CN112315984A - 海洋来源磷脂在促进血管生成方面的应用 - Google Patents
海洋来源磷脂在促进血管生成方面的应用 Download PDFInfo
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Abstract
本发明涉及海洋来源磷脂在促进血管生成方面的应用,属于生物医药技术领域。本发明通过将海洋磷脂应用于斑马鱼模型,评价其对血管内皮细胞生长因子受体抑制剂(PTK787)诱导的斑马鱼体节间血管(ISVs)损伤和对斑马鱼肠下静脉(SIVs)生长情况的影响。本发明首次发现,该磷脂可以逆转PTK787诱导的斑马鱼ISVs损伤,并对斑马鱼SIVs生长有显著的促进作用,能明显的增加血管的生成数量和血管长度,且毒副作用小。本发明可为制备缺血性疾病药物提供候选,应用前景良好。
Description
技术领域
本发明涉及海洋来源磷脂在促进血管生成方面的应用,属于生物医药技术领域。
背景技术
血管生成是指基于原始血管丛或现有血管使新的血管系统萌芽的过程,它是维持循环系统功能并确保能量供应的必要机制。许多重要的人类疾病,例如癌症、心血管和脑血管疾病以及糖尿病并发症,与血管生成密切相关。当血管生成不足时,会引起心肌缺血、脑缺血、创伤和骨折愈合缓慢、闭塞性血管炎、糖尿病性周围血管病变等疾病。寻找具有促进血管生成作用的治疗药物是治疗这些缺血性疾病的重要策略,这也是当今临床研究的热点。
血管生成药物的发现需要借助血管生成模型,常见的血管生成模型包括体外模型(内皮细胞模型和大鼠动脉环模型)、体内模型(角膜微囊、鸡胚绒毛尿囊膜、圆盘血管生成模型和海绵-基质胶模型等)、以及整体动物模型(斑马鱼模型和爪蟾蝌蚪模型)。斑马鱼具有个体小、产卵多、发育快、胚胎透明、饲养简便等诸多特点,具有细胞的高通量性和整体动物的高内涵性,在国际上已成为药物筛选评价体系中衔接细胞和哺乳动物模型的重要环节。而标记绿色血管荧光的转基因斑马鱼使得在活体胚胎中观察样品作用于血管生成的过程更加的直观而快捷。因此,斑马鱼模型是血管生成活性评价的优势模型。
海洋磷脂因富含二十二碳六烯酸(Docosahexaenoic acid,DHA)、二十碳五烯酸(Eicosapentaenoic acid,EPA)等多不饱和脂肪酸侧链,具有比大豆磷脂、蛋黄磷脂等陆地来源磷脂更广泛的生物活性和保健功能,例如,降血脂、抗衰老、促进神经传导、提高大脑活力、预防心脑血管疾病、保肝、增强免疫力、抗疲劳、促进血液循环、增强细胞膜透过性、抗癌、改善氧化稳定性、抗炎等。研究人员曾发现补充长链多不饱和脂肪酸可以促进新生血管性黄斑变性小鼠模型脉络膜新生血管的消退,提示增加膳食中多不饱和脂肪酸含量有益于体内病理性血管生成的减弱(Ryoji Yanai等,Cytochrome P450-generated metabolitesderived fromω-3fatty acids attenuate neovascularization,PNAS,2014,111,9603-9608)。但以血管生成作为靶点评价海洋磷脂的生物活性,目前尚未见报道。
中国文献《溶血磷脂酰胆碱对血管平滑肌细胞的增殖作用及其信号通路》(包良,内蒙古大学,硕士论文,2010年6月)讲述了溶血磷脂酰胆碱可以刺激血管平滑肌细胞的增殖。但血管生成机制复杂,主要跟内皮细胞的增殖、迁移有关。血管平滑肌细胞的增殖可使血管壁增厚,增加血管的强度和韧性。血管平滑肌细胞增殖与血管生成并非呈直接对应关系。另外,本发明中涉及的海洋磷脂组成成分复杂,其促血管生成作用是多种磷脂类型共同作用的结果,主要评价指标是斑马鱼模型的血管生成长度。且磷脂来源不同,即使同类型磷脂的脂肪酸侧链长度、不饱和键的数量和位置也不一样,结构差异较大。
