CN110302362A - 一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用 - Google Patents
一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用 Download PDFInfo
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Abstract
本发明涉及sDSS1蛋白在制备预防和治疗糖尿病并发症的药物中的应用。sDSS1能够有效的结合晚期糖化蛋白产物并屏蔽由它们引起的细胞毒性,缓解糖尿病并发症模型动物的疾病症状。sDSS1蛋白极具临床应用价值。
Description
技术领域
本发明属于医药技术领域,涉及sDSS1蛋白在制备预防和治疗糖尿病并发症的药物中的应用。
背景技术
糖尿病是以慢性高血糖为特征的一种代谢性疾病。据国际糖尿病联盟(IDF)调查数据显示(http://www.diabetesatlas.org),全世界每11个人中就有1个人患有糖尿病,2017年总计患病人数达4.25亿,其中中国大陆成人糖尿病患者约占1.144亿,位于全球第一。糖尿病病人并发心血管疾病的概率是非糖尿病病人的2~3倍。糖尿病病人终末期肾病的发病率是非糖尿病病人的10倍之多。世界每30秒钟就会有人因为糖尿病而失去下肢或下肢的一部分。并发症一旦产生,药物治疗很难逆转。因此尽早预防或延缓糖尿病并发症,保护糖尿病累及的终末器官,提高病人生活质量,降低社会经济负担,已经成为治疗糖尿病的终极目标。
长期的血糖升高使大血管、微血管受损并危及心、脑、肾、周围神经、眼睛、足等。尿微量白蛋白是糖尿病肾病最早发生改变的标记物。尿微量白蛋白还是心血管并发症的强有力的预测指标[1],其排泄量与糖尿病晚期并发症呈正相关[2]。因此尿微量白蛋白临床上被推荐为早期筛选糖尿病并发症的标记物(IDF DIABETES ATLAS Eighth edition 2017)。糖尿病并发症发病机制复杂,目前的研究结果提示代谢紊乱、氧化应激、血流动力学改变、慢性低度炎性反应、遗传等多种因素均参与了疾病的进程[3]。
终末糖基化产物(AGEs)是一类蛋白质、脂质或核酸等大分子在非酶促条件下自发的与过高的葡萄糖或其他还原性单糖反应所生成的稳定的共价加成物。可视为氧化应激助动的氧化及/或糖化复合物。一方面,循环中的AGEs可作用于RAGE受体进而引起氧化应激、炎症和细胞凋亡;另一方面长期的高糖及氧化压力可使细胞外基质蛋白发生糖基化交联,使血管硬化;第三,细胞内的过多糖基化修饰会干扰多种蛋白、酶或受体发挥其生物学功能[3,4]。I型及II型糖尿病患者血液及组织中AGEs水平增加[5]。组织中AGEs累积程度与糖尿病并发症呈正相关[6]。因此,抑制AGEs通路可成为预防或治疗糖尿病并发症的方式之一[7]。目前已报导可通过多种方式干扰AGEs毒性,比如抑制AGEs生成、抑制AGEs交联,RAGE受体拮抗剂等[7-10]。代表性药物如氨基胍、Pyridoxamine、TTP488、sRAGE等目前均处在临床研究阶段。
Shfm1(split hand/split foot malformation type 1)基因是人蟹爪病中的关键基因之一,进化上高度保守,它所编码的蛋白DSS1是一种通用的内源多功能无序蛋白。参与到稳定基因组、同源基因重组、DNA损伤修复、RNA剪切、蛋白降解和细胞增殖等过程[11]。研究结果显示DSS1蛋白作为标签可以通过耗能的酶促反应添加到氧化蛋白上,帮助细胞清除氧化蛋白[12]。这些结果显示DSS1蛋白在生物活动中的重要作用。
1.Bakris GL,Molitch M.Microalbuminuria as a risk predictor indiabetes:the continuing saga.Diabetes Care.2014;37(3):867-75.
2.Fekete T,Bogdan E,C.Microalbuminuria,as predictor oflate diabetic complications.A prospective study.Medecine interne.1990;28(2):131-4.
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5.Nowotny K,Jung T,Weber D,Grune T.Advanced glycation endproducts and oxidative stress in type 2 diabetes mellitus.Biomolecules.2015;5(1):194-222.
6.Monnier VM,Sell DR,Genuth S.Glycation products as markers andpredictors of the progression of diabetic complications.Annals of the NewYork Academy of Sciences.2005;1043(1):567-81.
7.Younus H,Anwar S.Prevention of non-enzymatic glycosylation(glycation):Implication in the treatment of diabeticcomplication.International journal of health sciences.2016;10(2):261.
8.Lv M,Chen Z,Hu G,Li Q.Therapeutic strategies of diabeticnephropathy:recent progress and future perspectives.Drug Discov Today.2015;20(3):332-46.
9.Bongarzone S,Savickas V,Luzi F,Gee AD.Targeting the Receptor forAdvanced Glycation Endproducts(RAGE):A Medicinal Chemistry Perspective.J MedChem.2017;60(17):7213-32.
