CN102250210A - 人纤溶酶原Kringle 5短肽、其药物组合物、用途和编码该种短肽的多核苷酸 - Google Patents
人纤溶酶原Kringle 5短肽、其药物组合物、用途和编码该种短肽的多核苷酸 Download PDFInfo
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Abstract
本发明公开了一种人纤溶酶原Kringle 5短肽、其药物组合物、用途和编码该种短肽的多核苷酸,该种短肽为包含如SEQ ID NO:1所示氨基酸序列的多肽,其与人纤溶酶原Kringle 5相比较,具有更强的内皮细胞迁移及新生血管生成抑制作用,并具有合成简便、经济等显著优点。
Description
技术领域
本发明属于生物技术领域,具体涉及一种人纤溶酶原Kringle来源的小分子多肽、其药物组合物、用途和编码该种短肽的多核苷酸。
背景技术
在健康人体中,血管生成(vasculogenesis)主要发生在胚胎早期,成年机体的血管新生不如胚胎时期旺盛和普遍,血管新生的速度在生理状态下也远比病理状态下缓慢,这与体内存在血管新生抑制因子有关,这些抑制因子主要包括:血管新生抑制素(angiostatin),内皮生成抑素(endostatin),组织金属蛋白酶抑制物(tissue inhibitor ofmetalloproteinase,TIMP),凝血酶敏感蛋白(thrombospondin,TSP)、纤溶酶原(Plasminogen)和血小板因子4(platelet factor-4,PF-4)等。
新生血管异常增生是许多重大疾病的病因和/或重要病理特征,由其引起的全身组织及器官的损害是目前最为棘手的医学难题之一,包括角膜新生血管增生,糖尿病性视网膜病变甚至失明,肿瘤的新生血管异常增生,以及银屑病的启动因素真皮乳头微血管异常增生等,具体说明如下:
角膜新生血管增生:角膜新生血管依然是目前最常见的致盲原因之一。正常角膜组织透明,无血管,周围血管终止于角膜缘,形成血管网,营养成分由此扩散入角膜。无血管是角膜的主要特征,也是维持角膜透明的重要条件。在病理状态下,新生毛细血管由角膜缘处侵入角膜内,称为角膜新生血管,主要与角膜水肿、促血管生成因子增加、抑制血管生成因子减少、炎症反应、缺氧以及角膜神经损伤等有关。
糖尿病性视网膜病变:糖尿病是一种内分泌代谢病,白内障、视网膜病变、暂时性屈光不正、眼外肌麻痹等病变是糖尿病晚期严重并发症。据报道,病程在5年以下者眼底改变为38%~39%,病程5~10年者发病率为50%~56.7%,10年以上者发病率增至69%~ 90%。糖尿病可导致视网膜上出现广泛的新生血管,新增血管可以生长在视网膜表层或视神经上。这些未成长的血管比正常血管脆弱,容易破裂使血液流入玻璃体中视网膜反复出血,棉絮状渗出增多,严重损害视力。晚期或严重病例,可反复发生大量的玻璃体出血,引起视网膜脱离,最后导致失明。
肿瘤的新生血管异常增生:肿瘤的发生是多种原因作用的结果,而新生血管异常增生则大大促进了肿瘤的增殖速度及恶化程度。大体上,肿瘤生长的阶段可以分为以下两种:第一种是无血管期的肿瘤,主要依靠周围组织的弥散作用来获取营养物质和排泄代谢产物,所以明显限制了其生长速度;肿瘤直径不超过1~2mm,甚至会长时间地潜伏在组织中而无明显进展。第二种是血管期肿瘤,瘤内出现新生毛细血管并获得进一步生长的能力,肿瘤从而迅速生长并发生转移。
纤溶酶原由5个Kringle组成,在血液中每一个Kringle都具有较强的抑制新生血管生成活性,这其中,人纤溶酶原Kringle5(Human Plasminogen Kringle 5,K5)作为引人注目的抑制血管生成蛋白,是目前发现的抑制新生血管活性最强的纤溶酶原(Plasminogen)片段,其具有生物活性强、高度的细胞特异性、分子量小、性质比较稳定等诸多的显著优点(1~3),现已证实,K5在新生血管性疾病如实体瘤(4,5)、糖尿病性视网膜增生(6)等疾病治疗中具有潜在的应用前景,另,根据现有技术报道,K5的多肽序列PRKLYDY可能是K5蛋白与其受体GRP-78相互作用位点,提示其潜在的抗血管生成活性(7)。但是传统的K5存在酵母系统生产工艺复杂、生产成本昂贵、应用剂量大,伴随潜在的毒副作用等缺陷,严重制约着K5在临床中的应用。
参考文献:
1.Cao.Y,Chen.A.An,S.S.Ji,R.W,Davidson.D and Llinas.M.Kringle 5 of plasminogenis a novel inhibitor of endothelial cell growth.J Biol Chem,272(36):22924-22928,1997.