发明内容
本发明针对现有技术的不足,提供海洋来源磷脂在促进血管生成方面的应用。
术语说明
海洋磷脂:所述海洋磷脂为从鱼、虾、贝等各种水生生物中提取得到的天然磷脂类成分。
本发明技术方案如下:
海洋磷脂在促进血管生成方面的应用。
海洋磷脂作为药效成分在制备治疗缺血性疾病药物中的应用。
根据本发明优选的,所述缺血性疾病为因血管受损或血管生成受抑制导致的缺血性疾病。
海洋磷脂作为有效成分在制备促进血管生成相关药物中的应用。
海洋磷脂作为有效成分在制备保健食品、特医食品等功能性食品中的应用。
根据本发明优选的,所述海洋磷脂中总磷脂纯度按质量分数计为40%以上。
根据本发明优选的,所述海洋磷脂中的磷脂成分按相对含量计包括:磷脂酰胆碱(phosphatidylcholine,PC)占总磷脂的50%-80%,溶血性磷脂酰胆碱(lysophosphatidylcholine,LPC)占总磷脂的2%-30%,磷脂酰乙醇胺(phosphatidylethanolamine,PE)占总磷脂的5%-30%,溶血性磷脂酰乙醇胺(lysophosphatidylethanolamine,LPE)占总磷脂的0%-5%,磷脂酰肌醇(phosphatidylinositol,PI)占总磷脂的0%-10%,神经鞘磷脂(sphingomyelin,SM)占总磷脂的0%-10%。
根据本发明优选的,所述海洋磷脂来源于海洋中的甲壳类浮游动物和/或海洋中的软体动物。
根据本发明优选的,所述海洋磷脂来源于南极磷虾和/或鱿鱼。
所述海洋磷脂的制备方法,包括如下步骤:将海洋生物样品粉碎、匀浆,按质量体积比g/mL为1:(7-10)的量加入浓度为90%以上的乙醇溶液,搅拌提取获得提取液,将提取液固液分离,将分离的液体浓缩为粗磷脂,将粗磷脂用浓度为50%-70%的乙醇溶液溶解,静置12-24h后,固液分离,保留固态部分,用正己烷配成质量浓度为0.3-1.0g/mL的溶液,按所述溶液与丙酮的体积比为1:(5-15)加入丙酮,静置沉淀,然后固液分离获得最终固体,将最终固体干燥即得海洋磷脂。
根据本发明优选的,上述制备方法中,均匀搅拌提取两次,每次6h获得提取液,合并提取液,将提取液减压抽滤固液分离。
根据本发明优选的,上述制备方法中,将分离的液体经45℃减压浓度得到粗磷脂。
根据本发明优选的,上述制备方法中,加入丙酮后,于4℃条件下静置24h。
进一步优选的,将最终固体真空干燥获得海洋磷脂。
本发明技术方案的有益效果
本发明通过将海洋磷脂应用于斑马鱼模型,评价其对血管内皮细胞生长因子受体抑制剂(PTK787)诱导的斑马鱼体节间血管(Intersegmental vessel,ISV)损伤和对斑马鱼肠下静脉(Subintestinal vein vessel,SIV)生长情况的影响。检测后首次发现,该磷脂可以逆转PTK787诱导的斑马鱼ISVs损伤,并对斑马鱼SIVs生长有显著的促进作用,能明显的增加血管的生成数量和血管长度,且毒副作用小。本发明可为缺血性疾病药物提供候选,具有广阔的市场前景。
附图说明
图1为南极磷虾磷脂的液质分析基峰图;
图2为南极磷虾磷脂处理斑马鱼后,ISVs生长的照片;
图中60、80、100μg/mL分别表示对应浓度下南极磷虾磷脂的处理组。
图3为南极磷虾磷脂处理斑马鱼后,ISVs长度统计的柱状图;
图中60、80、100μg/mL分别表示对应浓度南极磷虾磷脂的处理组;
与模型组相比,##P<0.01,###P<0.001,####P<0.0001。
