CN113521255A - Iapp-ft在制备胰岛淀粉样多肽聚集和纤维化抑制剂中的应用 - Google Patents
Iapp-ft在制备胰岛淀粉样多肽聚集和纤维化抑制剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种胰岛淀粉样多肽类似物IAPP‑FT在制备IAPP淀粉样聚集和纤维化抑制剂中的应用。本发明基于IAPP中C端酰胺化对其淀粉样聚集和纤维化的重要性，设计了C端去酰胺化的IAPP类似物，即IAPP‑FT，并考察其作为潜在抑制剂，对IAPP聚集、纤维化和细胞毒性的抑制作用。首先，经TEM和ThT法检测表明所设计的类似物IAPP‑FT自身确无聚集能力，同时不会生成成熟的纤维。随后将该类似物与IAPP混合后，可有效抑制IAPP的聚集和纤维化，同时显著降低由寡聚体引起的细胞毒性，对发现和设计抑制IAPP聚集及由其引起的生理毒性的药物具有重要意义。
Description
技术领域
本发明属于生物医药技术，具体涉及一种IAPP-FT在制备胰岛淀粉样多肽聚集和纤维化抑制剂中的应用。
背景技术
许多疾病的发生发展与蛋白质的淀粉样聚集有关，例如：阿尔兹海默症，帕金森氏症等神经退行性疾病以及II型糖尿病等疾病。人胰岛淀粉样多肽(human islet amyloidpolypeptide,hIAPP)的淀粉样聚集和纤维生成与II型糖尿病的发生发展密切相关。在病理情况下，IAPP会发生错误折叠和聚集，在胰岛内形成的沉积会引起胰岛β细胞功能紊乱，同时通过诱导细胞内氧化应激以及线粒体损伤等方式引起胰岛β细胞的凋亡，加重II型糖尿病的病程。因此，针对抑制或延缓IAPP淀粉样聚集和纤维化的抑制剂开发对II型糖尿病的治疗具有重要意义。目前针对IAPP淀粉样聚集抑制剂的开发已取得重要进展，包括小分子抑制剂以及淀粉样蛋白抗体的应用等。此外，基于IAPP氨基酸序列或翻译后修饰的淀粉样蛋白肽段类似物的设计和研究也受到广泛关注。
发明内容
发明目的：针对上述现有技术，本发明提供了IAPP-FT在制备胰岛淀粉样多肽聚集和纤维化抑制剂中的应用。
技术方案：本发明基于IAPP序列C端酰胺化修饰对IAPP淀粉样聚集、纤维化及细胞毒性的影响，提出C端去酰胺化修饰的IAPP类似物(Free C-Terminal IAPP,IAPP-FT)作为抑制胰岛淀粉样多肽IAPP聚集和纤维化的潜在抑制剂的应用。
IAPP-FT与IAPP序列信息如下：
本发明类似物IAPP-FT与IAPP混合后可有效抑制IAPP的聚集和纤维化，同时显著降低由寡聚体引起的细胞毒性，可作为抑制IAPP聚集和纤维化的潜在抑制剂。在体外实验中，与IAPP混合比例为1:1时可延缓寡聚体生成，当混合比例达1:5时可有效抑制纤维生长。
所述IAPP-FT在制备治疗II型糖尿病的药物中的应用也在本发明的保护范围内。
与IAPP相比，IAPP-FT自身不形成淀粉样聚集，无成纤维能力，且对胰岛β细胞的细胞毒性显著降低。将其作为抑制剂与IAPP混合后，可延缓IAPP的聚集速度，无明显纤维生成，同时可显著降低由IAPP寡聚体引起的细胞毒作用，从而保护胰岛功能。
有益效果：相比较于现有技术，本发明提供了C端去酰胺化修饰的IAPP类似物IAPP-FT作为IAPP聚集和纤维化抑制剂的应用，其与IAPP混合后可有效抑制IAPP的聚集和寡聚体引起的细胞毒性，可作为IAPP聚集及纤维化的潜在抑制剂，可用于开发治疗II型糖尿病药物。