10.Abbas G,Al-Harrasi AS,Hussain H,Hussain J,Rashid R,ChoudharyMI.Antiglycation therapy:Discovery of promising antiglycation agents for themanagement of diabetic complications.Pharmaceutical biology.2016;54(2):198-206.
11.Kragelund BB,SM,Rebula CA,Panse VG,Hartmann-PetersenR.DSS1/Sem1,a multifunctional and intrinsically disordered protein.Trends inbiochemical sciences.2016;41(5):446-59.
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发明内容
在本发明中,我们(发明人)提供的sDSS1蛋白可以与AGEs结合,降低AGEs引起的细胞毒性。sDSS1蛋白长期给药可以降低糖尿病模型小鼠的尿白蛋白排泄,并且显著改善肾脏滤过功能。因此,sDSS1蛋白可以成为临床上制备预防和治疗糖尿病并发症的药物。
具体的技术方案如下:
一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用,所述的应用是把sDSS1蛋白用于制备预防和治疗糖尿病并发症的药物。
优选地,所述的糖尿病并发症包括糖尿病肾病、糖尿病眼部并发症、糖尿病足、糖尿病心脑血管并发症或糖尿病神经病变。
优选地,所述的糖尿病眼部并发症包括糖尿病性视网膜病变、糖尿病性黄斑水肿(DME)、糖尿病性白内障或青光眼。
优选地,所述的糖尿病心脑血管并发症包括糖尿病引起的冠状动脉疾病(CAD)、心绞痛、心肌梗死、动脉硬化、中风、脑萎缩、外周动脉疾病(PAD)或充血性心力衰竭等。
优选地,所述的糖尿病并发症包括I型糖尿病并发症和II型糖尿病并发症。
优选地,所述的sDSS1蛋白包括人、黑猩猩、倭黑猩猩、大猩猩、红毛猩猩、白颊长臂猿、川金丝猴、恒河猴、滇金丝猴、东非狒狒、安哥拉疣猴、白顶白眉猴、鬼狒或豚尾猴的任一sDSS1蛋白序列形成的基础蛋白,其中人sDSS1的氨基酸序列如SEQ ID NO:1,黑猩猩sDSS1的氨基酸序列如SEQ ID NO:2,倭黑猩猩sDSS1的氨基酸序列如SEQ ID NO:3,大猩猩sDSS1的氨基酸序列如SEQ ID NO:4,红毛猩猩sDSS1的氨基酸序列如SEQ ID NO:5,白颊长臂猿sDSS1的氨基酸序列如SEQ ID NO:6,川金丝猴sDSS1的氨基酸序列如SEQ ID NO:7,恒河猴sDSS1的氨基酸序列如SEQ ID NO:8,滇金丝猴sDSS1的氨基酸序列如SEQ ID NO:9,东非狒狒sDSS1的氨基酸序列如SEQ ID NO:10,安哥拉疣猴sDSS1的氨基酸序列如SEQ ID NO:11,白顶白眉猴sDSS1的氨基酸序列如SEQ ID NO:12,鬼狒sDSS1的氨基酸序列如SEQ ID NO:13,豚尾猴sDSS1的氨基酸序列如SEQ ID NO:14。
优选地,所述的sDSS1蛋白是任一与所述的基础蛋白相似度达到70%以上的第一种蛋白。
优选地,所述的sDSS1蛋白是任一以基础蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他多肽片段,用于融合的多肽片段的结构特征或氨基酸序列特征与基础蛋白碳端31个序列相同或相似的第二种蛋白。
优选地,所述的sDSS1蛋白是任一以基础蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他氨基酸片段,融合后的蛋白能实现跨膜转运功能的第三种蛋白。
优选地,所述的sDSS1蛋白是利用基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白与该蛋白自身、载体蛋白、抗体或其他任意长度氨基酸片段连接形成的融合蛋白。
优选地,所述的sDSS1蛋白是基于基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白进行的修饰产生的蛋白修饰物。
优选地,所述蛋白修饰物的修饰是针对氨基酸侧链上的氨基、氨基酸侧链上的羰基、氮端末端氨基、碳端末端羰基、半胱氨酸、酪氨酸、丝氨酸、色氨酸进行的特异性或非特异性的1-20个位点的化学修饰。
优选地,所述蛋白修饰物的修饰方法包括糖基化修饰、脂肪酸修饰、酰基化修饰、Fc片段融合、白蛋白融合、聚乙二醇修饰、右旋糖苷修饰、肝素修饰、聚乙烯吡咯烷酮修饰、聚氨基酸修饰、多聚唾液酸修饰、壳聚糖及其衍生物修饰、凝集素修饰、海藻酸钠修饰、卡波姆修饰、聚乙烯吡咯烷酮修饰、羟丙基甲基纤维素修饰、羟丙基纤维素修饰、乙酰化修饰、甲酰化修饰、磷酸化修饰、甲基化修饰或磺酸化修饰以及其他医药上可用的多肽/蛋白药物修饰方法的一种或一种以上。
优选地,所述的sDSS1蛋白是利用基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白的氨基酸序列为基础进行的20种基本氨基酸以外的氨基酸进行的1-31个任意氨基酸位点替换的非天然氨基酸替代蛋白。
