CN116983333B - 人参外泌体在制备治疗糖尿病溃疡血管病变的纳米药物中的应用 - Google Patents
人参外泌体在制备治疗糖尿病溃疡血管病变的纳米药物中的应用 Download PDFInfo
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Abstract
本发明公开了人参外泌体在制备治疗糖尿病溃疡血管病变的纳米药物中的应用，涉及生物医药领域。本发明通过实验验证了人参外泌体能够对高糖条件下的血管内皮细胞糖酵解过程进行重编程，并通过抑制细胞中乙酰辅酶A表达，减少活性氧簇物质产生，进而促进一氧化氮合酶表达，从而减少或逆转高糖诱导下的细胞凋亡。将人参外泌体作为纳米药物，对糖尿病溃疡微血管病变具有预防、治疗或改善的作用。本发明中的人参外泌体制备工艺简单，细胞毒性低，递送效率高，稳定性良好，可作为一种新型的纳米药物用于糖尿病血管病变相关疾病的预防、治疗或改善。
Description
技术领域
本发明属于生物药物领域，具体涉及人参外泌体的新应用，特别涉及人参外泌体在制备治疗糖尿病溃疡血管病变的纳米药物中的应用。
背景技术
随着人口老龄化进程加剧和人民生活方式的改变，糖尿病、肥胖和代谢综合征等代谢类疾病的发生和发展正在与日俱增，已成为全球性公共健康问题。其中，糖尿病是一种由血糖浓度过高引起组织结构和器官功能异常的终身性糖代谢疾病，根据2021年国际糖尿病联盟(International Diabetes Federation，IDF)统计数据显示，全球大约有5.37亿成年人(20-79岁)患有糖尿病。糖尿病的并发症极多，其中长期高血糖环境易引起视网膜病变、动脉硬化、心血管及小血管等血管病变，且发生并发症后多数药物治疗效果甚微，难以逆转。然而截至目前，糖尿病的血糖控制率仅为49.4％，这使得多数药物无法从糖代谢调控的根本问题出发发挥治疗作用，因此新型药物的研发依然是抗糖尿病及其并发症治疗领域亟待解决的问题和研究热点。
其中，高血糖代谢障碍引发的血管内皮细胞连锁反应，从而造成血管新生不足是引起糖尿病溃疡血管病变及血管新生障碍的根本原因。在正常情况下，葡萄糖进入细胞后经过糖酵解和线粒体代谢产生物质和能量供给，以支持细胞的各种生理功能；而处于高血糖环境中的血管内皮细胞将发生一系列异常代谢过程：1.过量糖代谢中间产物乙酰辅酶A分子转化为酰基肉碱，酰基肉碱的积累会造成细胞毒性；2.过量底物负载造成细胞氧化磷酸化失败，ATP产生损失和ROS水平增加，随后导致线粒体衰竭；3.形成高级糖基化终产物，导致蛋白质功能改变或丧失，促进ROS的释放并引发一系列细胞信号传导的改变；4.过量葡萄糖还通过多元醇和己糖胺途径进行代谢，这分别导致ROS和炎症水平增加。因此如何在高血糖环境中对血管内皮细胞进行有效的糖代谢调控治疗是此领域的研究热点。
在目前针对糖尿病溃疡血管病变，尤其是糖尿病溃疡血管新生不足的治疗中，口服降糖药物实施全身治疗最为普遍，如二甲双胍、噻唑烷二酮类、α糖苷酶抑制剂，但长期使用会引发体重增加、心血管毒性、肝脏毒性和胃肠道反应等副作用，且针对血管治疗缺乏针对性；在全身降血糖处理的同时，口服或注射抗氧化应激药物(如α-硫酸锌)、扩张血管药物(如前列地尔)、局部使用各类生长因子制剂(如VEGF)是目前最普遍的治疗手段，这些药物虽具有一定的效果与使用价值，但普遍存在难以从疾病发生根源，即内皮细胞糖酵解紊乱角度解决治疗问题，且生物利用度低、功能单一，因而使用受限。
中医药防治糖尿病拥有几千年的临床实践，经过不断发展与创新，逐渐形成系统的理论学术体系。大量临床前研究及实践表明，中药植物及其有效成分在调节糖代谢紊乱疾病治疗中具有切实的疗效和巨大的发展前景，例如：玄参科植物提取物地黄寡糖、肉桂多酚、益母草碱和黄连的有效成分小檗碱等均被证明可通过增加葡萄糖激酶(GK)mRNA的表达或活性以促进葡萄糖代谢；凤丹皮多糖、蚕蛹油、芦荟提取物等可改善糖代谢紊乱及胰岛素抵抗；黄芪甲苷、人参皂苷分别通过抑制糖酵解过程中的糖原磷酸化酶(GP)和酸烯醇式丙酮酸羧激酶(PEPCK)减少糖原的降解、减少肝脏葡萄糖的生成，并抑制糖异生的过度活化，发挥降糖作用。