图4为南极磷虾磷脂处理斑马鱼后,SIVs生长的照片;
图中60、80、100μg/mL分别表示对应浓度南极磷虾磷脂的处理组。
图5为南极磷虾磷脂处理斑马鱼后,SIVs长度统计的柱状图;
图中60、80、100μg/mL分别表示对应浓度南极磷虾磷脂的处理组;
与空白对照组相比,#P<0.05,####P<0.0001。
图6为鱿鱼磷脂的液质分析基峰图。
图7为鱿鱼磷脂处理斑马鱼后,ISVs生长的照片;
图中60、80、100μg/mL分别表示对应浓度鱿鱼磷脂的处理组。
图8为鱿鱼磷脂处理斑马鱼后,ISVs长度统计的柱状图;
图中60、80、100μg/mL分别表示对应浓度鱿鱼磷脂的处理组;
与模型组相比,#P<0.05,###P<0.001,####P<0.0001。
图9为鱿鱼磷脂处理斑马鱼后,SIVs生长的照片;
图中60、80、100μg/mL分别表示对应浓度鱿鱼磷脂的处理组。
图10为鱿鱼磷脂处理斑马鱼后,SIVs长度统计的柱状图;
图中60、80、100μg/mL分别表示对应浓度鱿鱼磷脂的处理组;
与空白对照组相比,##P<0.01,####P<0.0001。
图11为DHA、EPA处理斑马鱼后,ISVs生长的照片。
图12为DHA、EPA处理斑马鱼后,ISVs长度统计的柱状图;
与空白对照组相比,##P<0.01,###P<0.001。
图13为DHA、EPA处理斑马鱼后,SIVs生长的照片。
图14为DHA、EPA处理斑马鱼后,SIVs长度统计的柱状图。
具体实施方式
下面结合具体试验方法和附图对本发明的技术方案及其所产生的技术效果进行进一步的阐述。下述说明仅是为了解释本发明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。
实施例中未注明具体条件的内容,按照常规条件进行;所用试剂或仪器未注明生产厂商的,均为普通市售产品。
材料的来源
实施例中所述南极磷虾和鱿鱼生殖腺由山东海都海洋食品有限公司提供,为普通市售产品,斑马鱼为山东省科学院生物研究所药物筛选研究室提供,为普通市售产品,链酶蛋白酶E、PTK787、丹参素钠、DHA、EPA等均购自sigma公司,丹红注射液(10mL/支)由山东步长制药股份有限公司生产,为普通市售产品。
实验例1
磷脂含量的测定
本发明采用分光光度法(钼蓝比色法)测定磷脂的含量。做出的磷酸二氢钾的标准曲线方程为:y=0.8458x+0.0057(R2=0.9998)。
准确量取0.3mL磷脂样品溶液(以二氯甲烷作为溶剂,将磷脂样品溶于二氯甲烷中,制得质量浓度为0.5mg/mL的磷脂样品溶液)置于10mL的刻度试管中(空白对照量取0.3mL二氯甲烷溶剂),水浴挥干溶剂,加入4滴浓硫酸、3滴高氯酸于电炉上消化至无色澄清,冷却后补水至2mL,加入1滴酚酞指示剂,用50%氢氧化钠溶液中和至溶液显红色,缓慢滴加稀硫酸(5/200,v/v)使红色消失,补水至5mL,摇匀,依次加入1.0mL调节酸度的硫酸,摇匀,1.0mL钼酸铵溶液,摇匀,0.6mL抗坏血酸溶液,补水至10mL,加塞混匀,迅速放入70℃的水浴中显色30min取出置冷水中冷却10min,以0mL为参比,在波长820nm处测定吸光值,代入标准曲线,得到无机磷的含量,再乘以系数26.3即得总磷脂的含量。
实验例2
采用UPLC-Q-Exactive Orbitrap/MS方法测定海洋磷脂样品中各类磷脂的组成和相对含量,所述相对含量的计算方法具体参考文献为:Xiaobin Li,et al.Lipidfingerprinting of different material sources by UPLC-Q-Exactive Orbitrap/MSapproach and their zebrafish-based activities comparison,Journal ofAgricultural and Food Chemistry,2020,68,2007-2015。