优选地,所述非天然氨基酸替代蛋白的氨基酸替换包括羟脯氨酸、羟赖氨酸、硒代半胱氨酸、D-型氨基酸或人工合成的非天然氨基酸及其衍生物。
优选地,所述的sDSS1蛋白是把基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物或非天然氨基酸替代物与医药上可应用的药物载体形成的部分或全部复合体。
优选地,所述药物载体包含肠溶衣制剂、胶囊、微球/囊、脂质体、微乳液、复乳液、纳米颗粒、磁颗粒、明胶或凝胶中的一种或一种以上。
优选地,所述的sDSS1蛋白是以个体自身sDSS1蛋白为靶点,通过外源药物影响个体自身sDSS1蛋白的水平。
优选地,所述的药物是以sDSS1蛋白、sDSS1蛋白的基因、sDSS1的基因的调控元件或sDSS1的基因的转录产物为药物作用靶点。
优选地,所述的药物是通过影响血液中蛋白酶/肽酶活性从而调节sDSS1蛋白在血液中的含量。
优选地,所述的药物是化学小分子药物、抗体、多肽/蛋白药物、核酸药物或纳米药物。
优选地,所述的sDSS1蛋白是以基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代物、复合体和药物中的任一一种成分中的两种或多种的药物组合。
优选地,所述的sDSS1蛋白是基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代物、复合体和药物中的任一一种成分中的一种、两种或多种与医药上可用的赋形剂形成的药物组合。
优选地,所述的sDSS1蛋白是通过表达体系把编码基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白和融合蛋白中任一蛋白的核苷酸序列导入体内并表达获得的蛋白。
优选地,所述的表达体系是真核表达质粒载体、腺病毒、腺相关病毒、慢病毒、逆转录病毒、杆状病毒、疱疹病毒、伪狂犬病毒、ZFN基因编辑技术、TALEN基因编辑技术、CRISPR/Cas基因编辑技术及其他医疗上可用的基因编辑技术或病毒载体。
优选地,所述的sDSS1蛋白是通过移植细胞在个体体内获得的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白和融合蛋白中的任一蛋白。
优选地,所述的细胞是任意一种人的干细胞、前体细胞或成体细胞。
优选地,所述的干细胞是胚胎干细胞、诱导多能干细胞、转分化得到的细胞,或者来源于原代培养的干细胞、由母细胞分化得到的多能或单能干细胞。
优选地,所述的sDSS1蛋白是通过血清、组织间液输注引入个体体内的sDSS1蛋白。
优选地,所述的sDSS1蛋白是通过移植组织或器官在个体体内获得的基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白。
优选地,组织或器官移植中,所述的组织是脑、肝、肾、脾、胰岛的完整器官或部分组织块,或血液、脂肪、肌肉、骨髓、皮肤。
优选地,所述的预防药物是包含基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代蛋白、复合体、药物组合、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品或食品添加剂。
优选地,所述的治疗药物是包含基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代蛋白、复合体、药物组合、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品或食品添加剂。
一种蛋白在改善糖尿病并发症护理设备中的应用,其是利用上述任一方案在制备预防和治疗糖尿病并发症药物中的应用,再将制备的药物用于提高糖尿病并发症护理相关的医疗器械的性能。
优选地,所述的医疗器械包括包括输采血设备和耗材、血液净化设备和耗材、血液净化设备辅助装置和耗材、体液处理设备和耗材、肾透析设备和耗材、腹膜透析设备和耗材、血液灌流器、输液器具、注射器具、缓释器具、人工肾脏中的一种或多种。
本发明的特点和/或有益效果有:
1.本发明提供的sDSS1蛋白与AGEs结合,有效缓解AGEs引起的细胞毒性。
2.本发明提供的sDSS1蛋白在db糖尿病模型小鼠上显著降低了糖尿病并发症的标志物之一尿微量白蛋白水平。
3.本发明提供的sDSS1蛋白在db糖尿病模型小鼠上改善了肾小球滤过率的代偿增加,提高了肾功能并降低血液糖化血红蛋白水平。
4.本发明提供的sDSS1蛋白是人和其他灵长类动物所具有的内源蛋白或其衍生物,分子量相对较小,免疫原性低,并且体内存在天然的蛋白降解机制,因此,临床应用中引起明显的免疫反应或其他的毒副效应的概率不高,安全可靠。
综上,本发明提供了一种sDSS1蛋白用于糖尿病并发症预防和治疗的方式,通过分子水平、细胞水平和动物水平的实验验证,sDSS1蛋白可以与AGEs结合,降低AGEs引起的细胞损伤。sDSS1蛋白可有效降低db糖尿病小鼠尿微量白蛋白的排泄,改善肾功能,降低糖化血红蛋白水平,缓解疾病症状。sDSS1蛋白具备用于临床上制备预防和治疗糖尿病并发症药物的潜力。
附图说明
下面结合附图,对本发明做进一步详细的阐述,以使本发明能够清楚、完整,但不是为了限制本发明的保护范围。