外泌体是一类由细胞释放的直径在30-150nm，并具有脂质双分子层膜结构的球状囊泡，可通过密度梯度离心、切向流过滤等方法大规模制备，并以浓缩液或冻干粉的形式在低温下长期保存。除表面脂质外，外泌体内部所含的蛋白与核酸(包括mRNA、miRNA、lncRNA、circle RNA)是其重要的功效物质，对多种生物过程与疾病治疗具有重要的调控作用。其中，从中药植物中提取的外泌体不仅具有显著的治疗活性，且毒副作用小、免疫原性低，已经成为了外泌体领域研究的新热点。目前，葡萄来源的外泌体正在开展临床试验治疗头颈癌化疗引起的口腔粘膜炎(NCT01668849)；生姜和芦荟来源的外泌体正在临床试验治疗多囊卵巢综合征(NCT03493984)；采用植物外泌体作为纳米载体递送姜黄素治疗结肠癌已进入临床试验阶段(NCT01294072)(https://beta.clinicaltrials.gov/)。
虽然外泌体治疗的有效性和中药植物调节糖代谢的临床依据都具有广泛的研究基础，然而截至目前，植物外泌体调控糖尿病中内皮细胞糖代谢过程用于糖尿病血管病变的治疗未见报道，尤其在治疗糖尿病溃疡处的血管新生障碍的应用尚无涉及。
发明内容
为克服现有治疗药物的不足之处，本发明提供一种高效安全的调节血管内皮细胞葡萄糖代谢过程的人参外泌体纳米药物，尤其提供人参外泌体在制备治疗糖尿病血管病变的纳米药物中的应用。本发明首次发现，人参外泌体对高糖环境中的病态血管内皮细胞具有治疗能力，能够通过调节其糖酵解过程，促进葡萄糖代谢，同时抑制细胞内乙酰辅酶A的产生，进而下调ROS水平，并上调一氧化氮合酶的表达，促进高糖中内皮细胞的存活、增殖，并恢复其正常的生理功能和活动。本发明对包含人参外泌体在内的各类中药植物外泌体的进一步开发和应用，具有重要的价值和意义。
为实现上述目的，本发明采用如下技术方案：
人参外泌体在制备治疗糖尿病血管病变的纳米药物中的应用。
进一步的，所述人参外泌体采用超速离心法、蔗糖密度梯度离心法、聚合物沉淀法或超滤结合尺寸排阻色谱法制备纯化。
更进一步的，所述人参外泌体采用超速离心法制备纯化；当采用超速离心法提取时，离心顺序及条件为:2000-6000g下20-50min，取上清液；7000-15000g下1h，取上清液；100000-200000g下1-3h，取沉淀即为提取所得人参外泌体。
更进一步的，所述人参外泌体中的活性物质来源于其内含的蛋白质、脂质、mRNA、小RNA中的一种或多种；所述小RNA为miRNA、lncRNA和circle RNA中的一种或多种。
进一步的，所述糖尿病血管病变为糖尿病皮肤溃疡中的微血管病变和/或血管新生障碍。
进一步的，所述纳米药物为以人参外泌体为活性成分的制剂或以人参外泌体为组成成分的复方制剂。
更进一步的，所述复方制剂为软膏、凝胶、贴剂中的一种或多种。
本发明的有益效果包括：
(1)本发明提供的植物外泌体，能显著重编程高糖或糖尿病诱导下血管内皮细胞的糖代谢过程，上调基因PFKM、PGK1、ENO1的表达以促进糖酵解过程，同时减少乙酰辅酶A的产生，降低内皮细胞内ROS水平，促进内皮细胞表达一氧化氮合酶。
(2)本发明提供的植物外泌体，能显著减少血管内皮细胞凋亡，特别是高糖诱导下或糖尿病动物皮肤溃疡局部的细胞凋亡，逆转高糖处理或糖尿病对内皮细胞的损伤，并促进细胞增殖。
(3)本发明提供的植物外泌体，能帮助糖尿病动物皮肤溃疡局部血管内皮细胞维持正常的生理状态，促进糖尿病溃疡处血管新生和微血管网络形成。
(4)本发明用于制备糖尿病血管病变的预防及治疗纳米药物，能起到保障糖尿病患者的健康、延长其寿的功效。
附图说明
图1为本发明一实施例所得人参外泌体的粒径分布图和电位值；
图2为本发明一实施例所得人参外泌体的TEM图；