实施例1
南极磷虾磷脂对斑马鱼ISVs生成的促进作用
(1)南极磷虾磷脂制备
称取冷冻保存30天的南极磷虾100g,粉碎,匀浆,加入浓度为90%的乙醇-水溶液800mL,匀速搅拌提取2次,每次6h,合并提取液,减压抽滤后,滤液经45℃减压浓缩,得到粗磷脂。粗磷脂加浓度为60%的乙醇-水溶液作为溶剂溶解,溶剂体积为200mL,静置24h后,离心15min,保留沉淀部分。用正己烷配成0.5g/mL的溶液,加入56mL丙酮,于4℃冰箱内静置使其沉淀24h。离心15min去除上清液,最终不溶物真空干燥,得到南极磷虾磷脂,其收率为0.5%(对投料冷冻南极磷虾计),总磷脂纯度按质量分数计为75.52%,其中磷脂成分按相对含量计包括:PC占总磷脂的72.35%,LPC占总磷脂的6.86%,PE占总磷脂的8.99%,PI占总磷脂的4.06%、SM占总磷脂的2.59%,LPE占总磷脂的0.53%。
南极磷虾磷脂的液质分析检测图谱见图1。
(2)斑马鱼前处理
本发明所用实验动物为血管荧光转基因Tg(fli-1:EGFP)系斑马鱼,在28℃、10h黑暗/14h照明周期的条件下饲养,每天定时定量喂食丰年虾。按雌雄1:2的比例取健康成熟的斑马鱼放入交配缸内,中间放置隔板,置于黑暗环境中。次日亮灯前抽去隔板,光照刺激使其排卵,排卵1h后收集受精卵,并用新的斑马鱼胚胎培养液冲洗3遍。为避免发育过程中的色素沉着影响结果,在胚胎的养鱼水中加入3%的1-苯基-2-硫脲(1-phenyl-2-thiourea,PTU,Sigma),放入28℃光照培养箱中控光培养,中间每24h换约1/3培养液,并及时吸出死亡胚胎。
(3)斑马鱼ISVs生成实验
将发育至24hpf(hours post fertilization)的斑马鱼胚胎,用1mg/mL的链酶蛋白酶E溶液脱去卵膜。将胚胎随机分为6组,即空白对照组、模型组、阳性对照组和南极磷虾磷脂三个剂量组,置于24孔板中每组3个平行孔,每孔10枚胚胎。空白对照组加2mL培养液,造模组2mL培养液中加入终浓度为0.25μg/mL的PTK787(N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine succinate),阳性对照组2mL培养液中同时加入PTK787(终浓度为0.25μg/mL)和丹参素钠(终浓度100μg/mL),南极磷虾磷脂三个剂量组2mL培养液中分别同时加入PTK787(终浓度为0.25μg/mL)和南极磷虾磷脂(终浓度分别为60、80、100μg/mL)。将以上各组分别放入28℃恒温培养箱中,培养24h。
(4)数据处理
给药24h后,利用荧光显微镜观察斑马鱼ISVs生成情况并采集其图像,采用ImagePro Plus 5.0软件测量ISVs长度后,使用GraphPad Prism 6.01软件进行数据统计。
实验结果如图2、图3所示。由实验结果可知,与空白对照组相比,模型组的斑马鱼ISVs长度显著性降低(P<0.0001),表明模型药显著抑制了斑马鱼的血管生成,造模成功。与模型组相比,各剂量的南极磷虾磷脂组中斑马鱼ISVs长度明显增加,具有统计学差异,证明南极磷虾磷脂可有效促进ISVs生长,且随着剂量的增加,磷脂促血管生成活性呈增强趋势。
实施例2
南极磷虾磷脂对斑马鱼SIVs生成的促进作用