图1A-1B.图1A.分子水平实验显示sDSS1与AGEs发生相互作用。AGEs蛋白或AGEs蛋白与sDSS1孵育产物经SDS-PAGE分离并用考马斯亮蓝染色,结果显示AGEs可以与sDSS1蛋白发生相互作用,SDSS1蛋白条带颜色变浅(L3vs L2,L6vs L5)。图1B.AGEs蛋白或AGEs蛋白与sDSS1孵育产物经SDS-PAGE分离并用抗AGEs抗体标记,在暗房中通过显影液和定影液显色,结果表明sDSS1与AGEs发生相互作用后抗体识别的AGEs蛋白条带颜色变浅,并且,变浅的程度与sDSS1蛋白浓度成正比,呈现明显的浓度依赖。
图2.sDSS1蛋白屏蔽AGEs导致的细胞毒性。在大鼠肾细胞培养物中添加50μM AGEs可以显著降低细胞活力水平,添加不同浓度的sDSS1蛋白后,细胞活力被挽回,随着sDSS1蛋白浓度增加,细胞活力逐渐上升,且该效应呈现典型的剂量依赖效应。75μM sDSS1蛋白可以完全屏蔽50μM AGEs导致的细胞活力水平下降。数据经ANOVA分析,#,空白对照组vs仅添加AGEs组,*,所有添加AGEs和sDSS1组vs仅添加AGEs组;*,p-value<0.05;**,p-value<0.01;###,p-value<0.001;****,p-value<0.0001。
图3A-3B.分子水平实验显示sDSS1与CML-BSA发生相互作用。图3A.SDS-PAGE分离并用考马斯亮蓝染色后显示,CML-BSA可以与sDSS1蛋白发生相互作用,sDSS1蛋白对应位置处条带颜色变浅(L3vs L2,L5vs L4)。图3B.蛋白免疫印迹法显示sDSS1与CML-BSA发生相互作用后抗体识别的CML-BSA蛋白数量减少,表现为条带变浅。并且变浅的程度与sDSS1蛋白浓度成正比,呈现明显的浓度依赖。
图4A-4B.sDSS1蛋白屏蔽CML-BSA导致的细胞毒性。图4A.在大鼠肾细胞培养物中添加33μg/mL CML-BSA,显微镜下观察并拍照记录细胞状态。当培养液中加入33μg/mL CML-BSA时,细胞数量明显减少,细胞变圆,细胞间连接基本消失,随着加入sDSS1蛋白浓度增加,细胞状态逐渐恢复正常。图4B.通过检测细胞活力,在大鼠肾细胞培养物中添加33μg/mLCML-BSA可以显著降低细胞活力水平,添加不同浓度的sDSS1蛋白后,细胞活力被挽回,随着sDSS1蛋白浓度增加,细胞活力逐渐上升。30μM sDSS1蛋白可以基本屏蔽33μg/mL CML-BSA导致的细胞活力水平下降,且该效应呈现典型的剂量依赖效应。数据经ANOVA分析,#,空白对照组vs仅添加CML-BSA组,*,所有添加CML-BSA和sDSS1组vs仅添加CML-BSA组;#、*,p-value<0.05;##、**,p-value<0.01;###、***,p-value<0.001。
图5.sDSS1改善糖尿病小鼠尿白蛋白水平。db小鼠尿白蛋白相比野生同窝小鼠尿白蛋白排泄增加,给予sDSS1后,尿白蛋白排泄症状得到好转,10mpk(mg protein perkilogram body weight,mg蛋白每千克体重)剂量组随着给药时间延长效果增加,在给药40天后显著的降低了尿白蛋白排泄量。数据经ANOVA分析,*P<0.05,**P<0.01vs.db/db。
图6.sDSS1改善糖尿病并发肾病症状。db小鼠肾小球滤过功能相比野生同窝小鼠代偿性增加,给予sDSS1后,sDSS1能够剂量依赖性的缓解这一代偿增加。数据经ANOVA分析,*P<0.05,**P<0.01vs.db/db。
图7.sDSS1改善糖尿病小鼠糖化血红蛋白水平。db小鼠血液糖化血红蛋白相比野生同窝小鼠糖化血红蛋白大幅增加,给予sDSS1后,10mpk剂量降低了约1.3%的糖化血红蛋白比例。数据经ANOVA分析,*P<0.05,****P<0.0001vs.db/db。
具体实施方式
以下内容将结合实例对本发明中的优选方案进行说明和验证,不是对本发明的范围进行限定。本发明的所有范围限定以权利要求书中的限定为准。
下述实施案例中所用的实验方法如无特殊说明,均为常规实验方法。
下述实施案例中所用的sDSS1蛋白为本公司自行生产并进行质量控制,经检测蛋白纯度大于95%,内毒素和其他杂质残留符合标准,可用于动物实验而不引起明显的动物毒性反应。
下述实施案例中材料和试剂,除了sDSS1蛋白外,其他均可以通过商业途径获取。
实施例1.sDSS1蛋白与AGEs存在相互作用。
1.1.实验材料与方法
材料:牛血清白蛋白(阿拉丁,A104912),核糖(阿拉丁,D1608050),anti-AGEs抗体购自TransGenic公司,50489-M08H。
方法:将牛血清白蛋白和核糖在PBS缓冲液中共同孵育14天,制备得到AGEs蛋白。分别将2μg的AGEs、4μg的sDSS1、2μg的AGEs与4μg的sDSS1、2μg的AGEs、8μg的sDSS1、2μg的AGEs与8μg的sDSS1加入1.5mL EP管,37℃反应过夜。孵育产物加入上样缓冲液后,混匀,100℃变性处理10分钟制成上样样品。样品用聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,PAGE胶进行考马斯亮蓝染色显示蛋白条带。
分别将2μg的AGEs、2μg的AGEs与4μg的sDSS1、2μg的AGEs与10μg的sDSS1、2μg的AGEs与20μg的sDSS1加入1.5mL EP管,37℃反应过夜。孵育产物加入上样缓冲液后,混匀,100℃变性处理10分钟制成上样样品。样品用聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,分离后转印于PVDF膜,并用5%脱脂牛奶封闭。然后进行一抗4℃孵育(anti-AGEs)过夜。用TBST洗膜后加入辣根过氧化物酶(HRP)偶联的山羊抗鼠二抗进行孵育。随后用TBST洗膜,ECL超敏发光液淋洗PVDF膜,并对X-光片进行显影、定影,显示目的蛋白条带。