附图说明
图1为透射电子显微镜TEM对纤维形貌的表征；
图2为ThT法对IAPP，IAPP-FT及两者混合物聚集动力学的表征；
图3为IAPP，IAPP-FT及两者混合物与细胞共孵育24小时后，MTT法测试细胞活力结果图。
具体实施方式
下面结合具体实施例对本申请作出详细说明。
本发明中所用IAPP-FT的设计参考文献Chem.Commun.,2013,49,1799，委托南京肽业有限公司合成，纯度为98％。具体合成步骤如下：
1.选Fmoc-Tyr(tbu)-Wang-Resin树脂用二氯甲烷(DCM)溶胀半小时。
2.N，N-二甲基甲酰胺(DMF)洗三遍，哌啶(DBLK)去保护15分钟，DMF洗六遍。
3.偶联Fmoc-Thr(tbu)-OH反应半小时。
4.以溶液亮黄，树脂透明为检测合格，随后重复步骤2。
5.偶联Fmoc-Asn(Trt)-OH反应半小时，检测合格(溶液亮黄，树脂淡黄)后重复步骤2。
6.偶联Fmoc-Ser(tbu)-OH，反应半小时检测合格(溶液亮黄，树脂透明)后重复步骤2。
7.偶联至最后一个氨基酸Fmoc-Lys(Boc)-OH。
8.用DMF,DCM和甲醇各洗3遍收缩。
9.切割后空气氧化后进行纯化。
实施例1
采用TEM表征IAPP和IAPP-FT的纤维生成，用于考察两者的成纤维能力。
IAPP样品制备：精密称量IAPP粉末，溶于HFIP溶液中，母液浓度为2mM，超声5min使其溶解。将一定量的IAPP母液挥干，用25mM PBS缓冲液(含有50mM NaCl,pH 7.4)复溶至终浓度100μM，孵育两天。
IAPP-FT样品制备：与上述IAPP样品制备方法相同。
IAPP和IAPP-FT混合物制备：将IAPP和IAPP-FT的母液以1：5的体积比混合，挥干，用25mM PBS缓冲液(含有50mM NaCl,pH 7.4)复溶至IAPP终浓度100μM，孵育两天。
电镜样品制备：待测试溶液20μL滴加到超薄镀碳铜网表面，静置5min，将剩余液体吸走。在沉积样品的铜网表面滴20μL 2％磷钨酸铵水溶液，沉积5min，将剩余液体吸走。制备好的样品干燥2min，利用TEM(日立HT7700)成像。
TEM可用于表征IAPP和IAPP-FT的纤维形成。如图1A为培养2天后IAPP所形成纤维的形貌，所示(标尺为2μm)，孵育两天后的IAPP溶液中已经产生大量的成熟纤维，具有典型淀粉样纤维的长直线型形貌。相反，如图1B为培养2天后IAPP-FT所得无定型聚合物的形貌，在IAPP-FT样本中并未观察到具有相同形貌的成熟纤维，而视野中的球状物质推测为样本制备后部分蛋白颗粒沉积形成。由此可确证IAPP-FT无IAPP的淀粉样聚集和成纤维能力。在此基础上，将IAPP-FT与IAPP混合孵育后，结果如图1C所示。在视野范围内未观察到典型纤维或沉淀的生成，表明所述类似物IAPP-FT能显著抑制IAPP的纤维化。
实施例2
基于ThT法，采用荧光分光光度计对IAPP，IAPP-FT及两者混合物聚集动力学的表征。
IAPP样品制备：精密称量IAPP粉末，溶于HFIP溶液中，超声5min使其溶解，将多肽的HFIP溶液用25mM PBS缓冲液(含有50mM NaCl,pH 7.4)稀释至终浓度5μM，HFIP含量为1％(v/v)。
IAPP-FT样品制备：与上述IAPP样品制备方法相同。