图3为本发明一实施例中正常糖条件下不同浓度人参外泌体对血管内皮细胞增殖的影响结果图；
图4为本发明一实施例中高糖条件下，8ug/mL人参外泌体对血管内皮细胞增殖的影响结果图；
图5为本发明一实施例中8ug/mL人参外泌体治疗前后血管内皮细胞基因组中糖酵解通路的GSEA富集分析图和差异表达基因；
图6为本发明一实施例中8ug/mL人参外泌体治疗前后血管内皮细胞中糖酵解基因PFKM、PGK1、ENO1 Western blot表达分析图；
图7为本发明一实施例中8ug/mL人参外泌体治疗前后细胞代谢组学中糖酵解相关通路及差异性表达的代谢物；
图8为本发明一实施例中高糖条件下8ug/mL人参外泌体对细胞表达乙酰辅酶A的影响结果图；
图9为本发明一实施例中高糖条件下，8ug/mL人参外泌体对细胞ROS水平的影响结果图；
图10为本发明一实施例中高糖条件下，8ug/mL人参外泌体对细胞一氧化氮合酶表达水平的影响结果图；
图11为本发明一实施例中糖尿病小鼠局部应用人参外泌体后，皮肤溃疡局部组织中糖酵解基因PFKM、PGK1、ENO1表达的Western blot影响结果图；
图12为本发明一实施例中糖尿病小鼠局部应用人参外泌体后，皮肤溃疡局部组织中乙酰辅酶A含量的变化图；
图13为本发明一实施例中糖尿病小鼠局部应用人参外泌体后，皮肤溃疡局部组织中一氧化氮合酶含量的变化图；
图14为本发明一实施例中糖尿病小鼠局部应用人参外泌体后，皮肤溃疡局部微血管网络形成的体式显微镜图片。
具体实施方式
下面通过实施例的方式进一步说明本发明。
实施例1超速离心法提取人参外泌体
人参使用去离子水洗净，去皮，切块，通过榨汁机榨汁。将得到的汁液，通过差速离心，根据不同中药植物药用部位的特征将汁液先用台式冷冻离心机在2000-6000g下离心20-50min，以除去较大的植物残渣，取上清液；然后在7000-15000g下离心1h，以除去可能存在的微小的植物纤维及碎屑，取上清液；然后于落地超速离心机(100000-200000g，1-3h)离心，取底部沉淀物，用少量磷酸盐缓冲液(PBS)重悬，得到人参外泌体溶液。
实施例2蔗糖密度梯度离心法提取人参外泌体
人参使用去离子水洗净，去皮，切块，通过榨汁机榨汁。将所得汁液冷冻离心，离心条件为5000g-12000g下1h，重复两次，均取上清液，然后将上清液于120000-180000g下超速离心1-3h，取沉淀物，使用PBS重悬，将重悬液使用蔗糖密度梯度离心法进行纯化。制备具有8％，30％，45％和60％四种不同浓度的蔗糖密度梯度溶液，将重悬液通过注射器加入至密度梯度溶液上方，然后于落地超速离心机(100000-200000g，1-3h)离心。离心之后，将富集在30/45％界面间的外泌体进行回收。然后通过台式超速离心机(100000-200000g，1-3h)除去蔗糖溶液，将底部沉淀用少量PBS重悬，得到纯化后的人参外泌体溶液。
实施例3聚合物沉淀法提取人参外泌体
人参使用去离子水洗净，去皮，切块，通过榨汁机榨汁，然后通过聚合物沉淀法进行纯化。对于基于聚乙二醇(PEG)的沉淀，将含PEG的溶液以1:5-1:1的体积比(v/v)与植物汁液混合，并将混合物在4℃下孵育过夜。然后将混合物在4℃下使用1000-3000g转速离心20-50分钟，而后弃去上清液，重悬含有外泌体样纳米囊泡的沉淀。然后使用超速离心将外泌体沉淀悬浮液在100000-1500000g离心力下离心1-4h，除去上清液，将底部沉淀用少量PBS重悬，得到纯化后的人参外泌体溶液。
实施例4超滤结合尺寸排阻色谱法提取人参外泌体
人参使用去离子水洗净，去皮，切块，通过榨汁机榨汁，然后通过超滤结合尺寸排阻色谱法进行纯化。对于尺寸排阻色谱法，使用商业色谱柱(Izon Science,Canterbury,New Zealand)进行分离。首先用70mL PBS对柱进行调节。然后装上10mL透明粗提植物汁液，收集10mL洗脱液作为组分1。然后在柱中加入20mLPBS，收集20mL洗脱液作为组分2。最后，向柱中加入50mL PBS，收集20mL洗脱液作为组分3。分析不同组分中植物外泌体的丰度，选择丰度最高的组分，使用10000MWCO膜进一步浓缩，得到纯化后的人参外泌体溶液。