1.2.实验结果
在染色后的PAGE胶上可以看到,制得的AGEs蛋白分子量75KD-150KD不等,形成弥散的条带(L1,图1A)。当把AGEs与sDSS1蛋白进行混合孵育后,可以看到sDSS1蛋白的条带颜色变浅提示AGEs与sDSS1蛋白发生了相互作用并使sDSS1蛋白数量减少。在Westen-Blot实验中,当把AGEs与sDSS1蛋白进行混合孵育后,可以看到AGEs蛋白的两条条带颜色明显变浅,而且,随着sDSS1比例增高,条带颜色越来越浅(L2-L4,图1B)。这些结果说明,sDSS1蛋白可以与AGEs发生相互作用。
实施例2.sDSS1蛋白屏蔽AGEs引起的细胞毒性。
2.1.实验材料与方法
材料:NRK-52E细胞株(中国科学院典型培养物保藏委员会细胞库,目录号:GNR8),DMEM培养基(HyClone,AC10210629),SpectraMax Plus384多功能酶标仪(MolecularDevices公司),细胞增殖/毒性检测试剂盒(Dojindo,CK04)。
方法:NRK-52E细胞按照2ⅹ104细胞每孔接种到96孔板。细胞贴壁过夜后换成无血清培养基饥饿处理24小时。各组分别加入空白培养基、50μM AGEs、50μM AGEs和25μMsDSS1、50μM AGEs和50μM sDSS1、50μM AGEs和75μM sDSS1、50μM AGEs和100μM sDSS1。48小时后使用细胞增殖毒性检测试剂盒进行细胞活力水平检测。
2.2.实验结果
细胞活力检测结果显示,当NRK-52E细胞中只加入10μMAGEs时,细胞活性水平显著降低,只有对照细胞的20%左右。当sDSS1蛋白加入培养基之后,细胞活力逐渐提高。在25μM-75μM范围内,随着sDSS1蛋白浓度增加,细胞活性增强愈加显著,呈现浓度依赖性(图2)。这些结果说明,sDSS1蛋白能够屏蔽AGEs引起的细胞活力下降。
实施例3sDSS1可以与CML-BSA发生相互作用。
3.1实验材料与方法
材料:CML-BSA购自Cell Biolab(STA-314);anti-AGEs抗体购自TransGenic公司(50489-M08H)。
方法:将1μg的CML-BSA与0.5、5μg的sDSS1混合。同时包含单独的CML-BSA与sDSS1对照。所有样品在EP管中37度过夜孵育后,孵育产物加入上样缓冲液,混匀,100℃变性处理10分钟制成上样样品。样品用聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,PAGE胶进行考马斯亮蓝染色显示蛋白条带。
同时样品用SDS-PAGE进行分离后转印于PVDF膜,并用5%脱脂牛奶封闭。然后进行一抗孵育(anti-AGEs),4℃过夜。依次洗膜,加入二抗,洗膜,显色,显影以及定影显示目的蛋白条带。
3.2实验结果
CML是AGEs复合成分的代表之一。当把CML-BSA与sDSS1蛋白进行混合孵育时,可以看到sDSS1蛋白条带颜色变浅(图3A)。后续Western-Blot结果显示对应CML-BSA蛋白的两条条带颜色明显变浅,而且,随着sDSS1比例增高,条带颜色越来越浅(图3B)。这些结果说明,sDSS1蛋白可以与CML-BSA发生相互作用并使抗体识别的AGE蛋白数量减少。
实施例4.sDSS1蛋白屏蔽CML-BSA引起的细胞毒性。
4.1.实验材料与方法
材料:NRK-52E细胞株,DMEM培养基(HyClone,AC10210629),SpectraMax Plus384多功能酶标仪(Molecular Devices公司),细胞增殖/毒性检测试剂盒(Dojindo,CK04),CML-BSA(Cell Biolabs公司,STA-314)。
方法:NRK-52E细胞按照2ⅹ104细胞每孔接种到96孔板。细胞贴壁后饥饿处理24小时。各组分别加入空白培养基、33μg/mL CML-BSA、33μg/mL CML-BSA和3μM sDSS1、33μg/mLCML-BSA和10μM sDSS1、33μg/mL CML-BSA和30μM sDSS1。完成后,细胞继续处理48小时。处理完成的细胞显微镜下观察细胞形态,并使用细胞增殖毒性检测试剂盒进行细胞活力水平检测。
4.2.实验结果
当NRK-52E细胞中只加入33μg/mL CML-BSA时,在显微镜下观察细胞数量明显减少,细胞变圆,细胞间连接基本消失;与空白组正常的NRK-52E细胞的梭形形态呈现显著差异(图4A)。且细胞活力检测结果显示,细胞活性水平显著降低,只有对照细胞的30%左右(图4B)。当sDSS1蛋白加入培养基之后,细胞的形态及活力均得到挽救。在加入3μM sDSS1蛋白时,细胞活力略有提高,细胞形态无明显改善,但呈现出细胞数量增加的趋势;随着sDSS1蛋白浓度增加达到10μM-30μM时,细胞活性增强愈加显著,显微镜下细胞形态也已经恢复至正常状态,该效应呈现浓度依赖性。这些结果说明,sDSS1蛋白可以屏蔽CML-BSA引起的细胞毒性,保护细胞活力。
实施例5.sDSS1可以缓解db糖尿病小鼠并发症症状
5.1.实验材料与方法