2.W.R.Ji,L.G.Barrientos,M.Llinás,H.Gray,X.Villarreal,M.E.DeFord,F.J.Castellino,R.A.Kramer and P.A.Trail.Selective inhibition by kringle 5 of human plasminogen onendothelial cell migration,an important process in angiogenesis.Biochem Biophys ResCommun,247(2):414-419,1998.
3.Soff,G.A.Angiostatin and angiostatin-related proteins.Cancer Metastasis Rev,19(1-2):97-107,2000.
4.Davidson DJ,Haskell C,Majest S,et al.Kringle 5 of human plasminogen inducesapoptosis of endothelial and tumor cells through surface-expressed glucose-regulated protein 78.Cancer Res,65:4663-72,2005.
5.Perri SR,Nalbantoglu J,Annabi B,et al.Plasminogen kringle 5-engineered glioma cellsblock migration of tumor-associated macrophages and suppress tumor vascularization andprogression.Cancer Res,65:8359-65,2005.
6.Zhihong Zhang,Jian-xing Ma,Guoquan Gao,Chaoyang Li,Lihui Luo,MeiZhang,Wenzhao Yang,Aihua Jiang,Wenhui Kuang,Liying Xu,Jiaqi Chen,and Zuguo Liu.Plasminogen Kringle 5 Inhibits Alkali-Burn-Induced Corneal Neovascularization.InvestOphthalmol Vis Sci.46:4062-4071,2005.
7.Davidson DJ,Haskell C,Maj est S,Kherzai A,Egan DA,Walter KA,Schneider A,Gubbins EF,Solomon L,Chen Z,Lesniewski R,Henkin J.Kringle 5 of Human Plasminogeninduces Apoptosis of Endothelial and Tumor Cells through Surface-ExpressedGlucose-Regulated Protein 78.Cancer Res.2005 Jun 1;65(11):4663-72.
发明内容
本发明的目的在于提供一种人纤溶酶原Kringle 5短肽,以解决现有技术中存在的上述问题。本发明使用小片段多肽合成来代替繁琐的K5蛋白表达纯化工艺,并获得了一种高活性、经济的新型多肽类药物。
本发明提供的技术方案如下:
一种人纤溶酶原Kringle 5短肽,其特征在于:它包含如SEQ ID NO:1所示氨基酸序列的多肽。