南极磷虾磷脂制备与斑马鱼前处理同实施例1。
(1)斑马鱼SIVs生成实验
将发育至72hpf的斑马鱼胚胎随机分为5组,即空白对照组、阳性对照组和南极磷虾磷脂三个剂量组,置于24孔板中每组3个平行孔,每孔10枚胚胎。空白对照组加2mL培养液,阳性对照组2mL培养液中加入丹红注射液(终浓度9μL/mL),南极磷虾磷脂三个剂量组2mL培养液中分别加入南极磷虾磷脂样品(终浓度分别为60、80、100μg/mL)。将以上各组分别放入28℃恒温培养箱中,培养24h。
(2)数据处理
给药24h后,利用荧光显微镜观察斑马鱼SIVs生成情况并采集其图像,采用ImagePro Plus 5.0软件测量ISVs长度后,使用GraphPad Prism 6.01软件进行数据统计。
实验结果如图4、图5所示。由实验结果可知,与空白对照组相比,各剂量的南极磷虾磷脂组中斑马鱼SIVs长度明显增加,具有显著性差异,证明南极磷虾磷脂具有诱导斑马鱼SIV生成的活性,且随着剂量的增加,磷脂促血管生成活性呈增强趋势。
实施例3
鱿鱼磷脂对斑马鱼ISVs生成的促进作用
斑马鱼前处理、斑马鱼ISVs生成实验和数据处理同实施例1。
鱿鱼磷脂制备:
称取冷冻保存25天的鱿鱼生殖腺100g,粉碎,匀浆,加入浓度为90%的乙醇-水溶液800mL,匀速搅拌提取2次,每次6h,合并提取液,减压抽滤后,滤液经45℃减压浓缩,得到粗磷脂。粗磷脂加浓度为60%的乙醇-水溶液作为溶剂溶解,溶剂体积为200mL,静置24h后,离心15min,保留沉淀部分。用正己烷配成0.5g/mL的溶液,加入70mL丙酮,于4℃冰箱内静置使其沉淀24h。离心15min去除上清液,最终不溶物真空干燥,得到鱿鱼磷脂,其收率为1.2%(对投料冷冻鱿鱼生殖腺计),总磷脂纯度按质量分数计为83.29%,其中磷脂成分按相对含量计包括:PC占总磷脂的55.16%,LPC占总磷脂的8.61%,PE占总磷脂的19.24%,SM占总磷脂的4.83%,LPE占总磷脂的2.15%,PI占总磷脂的1.56%。
鱿鱼磷脂的液质分析检测图谱见图6。
实验结果如图7、图8所示。由实验结果可知,与空白对照组相比,模型组的斑马鱼ISVs长度显著性降低(P<0.0001),表明模型药显著抑制了斑马鱼的血管生成,造模成功。与模型组相比,各剂量的鱿鱼磷脂组中斑马鱼ISVs长度明显增加,具有统计学差异,证明鱿鱼磷脂可有效促进ISVs生长,且随着剂量的增加,磷脂促血管生成活性呈增强趋势。
实施例4
鱿鱼磷脂对斑马鱼SIVs生成的促进作用
鱿鱼磷脂制备和斑马鱼前处理同实施例3。
斑马鱼SIVs生成实验和数据处理同实施例2。
实验结果如图9、图10所示。由实验结果可知,与空白对照组相比,各剂量的鱿鱼磷脂组中斑马鱼SIVs长度明显增加,具有显著性差异,证明鱿鱼磷脂具有诱导斑马鱼SIV生成的活性,且随着剂量的增加,磷脂促血管生成活性呈增强趋势。
对比例1
DHA和EPA对斑马鱼ISVs生成的影响
斑马鱼前处理和数据处理同实施例1。
斑马鱼ISVs生成实验
将发育至24hpf的斑马鱼胚胎,用1mg/mL的链酶蛋白酶E溶液脱去卵膜。将胚胎随机分为3组,即空白对照组、DHA组和EPA组,置于24孔板中每组3个平行孔,每孔10枚胚胎。空白对照组加2mL培养液,DHA组2mL培养液中加入DHA(终浓度为80μg/mL),EPA组2mL培养液中加入EPA(终浓度为80μg/mL)。将以上各组分别放入28℃恒温培养箱中,培养24h。
实验结果如图11、图12所示。由实验结果可知,与空白对照组相比,DHA组和EPA组中斑马鱼ISVs长度明显减少,具有显著性差异,说明DHA和EPA可抑制斑马鱼的ISV生长。