C57BLKS/J背景的db/db小鼠,购自南京大学-南京生物医药研究院,雄性。小鼠适应性饲养后根据体重和血糖分组,之后每两天静脉给予溶剂对照或sDSS1蛋白。同时监测体重和饮食。给药21天时,将小鼠放入代谢笼中收集24小时尿液,检测尿白蛋白浓度。给药37天后,静脉注射FITC标记的菊粉,分别于3/7/10/15/35/70分钟检测血浆菊粉浓度,计算肾小球滤过率。给药40天时,再次将小鼠放入代谢笼中收集24小时尿液,检测尿白蛋白浓度。给药满6周后安乐处死小鼠,收集血浆、心脏、眼球、肾脏、以及脑。小鼠尿白蛋白检测试剂盒购自Bethyl Laboratories,Inc.小鼠糖化血红蛋白Elisa试剂盒购自上海朗顿生物技术有限公司(货号:BPE20512)。
5.2.实验结果
根据24h尿白蛋白检测结果,db糖尿病小鼠相比其同窝野生对照小鼠尿白蛋白排泄增加,提示肾功能受到损害。给药21天时,sDSS1有改善尿白蛋白排泄的趋势,但是还没有统计差异。给药至37天时,sDSS1 10mpk能显著改善尿白蛋白排泄,而且sDSS1 10mpk的效果强于3mpk的效果(图5)。检测肾小球滤过率功能,结果显示db小鼠相比其野生对照小鼠肾小球滤过功能代偿性的增加。而sDSS1蛋白3mpk和10mpk能缓解db小鼠肾小球滤过率的代偿增加(图6)。血中糖化血红蛋白数据显示,10mpk剂量降低了约1.3%的糖化血红蛋白比例,提示sDSS1能够影响到糖基化血红蛋白水平(图7)。总结这些数据,sDSS1能改善db糖尿病小鼠的肾脏并发症症状,且这一改善具有时效及量效关系。
序列表
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Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu
20 25 30
Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val
35 40 45
Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Lys
50 55 60
Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly
65 70 75 80
Arg Met Cys Ser Ala Phe Leu Cys Cys
85
<210> 11
<211> 89
<212> PRT
<213> 安哥拉疣猴(Cercocebusatys)
<400> 11
Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp
1 5 10 15
Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu
20 25 30
Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val
35 40 45
Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile
50 55 60
Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly
65 70 75 80
Arg Met Cys Ser Ala Phe Leu Cys Cys
85
<210> 12
<211> 89
<212> PRT
<213> 白顶白眉猴(M.leucophaeus)
<400> 12
Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp
1 5 10 15
Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu
20 25 30
Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val
35 40 45
Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile
50 55 60
Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly
65 70 75 80
Arg Met Cys Ser Ala Phe Leu Cys Cys
85
<210> 13
<211> 89
<212> PRT
<213> 鬼狒(Macacanemestrina)
<400> 13
Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp
1 5 10 15
Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu
20 25 30
Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val
35 40 45
Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile
50 55 60
Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly
65 70 75 80
Arg Met Cys Ser Ala Phe Leu Cys Cys
85
<210> 14