我们通过生物信息学的方法,研究纤溶酶原(Plasminogen)结构与功能的关系,了解多肽中哪些氨基酸系列是活性所必需的,申请人通过查询NCBI数据库,找出人类(Homosapiens),小鼠(Mus musculus),普通牛(Bos taurus),褐家鼠(Rattus norvegicus), 猕猴(Macaca mulatta),黑猩猩(Pan troglodytes),斑马鱼(Danio rerio),原鸡(Gallusgallus),家犬(Canis familiaris)等八种种属的纤溶酶原(Plasminogen)的全长序列,并分别比较八种种属间五个kringle的同源性,发现各种种属间都分别具有保守的Asn-Tyr-Cys-Arg-Asn-Pro-Asp(即NYCRNPD)序列,具体如图1中所示。另外,申请人还通过比较人纤溶酶原(Plasminogen)的五种kringle之间同源性,发现氨基酸序列NYCRNPD在人的五个kringle环中也是高度保守,具体如图2中所示。另外,此段序列位于kringle环二级结构的中心位置,对纤溶酶原蛋白维持活性构象的形式起着重要作用。NYCRNPD序列的结构式如图3中所示。
上述人纤溶酶原Kringle 5短肽NYCRNPD可利用美国PTZ仪器总公司的多肽合成仪(PS3)合成,其脱保护剂为哌啶与DMF的混合液,活化剂为0.4当量的NMM的DMF溶液;裂解液为TFA、苯甲硫醚、水等的混合溶液。合成的多肽用乙醚反复冲洗沉淀。
合成后的短肽NYCRNPD采用以下的步骤对其进行氨基酸序列分析:首先使用苯异硫氰酸酯(PITC)在pH9.0的碱性条件下对蛋白质或多肽进行处理,PITC与肽链的N-端的氨基酸残基反应,形成PTC-肽。然后PTC-肽用三氟乙酸处理,N-端氨基酸残基肽键被有选择地切断,释放出该氨基酸残基的噻唑啉酮苯胺衍生物。接下来将该衍生物用有机溶剂从反应液中萃取出来,而去掉了一个N-端氨基酸残基的肽仍留在溶液中。萃取出来的噻唑啉酮苯胺衍生物经酸作用,再进一步环化,形成一个稳定的PTH-氨基酸。留在溶液中的减少了一个氨基酸残基的肽再重复进行上述反应过程,整个测序过程都是通过测序仪自动进行。
我们测定了短肽NYCRNPD序列的HMNR、HPLC、MS图谱,具体如图4、5和6中所示。
本发明的另一目的是提供一种多核苷酸,该多核苷酸包含编码如SEQ ID NO:1所示氨基酸序列的多核苷酸。
本发明的又一目的是提供一种药物组合物,该药物组合物含有治疗或预防有效量的如SEQ ID NO:1所示氨基酸序列的多肽和含有至少一种的药学上可接受的载体。
本发明的再一目的是提供上述人纤溶酶原Kringle 5短肽在用于制备抗新生血管生成 药物中的用途。
涉及上述人纤溶酶原Kringle 5短肽在应用于制备治疗角膜新生血管增生、视网膜新生血管及肿瘤等血管生成性疾病药物中的用途。
除非特别指明,这里所使用的所有技术和科学术语的含义与本发明所属技术领域一般技术人员通常所理解的含义相同。同样,所有在此提及的出版物、专利申请、专利及其他参考资料均引入本发明作为参考。
与现有技术相比,本发明具有以下的特点:
1)本发明使用小片段多肽合成来代替繁琐的K5蛋白表达纯化工艺,并获得了一种高活性、经济的新型多肽类药物;
2)本发明通过生物信息学方法模拟纤溶酶原Kringle中保守的氨基酸序列,合成短肽NH3-NYCRNPD-COOH,并通过人脐静脉血管内皮细胞(HUVEC)增殖实验证明,NYCRNPD多肽可以有效抑制内皮细胞增殖(见图7),并通过HUVEC细胞迁移实验证明,NYCRNPD多肽可以有效抑制内皮细胞迁移(见图8),此外,还通过鸡胚尿囊膜(CAM)实验证明,NYCRNPD多肽可以明显抑制尿囊膜新生血管生成(见图9)。与人纤溶酶原Kringle 5比较,发现其具有更强的内皮细胞迁移及新生血管生成抑制作用,具有合成简便、经济等显著优点。
附图说明
图1.DNAMAN对纤溶酶原八种种属间五个kringle环的同源性分析;
图2.DNAMAN对人纤溶酶原中五个kringle序列的同源性分析;
图3为NYCRNPD序列的结构式;
图4为NYCRNPD序列的HMNR图谱;
图5为NYCRNPD序列的HPLC图谱;
图6为NYCRNPD序列的MS图谱;