对比例2
DHA和EPA对斑马鱼SIVs生成的影响
斑马鱼前处理、斑马鱼SIVs生成实验和数据处理同实施例2,不同之处在于,没有阳性对照组,且南极磷虾三个剂量组更换为DHA组和EPA组,DHA和EPA的实验终浓度均为80μg/mL。实验结果如图13、图14所示。由实验结果可知,与空白对照组相比,DHA组和EPA组中斑马鱼SIVs长度显著减少,具有统计学意义,说明DHA和EPA对斑马鱼的SIV生长有抑制作用。
综上,本发明首次发现,海洋磷脂可以逆转PTK787诱导的斑马鱼ISVs损伤,并对斑马鱼SIVs生长有显著的促进作用,能明显的增加血管的生成数量和血管长度,且毒副作用小。本发明可为缺血性疾病药物提供候选,具有广阔的市场前景。
以上所述,仅是本发明的优选实施方式,并非是对本发明作其它形式的限制,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,依据本发明的技术实质对以上实施例还可以做出若干改进和润饰,这些改进和润饰也属于本发明的保护范围。
Claims (10)
1.海洋磷脂在促进血管生成方面的应用。
2.海洋磷脂作为药效成分在制备治疗缺血性疾病药物中的应用。
3.如权利要求2所述应用,其特征在于,所述缺血性疾病为因血管受损或血管生成受抑制导致的缺血性疾病。
4.海洋磷脂作为有效成分在制备促进血管生成相关药物中的应用。
5.海洋磷脂作为有效成分在制备保健食品、特医食品等功能性食品中的应用。
6.如权利要求1-5任一项应用所述,其特征在于,所述海洋磷脂中总磷脂纯度按质量分数计为40%以上;
优选的,所述海洋磷脂来源于海洋中的甲壳类浮游动物和/或海洋中的软体动物;
优选的,所述海洋磷脂来源于南极磷虾和/或鱿鱼。
7.如权利要求6所述应用,其特征在于,所述海洋磷脂中的磷脂成分按相对含量计包括:磷脂酰胆碱(phosphatidylcholine,PC)占总磷脂的50%-80%,溶血性磷脂酰胆碱(lysophosphatidylcholine,LPC)占总磷脂的2%-30%,磷脂酰乙醇胺(phosphatidylethanolamine,PE)占总磷脂的5%-30%,溶血性磷脂酰乙醇胺(lysophosphatidylethanolamine,LPE)占总磷脂的0%-5%,磷脂酰肌醇(phosphatidylinositol,PI)占总磷脂的0%-10%,神经鞘磷脂(sphingomyelin,SM)占总磷脂的0%-10%。
8.权利要求1-5任一项所述海洋磷脂的制备方法,其特征在于,所述海洋磷脂的制备方法,包括如下步骤:将海洋生物样品粉碎、匀浆,按质量体积比g/mL为1:(7-10)的量加入浓度为90%以上的乙醇溶液,搅拌提取获得提取液,将提取液固液分离,将分离的液体浓缩为粗磷脂,将粗磷脂用浓度为50%-70%的乙醇溶液溶解,静置12-24h后,固液分离,保留固态部分,用正己烷配成质量浓度为0.3-1.0g/mL的溶液,按所述溶液与丙酮的体积比为1:(5-15)加入丙酮,静置沉淀,然后固液分离获得最终固体,将最终固体干燥即得海洋磷脂。
9.如权利要求8所述的制备方法,其特征在于,所述制备方法中,均匀搅拌提取两次,每次6h获得提取液,合并提取液,将提取液减压抽滤固液分离;
优选的,所述制备方法中,将分离的液体经45℃减压浓度得到粗磷脂。
10.如权利要求8所述的制备方法,其特征在于,所述制备方法中,加入丙酮后,于4℃条件下静置24h;
优选的,将最终固体真空干燥获得海洋磷脂。
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