低CaM的降解速率而上调CaM的表达水平
进一步我们探究了LMWF上调高糖下CaM表达水平的机制。首先在蛋白酶抑制剂CHX下探究了 LMWF对高糖下CaM半衰期的影响。由A，B图我们可知，高糖明显提高了CaM降解速率，缩减了其在细胞内的半衰期。而LMWF可通过减少CaM降解速率延长其在细胞内的半衰期。基于报道CaM在细胞内主要经泛素-蛋白酶体代谢，如图C对高糖下应用蛋白酶体抑制剂MG132后的CaM泛素化水平进行测定发现，高糖促进了CaM的泛素化，而LMWF减少了高糖下CaM的泛素化。由此我们认为LMWF通过减少CaM的泛素-蛋白酶体降解而上调CaM的蛋白表达水平，进而促进血管新生而改善下肢缺血症状和结构的改变。
本发明结论
本发明研究发现：
(1)由图1可知，证明本发明糖尿病血管内皮细胞及BM-EPCs中CaM的蛋白表达下调；
(2)由图2可知，证明本发明质粒过表达CaM可以改善db/db小鼠BM-EPCs的功能；
(3)由图3可知，证明本发明糖尿病减弱骨骼肌血管内皮eNOS与CaM相互作用；
(4)由图4可知，证明本发明CaM作为新的治疗靶点能善db/db小鼠缺血肢足底血流的恢复；
(5)由图5可知，证明本发明CaM作为新的治疗靶点能明显改善缺血造成的糖尿病小鼠腓肠肌组织病理改变；
(6)由图6可知，证明本发明CaM作为新的治疗靶点促进db/db小鼠缺血肢的血管新生；
(7)由图7可知，证明本发明LMWF能逆转高糖诱导HUVECs中CaM表达的下调。
(8)由图8可知，证明本发明LMWF能增强高糖下内皮eNOS与CaM相互作用。
(9)由图9可知，证明本发明LMWF通过降低CaM泛素蛋白酶体降解而上调CaM的表达水平。
综上，本发明表明，糖尿病的病理环境降低了内皮CaM的蛋白表达水平，导致诱导eNOS/NO通路激活功能下降，从而诱发血管内皮细胞损伤和血管新生障碍。采用腺相关病毒携带CALM1基因(CaM-AAV) 进行局部治疗，可以使腓肠肌血管表达CaM，进而激活eNOS，生成NO和VEGF增加，促进内皮修复和血管新生。最终促进血流恢复，改善缺血性的病理损伤，有效防治糖尿病血管并发症的发生和发展，为糖尿病血管合并症的治疗提供了新的有效靶点。此外，本研究还发现昆布多糖，即低分子褐藻多糖(LMWF) 可通过抑制CaM的泛素化水平和降解速率，增加血管内皮细胞CaM的水平，促进CaM与eNOS的相互作用，激活eNOS的活性。因此，CaM是糖尿病及其血管并发症防治极其重要的新靶点，针对该靶点构建的基因疗法，CaM-AAV，具有疗效明确，作用维持时间长(0.5-1年)，AAV已作为载体在人体应用，毒、副反应低的优点，有望成为有效，低毒，维持作用时间长，用于糖尿病足具有广阔前景的新型药物。
Claims (9)
1.钙调蛋白（calmodulin, CaM）表达改变参与糖尿病血管内皮损伤病理生理学新的机制应用。
2.钙调蛋白（CaM）在作为糖尿病下肢外周血管疾病的防治靶点方面的应用。
3.钙调蛋白（CaM）作为靶点在制备糖尿病下肢外周血管疾病的防治试剂中的应用。
4.钙调蛋白（CaM）在作为增强糖尿病血管eNOS的活性，激活eNOS/NO/VEGF信号通路的靶点方面的应用。
5.钙调蛋白（CaM）在制备增强糖尿病血管eNOS的活性，激活eNOS/NO/VEGF信号通路的试剂中的应用。
6.所述的昆布多糖（LMWF）治疗糖尿病下肢外周血管疾病，其特征在于特异性的提高糖尿病血管内皮钙调蛋白（CaM）的表达水平。
7.所述的LMWF治疗糖尿病下肢外周血管疾病，其特征在于机制是LMWF通过降低钙调蛋白（CaM）的泛素-蛋白酶体的降解速率而上调糖尿病血管内皮钙调蛋白（CaM）的表达水平。
8.根据权利要求6所述的LMWF来源、组成、分子量及其应用等均在专利（CN 101912408）和（CN 105663154）中进行的详尽的描述。
9.一种编码人CALM1基因的腺相关病毒是增加腓肠肌局部血管内皮CaM表达的动物模型，其特征在于，该模型有效提高局部血管内皮CaM的蛋白表达水平，可用于糖尿病足的治疗用途。
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