图7显示了采用MTT法检测K5短肽对人脐静脉内皮细胞的增殖影响;
图8显示了采用迁移水平检测K5短肽对人脐静脉内皮细胞的影响;
图9显示了采用CAM assay法检测0.5nmolK5短肽对鸡胚尿囊膜血管生成的影响。
具体实施方式
下面结合实施例对本发明做进一步的描述,但不构成对本发明的任何限制。
实施例通过体外实验检测K5短肽NYCRNPD的抗血管生成活性,实验中又加入了已报道的另一段多肽PRKLYDY做为阳性对照。
(1)HUVEC增殖试验:原代培养的人脐静脉内皮细胞经胰酶消化后,接种在96孔板中(5000个/孔),于37℃培养箱培养4h,待其贴壁后,分别向培养基中加入500nM K5、NYCRNPD及PRKLYDY,PBS为阴性对照,培养箱孵育48h,用MTT标准方法检测存活的内皮细胞数量。结果如图7,与对照组相比,K5及其多肽均具有显著的抑制HUVEC细胞增殖作用,抑制率分别为65.3%、43.0%和46.8%,和对照组相比,三种蛋白均具有显著性差异(*P<0.05),各蛋白之间活性相似,无显著性差异。
(2)内皮细胞迁移实验:迁移实验所用的材料为美国millpore公司生产的transwell,上室加无血清培养基,下室加完全培养基。原代培养的人脐静脉内皮细胞经胰酶消化后,接种在transwell上室中(2.0万个/孔),分别向上室中加入K5、NYCRNPD及PRKLYDY,终浓度为250nM,PBS为阴性对照,于37℃培养箱培养24h。以棉花轻轻擦去上室中残留的未迁移细胞,结晶紫染色30min,显微镜下计数迁移至下层的HUVEC细胞数。结果如图8,与对照相比,K5、NYCRNPD及PRKLYDY均显著抑制HUVEC细胞迁移,抑制率分别为72.0%,77.4%,82.9%,和对照组相比,三种蛋白均具有显著性差异(*P<0.05),各蛋白之间活性相似,无显著性差异。
(3)鸡胚尿囊膜试验:种蛋尖端消毒,打开并吸出1.0~1.5ml蛋清,胶带封口,在37℃培养箱(保持一定的湿度)蛋架上孵育7天,鸡蛋每天旋转三次。孵育第7天,在种蛋钝端消毒后用无菌的眼科剪开一1.5×1.5cm窗口,剥除壳膜后暴露鸡胚尿囊膜,将6mm直径大小的灭菌滤纸片放置于血管部位,用微量加样器将10μl 0.05nM的K5及其短肽NYCRNPD及PRKLYDY滴入灭菌滤纸片中,阴性对照组用等体积无菌PBS缓冲液。用无菌透明胶带封窗后放入孵化箱,37℃继续孵化48小时。去除胶带,打开钝端蛋壳,加入2ml甲醇和丙酮等体积混合液,室温放置15min,剪下鸡胚尿囊膜拍照,分析给药区血管形成情况。结果如图9中所示,与对照组相比,K5及多肽NYCRNPD及PRKLYDY对 新生血管均有显著的抑制作用。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110>金光辉
<120>人纤溶酶原Kringle 5短肽、其药物组合物、用途和编码该种短肽的多核苷酸
<160>1
<210>1
<211>7bp
<212>PRT
<213>人工合成
<400>1
Asn Tyr Cys Arg Asn Pro Asp 7
Claims (5)
1.一种人纤溶酶原Kringle 5短肽,其特征在于:它包含如SEQ ID NO:1所示氨基酸序列的多肽。
2.一种多核苷酸,该多核苷酸包含编码如SEQ ID NO:1所示氨基酸序列的多核苷酸。
3.一种药物组合物,该药物组合物含有治疗或预防有效量的如SEQ ID NO:1所示氨基酸序列的多肽和含有至少一种的药学上可接受的载体。
4.权利要求1中的人纤溶酶原Kringle 5短肽在用于制备抗新生血管生成药物中的用途。
5.权利要求1中的人纤溶酶原Kringle 5短肽在用于制备治疗角膜新生血管增生、视网膜新生血管及肿瘤等血管生成性疾病药物中的用途。
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