进一步的，为减小人参外泌体提取方法带来的品质差异，以下实施案例5-8中扫描电镜表征、内含物定量定性分析、细胞实验、动物实验中所用的人参外泌体均为实施例1中超速离心法所得。
实施例5人参外泌体的表征及内含物定性定量分析
人参外泌体的表征：使用透射电镜观察实施例1中制备得到的人参外泌体的大小与形态，如图1。使用马尔文粒径电位仪测定该外泌体的粒径，如图2。如图1,2所示，透射电镜下观察到的形态均符合外泌体的特征，呈规则球形，大小较均一，粒径为117.7±6.3nm。
人参外泌体内含蛋白质的分离：人参外泌体所含蛋白质用SDT(4％SDS,100mMTris-HCl,pH7.6)缓冲液分离提取。每个样品取20μg蛋白，分别与5X上样缓冲液混合，煮沸5min，用4％-20％SDS-PAGE凝胶(恒压180V,45min)分离，使用考马斯蓝R-250染色显示蛋白条带。
人参外泌体内含核酸的分离分析：植物外泌体总RNA使用Trizol试剂(Invitrogen,Carlsbad,CA,USA)分离纯化，经裂解、标记、扩增、配对端测序、序列映射的程序分离分析植物外泌体中的核酸，包括mRNA和小RNA(miRNA、lncRNA、circle RNA)。
人参外泌体内含脂质的分离分析：采用MTBE法提取G-Exos的脂质。简单地说，样品(100μL)用内脂标品加标，然后利用反相液相色谱法分离，采用电喷雾电离法(ESI)检测正离子和负离子，最后人参外泌体内含脂质分子和内标脂质分子进行峰提取和鉴定。
实施例6人参外泌体对高糖环境中的细胞增殖影响实验
本实施例使用人脐静脉内皮细胞(HUVECs)作为细胞模型，对人参外泌体对于糖代谢紊乱的治疗作用及机制进行探究。HUVECs来源于中国科学院(上海)细胞库。HUVECs在含有10％牛血清(Gibco BRL)、l-谷氨酰胺、青霉素(50U/mL)和链霉素(50U/mL)的RPMI 1640培养基中培养。细胞保存在37℃，5％CO2。
为了首先探究不同浓度人参外泌体对细胞增殖的影响，将HUVECs接种于RPMI1640培养基中，并允许贴壁过夜。第二天，使用添加了不同浓度(0μg/mL，0.25μg/mL，0.5μg/mL，1μg/mL，2μg/mL，4μg/mL，8μg/mL，10μg/mL，12μg/mL)植物外泌体的RPMI 1640替代培养基培养细胞24h。然后使用细胞计数试剂盒(Beyotime)测定细胞增殖率。简单地说，每孔加入10μL CCK-8(100μL培养基)。37℃孵育2h后，用BioTek Cytation3 celllimaging多模式读取器测定450nm处吸光度。其结果如图3所示，结果显示，细胞增殖率随着人参外泌体给药浓度的增加而增加，当浓度大于8μg/mL后，增殖率无明显变化，因此后续细胞实验所使用的人参外泌体浓度均选用8μg/mL。
为了首先探究高糖条件下人参外泌体对细胞增殖的影响，将上述实验组更换为正常组、高糖组、高糖+人参外泌体组进行实验，其余步骤均不变，测定不同实验组中的细胞增殖率。本实施例中正常组中葡萄糖含量为16mmol/L，高糖组和高糖+人参外泌体组中葡萄糖含量为45mmol/L。其结果如图4所示，结果显示，高糖组由于细胞受损，其增殖率明显低于正常组，而高糖+人参外泌体组增殖率与正常组相当，说明人参外泌体治疗有效改善了高糖诱导的细胞凋亡。
实施例7人参外泌体对高糖环境中细胞内基因PFKM、PGK1、ENO1、乙酰辅酶A含量、ROS表达、一氧化氮合酶表达影响的实验