<211> 89
<212> PRT
<213> 豚尾猴(Papioanubis)
<400> 14
Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp
1 5 10 15
Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu
20 25 30
Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val
35 40 45
Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile
50 55 60
Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly
65 70 75 80
Arg Met Cys Ser Ala Phe Leu Cys Cys
85
Claims (35)
1.一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用,其特征在于,所述的应用是把sDSS1蛋白用于制备预防和治疗糖尿病并发症的药物。
2.根据权利要求1所述的应用,其特征在于,所述的糖尿病并发症包括糖尿病肾病、糖尿病眼部并发症、糖尿病足、糖尿病心脑血管并发症或糖尿病神经病变。
3.根据权利要求2所述的应用,其特征在于,所述的糖尿病眼部并发症包括糖尿病性视网膜病变、糖尿病性黄斑水肿(DME)、糖尿病性白内障或青光眼。
4.根据权利要求2所述的应用,其特征在于,所述的糖尿病心脑血管并发症包括糖尿病引起的冠状动脉疾病(CAD)、心绞痛、心肌梗死、动脉硬化、中风、脑萎缩、外周动脉疾病(PAD)或充血性心力衰竭等。
5.根据权利要求1所述的应用,其特征在于,所述的糖尿病并发症包括I型糖尿病并发症和II型糖尿病并发症。
6.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白包括人、黑猩猩、倭黑猩猩、大猩猩、红毛猩猩、白颊长臂猿、川金丝猴、恒河猴、滇金丝猴、东非狒狒、安哥拉疣猴、白顶白眉猴、鬼狒或豚尾猴的任一sDSS1蛋白序列形成的基础蛋白,其中人sDSS1的氨基酸序列如SEQ ID NO:1,黑猩猩sDSS1的氨基酸序列如SEQ ID NO:2,倭黑猩猩sDSS1的氨基酸序列如SEQ ID NO:3,大猩猩sDSS1的氨基酸序列如SEQ ID NO:4,红毛猩猩sDSS1的氨基酸序列如SEQ ID NO:5,白颊长臂猿sDSS1的氨基酸序列如SEQ ID NO:6,川金丝猴sDSS1的氨基酸序列如SEQ ID NO:7,恒河猴sDSS1的氨基酸序列如SEQ ID NO:8,滇金丝猴sDSS1的氨基酸序列如SEQ ID NO:9,东非狒狒sDSS1的氨基酸序列如SEQ ID NO:10,安哥拉疣猴sDSS1的氨基酸序列如SEQ ID NO:11,白顶白眉猴sDSS1的氨基酸序列如SEQ ID NO:12,鬼狒sDSS1的氨基酸序列如SEQ ID NO:13,豚尾猴sDSS1的氨基酸序列如SEQ ID NO:14。
7.根据权利要求6所述的应用,其特征在于,所述的sDSS1蛋白是任一与所述的基础蛋白相似度达到70%以上的第一种蛋白。
8.根据权利要求6所述的应用,其特征在于,所述的sDSS1蛋白是任一以基础蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他多肽片段,用于融合的多肽片段的结构特征或氨基酸序列特征与基础蛋白碳端31个序列相同或相似的第二种蛋白。
9.根据权利要求6所述的应用,其特征在于,所述的sDSS1蛋白是任一以基础蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他氨基酸片段,融合后的蛋白能实现跨膜转运功能的第三种蛋白。
10.根据权利要求6-9任一所述的应用,其特征在于,所述的sDSS1蛋白是利用基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白与该蛋白自身、载体蛋白、抗体或其他任意长度氨基酸片段连接形成的融合蛋白。
11.根据权利要求6-9任一所述的应用,其特征在于,所述的sDSS1蛋白是基于基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白进行的修饰产生的蛋白修饰物。
12.根据权利要求11所述的蛋白修饰物,其特征在于,所述蛋白修饰物的修饰是针对氨基酸侧链上的氨基、氨基酸侧链上的羰基、氮端末端氨基、碳端末端羰基、半胱氨酸、酪氨酸、丝氨酸、色氨酸进行的特异性或非特异性的1-20个位点的化学修饰。
13.根据权利要求11所述的多肽/蛋白修饰物,其特征在于,所述蛋白修饰物的修饰方法包括糖基化修饰、脂肪酸修饰、酰基化修饰、Fc片段融合、白蛋白融合、聚乙二醇修饰、右旋糖苷修饰、肝素修饰、聚乙烯吡咯烷酮修饰、聚氨基酸修饰、多聚唾液酸修饰、壳聚糖及其衍生物修饰、凝集素修饰、海藻酸钠修饰、卡波姆修饰、聚乙烯吡咯烷酮修饰、羟丙基甲基纤维素修饰、羟丙基纤维素修饰、乙酰化修饰、甲酰化修饰、磷酸化修饰、甲基化修饰或磺酸化修饰以及其他医药上可用的多肽/蛋白药物修饰方法的一种或一种以上。