为了首先探究不同浓度人参外泌体对细胞增殖的影响，将HUVECs接种于RPMI1640培养基中，并允许贴壁过夜。第二天，使用添加或不添加高糖或人参外泌体的RPMI1640替代培养基培养24h，即设置两个实验组：高糖组、高糖+人参外泌体组，使用的葡萄糖含量与实施例6中相同。然后分别使用Western blot实验、乙酰辅酶A含量测定试剂盒、ROS荧光探针试剂盒、一氧化氮合酶表达测定试剂盒对不同组别细胞进行定性定量分析，以确定人参外泌体对高糖环境中细胞ROS水平、基因和酶表达的影响。其结果如图5,6,7,8,9,10所示。结果显示，人参外泌体组细胞内中PFKM、PGK1、ENO1基因表达水平明显高于对照组(图5,6)，乙酰辅酶A含量明显低于对照组(图7,8)，ROS表达明显低于对照组(图9)，一氧化氮合酶表达明显高于对照组(图10)。表明人参外泌体促进了细胞糖酵解相关基因的表达，抑制了乙酰辅酶A的表达，降低了细胞ROS水平，提高了一氧化氮合酶的表达。
实施例8人参外泌体对糖尿病动物溃疡皮肤组织内基因PFKM、PGK1、ENO1、乙酰辅酶A含量、一氧化氮合酶表达影响的实验
本实施例使用糖尿病小鼠作为体内动物模型，并制造局部全皮组织缺损模型，对人参外泌体的作用机制进行探究。研究使用8-12周龄雄性B6.BKS(D)-Lepr db/J(db/db)小鼠，将小鼠全身麻醉，用手术刀在剃光毛发的背部皮肤上切除创面，创面直径为12mm，使用直径为15mm的圈装硅胶夹板固定创面以防止伤口收缩。将糖尿病小鼠随机分为2个实验组。创面分别用100μL PBS或人参外泌体溶液(100μg/mL-500μg/mL)处理，作为动物对照组和动物给药组，隔日一次。在伤口愈合期间，分别收集两组小鼠创面皮肤愈合组织，进行组织研磨或进行皮肤切片。对于组织匀浆，使用Western blot实验、乙酰辅酶A含量测定试剂盒测定匀浆中的PFKM、PGK1、ENO1基因表达和乙酰辅酶A含量；对于皮肤切片，使用一氧化氮合酶染色试剂盒对切片皮肤进行染色，以测定局部组织中一氧化氮合酶水平。其结果如图11,12,13所示，结果显示，动物给药组糖尿病小鼠溃疡组织中PFKM、PGK1、ENO1基因表达水平明显高于动物对照组(图11)，乙酰辅酶A含量明显低于动物对照组(图12)，一氧化氮合酶表达明显高于动物对照组(图13)。表明人参外泌体促进了糖尿病动物溃疡局部组织糖酵解相关基因的表达，抑制了乙酰辅酶A的表达，提高了一氧化氮合酶的表达。
实施例9人参外泌体对糖尿病动物溃疡皮肤微血管新生和形成影响的实验
使用实施例8所述的动物实验方案，构建糖尿病小鼠皮肤溃疡模型，并使用实施例8所述的人参外泌体给药方案进行治疗。在伤口愈合后期，分别收集两组小鼠创面皮肤愈合组织，并使用体式显微镜对皮肤组织内的微血管进行成像。其结果如图14所示，结果显示，动物给药组糖尿病小鼠溃疡处微血管网络密度显著高于动物对照组。表明人参外泌体局部给药显著促进了糖尿病动物溃疡皮肤处的血管新生和微血管形成。
Claims (4)
1. 人参外泌体在制备治疗糖尿病血管病变的纳米药物中的应用；所述人参外泌体采用超速离心法得到，离心顺序及条件为：2000-6000g下离心20-50min，取上清液；7000-15000g下离心1h，取上清液；100000-200000 g下离心1-3 h，取沉淀即为提取所得人参外泌体；所述糖尿病血管病变为糖尿病皮肤溃疡中的微血管病变和/或血管新生障碍。
2. 根据权利要求1所述的应用，其特征在于，所述人参外泌体中的活性物质来源于其内含的蛋白质、脂质、mRNA、小RNA中的一种或多种；所述小RNA为miRNA、lncRNA和circleRNA中的一种或多种。
3.根据权利要求1或2所述的应用，其特征在于，所述纳米药物为以人参外泌体为活性成分的制剂或以人参外泌体为组成成分的复方制剂。
4.根据权利要求3所述的应用，其特征在于，所述复方制剂为软膏、凝胶、贴剂中的一种或多种。
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