14.根据权利要求6-10任一所述的应用,其特征在于,所述的sDSS1蛋白是利用基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白的氨基酸序列为基础进行的20种基本氨基酸以外的氨基酸进行的1-31个任意氨基酸位点替换的非天然氨基酸替代蛋白。
15.根据权利要求14所述的应用,其特征在于,所述非天然氨基酸替代蛋白的氨基酸替换包括羟脯氨酸、羟赖氨酸、硒代半胱氨酸、D-型氨基酸或人工合成的非天然氨基酸及其衍生物。
16.根据权利要求6-15任一所述的应用,其特征在于,所述的sDSS1蛋白是把基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物或非天然氨基酸替代物与医药上可应用的药物载体形成的部分或全部复合体。
17.根据权利要求16所述的应用,其特征在于,所述药物载体包含肠溶衣制剂、胶囊、微球/囊、脂质体、微乳液、复乳液、纳米颗粒、磁颗粒、明胶或凝胶中的一种或一种以上。
18.根据权利要求6所述的应用,其特征在于,所述的sDSS1蛋白是以个体自身sDSS1蛋白为靶点,通过外源药物影响个体自身sDSS1蛋白的水平。
19.根据权利要求18所述的应用,其特征在于,所述的药物是以sDSS1蛋白、sDSS1蛋白的基因、sDSS1的基因的调控元件或sDSS1的基因的转录产物为药物作用靶点。
20.根据权利要求18所述的应用,其特征在于,所述的药物是通过影响血液中蛋白酶/肽酶活性从而调节sDSS1蛋白在血液中的含量。
21.根据权利要求18-20任一所述的应用,其特征在于,所述的药物是化学小分子药物、抗体、多肽/蛋白药物、核酸药物或纳米药物。
22.根据权利要求6-21任一所述的应用,其特征在于,所述的sDSS1蛋白是以基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代物、复合体和药物中的任一一种成分中的两种或多种的药物组合。
23.根据权利要求6-21任一所述的应用,其特征在于,所述的sDSS1蛋白是基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代物、复合体和药物中的任一一种成分中的一种、两种或多种与医药上可用的赋形剂形成的药物组合。
24.根据权利要求6-10任一所述的应用,其特征在于,所述的sDSS1蛋白是通过表达体系把编码基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白和融合蛋白中任一蛋白的核苷酸序列导入体内并表达获得的蛋白。
25.根据权利要求24所述的应用,其特征在于,所述的表达体系是真核表达质粒载体、腺病毒、腺相关病毒、慢病毒、逆转录病毒、杆状病毒、疱疹病毒、伪狂犬病毒、ZFN基因编辑技术、TALEN基因编辑技术、CRISPR/Cas基因编辑技术及其他医疗上可用的基因编辑技术或病毒载体。
26.根据权利要求6-10任一所述的应用,其特征在于,所述的sDSS1蛋白是通过移植细胞在个体体内获得的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白和融合蛋白中的任一蛋白。
27.根据权利要求26所述的应用,其特征在于,所述的细胞是任意一种人的干细胞、前体细胞或成体细胞。
28.根据权利要求27所述的应用,其特征在于,所述的干细胞是胚胎干细胞、诱导多能干细胞、转分化得到的细胞,或者来源于原代培养的干细胞、由母细胞分化得到的多能或单能干细胞。
29.根据权利要求6所述的应用,其特征在于,所述的sDSS1蛋白是通过血清、组织间液输注引入个体体内的sDSS1蛋白。
30.根据权利要求6所述的应用,其特征在于,所述的sDSS1蛋白是通过移植组织或器官在个体体内获得的基础蛋白、第一种蛋白、第二种蛋白和第三种蛋白中的任一蛋白。
31.根据权利要求30所述的应用,其特征在于,组织或器官移植中,所述的组织是脑、肝、肾、脾、胰岛的完整器官或部分组织块,或血液、脂肪、肌肉、骨髓、皮肤。
32.根据权利要求1-31任一所述的应用,其特征在于,所述的预防药物是包含基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代蛋白、复合体、药物组合、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品或食品添加剂。
33.根据权利要求1-31所述的应用,其特征在于,所述的治疗药物是包含基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、蛋白修饰物、非天然氨基酸替代蛋白、复合体、药物组合、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品或食品添加剂。
34.一种蛋白在改善糖尿病并发症护理设备中的应用,其特征在于,其是利用权利要求6-17任一所述的应用在制备预防和治疗糖尿病并发症药物中的应用,再将制备的药物用于提高糖尿病并发症护理相关的医疗器械的性能。
35.根据权利要求34所述的蛋白在改善糖尿病并发症护理设备中的应用,其特征在于,所述的医疗器械包括包括输采血设备和耗材、血液净化设备和耗材、血液净化设备辅助装置和耗材、体液处理设备和耗材、肾透析设备和耗材、腹膜透析设备和耗材、血液灌流器、输液器具、注射器具、缓释器具、人工肾脏中的一种或多种。
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