IAPP和IAPP-FT混合物制备：配制2倍浓度的IAPP和IAPP-FT的HFIP母液，分别取出等体积的溶液混合后，用25mM PBS缓冲液(含有50mM NaCl,pH 7.4)稀释至溶液中IAPP和IAPP-FT的终浓度均为5μM，HFIP含量为1％。
ThT检测方法：分别取1980μL上述各组待测溶液置于石英比色皿中，再加入20μLThT储液，吹打均匀。在Perkin Elmer 6500荧光分光光度计中，以450nm作为激发波长，收集486nm处的发射光信号，每30min采集一次荧光值。ThT法基于ThT染料与溶液中β-折叠结构特异性结合后产生荧光，且荧光强度与溶液中β-折叠含量成正比的原理，可对IAPP聚集动力学进行表征。
通过对所采集的不同时间点数据进行Boltzmann拟合分析，结果如图2所示。对于IAPP组(图2A)，荧光值随时间呈现“S”型曲线，延迟期约为8小时，符合典型纤维生长动力学特征。而对于IAPP-FT组(图2A)，在相同时间内荧光强度没有显著变化，表明溶液中无大量β-折叠结构生成，进一步确证IAPP-FT自身无聚集和成纤维能力。在此基础上，将IAPP-FT与IAPP共孵育后所得曲线(图2B)，延迟期延长为11小时。上述结果表明，IAPP-FT与IAPP混合后可延缓IAPP聚合物的生成速度，通过影响寡聚体的形成影响IAPP纤维化过程，具有抑制作用。
实施例3
基于MTT法对IAPP，IAPP-FT及两者混合物的细胞毒性检测。
样品制备：精密称量IAPP和IAPP-FT粉末，用HFIP溶解至相同浓度并分散过夜，取等体积的IAPP，IAPP-FT的HFIP溶液分别至1.5mL EP管中。另取相同体积的两种多肽溶液混合于1.5mL EP管中，制成IAPP和IAPP-FT的混合溶液。随后将样品中的HFIP挥干，并用不含血清的1640培养基复溶，使每管中多肽终浓度均为30μM。
细胞培养：收集处于对数生长期的大鼠胰岛瘤RIN-m5F细胞(广州吉妮欧生物科技有限公司)，以新鲜完全培养基重悬后，将细胞悬液稀释至合适的浓度，铺于96孔板中，使细胞密度约为每孔3000个。随后将96孔板置于37℃，5％二氧化碳培养箱中，培养24小时后去除培养基，并重新加入上述配置的各多肽溶液继续培养细胞。设置6个复孔，同时以不含血清的1640培养基培养的细胞为对照组。
MTT法：给药24小时后，在避光的条件下，向每孔中加入10μL 5mg/mL新鲜配制的噻唑蓝(MTT)溶液(溶解于无菌的PBS溶液)，继续培养4小时。终止培养并除去培养基，每孔加入100μL DMSO，置摇床上低速震荡10min。待结晶物完全溶解后，用酶标仪下检测吸光度，波长为490nm。以正常组的细胞存活为100％，计算给药组的细胞存活率。
MTT的结果如图3所示，结果显示，RIN-m5F细胞与IAPP孵育24小时后存活率只有68％，而与IAPP-FT孵育后细胞存活率为84％，表明IAPP-FT本身对细胞毒性较低。而当细胞与IAPP和IAPP-FT的混合物孵育24小时后，存活率上升至90％，存活率得到了显著的提高。此外，当细胞同时与IAPP和IAPP-FT一起孵育时，其存活率与对照组无显著性差异，表明IAPP-FT的加入可显著降低由IAPP寡聚体引起的细胞毒性。
Claims (4)
1.IAPP-FT在制备胰岛淀粉样多肽聚集抑制剂中的应用。
2.IAPP-FT在制备胰岛淀粉样多肽纤维化抑制剂中的应用。
3.IAPP-FT在制备降低由胰岛淀粉样多肽寡聚体引起的细胞毒性的药物中的应用。
4.IAPP-FT在制备治疗II型糖尿病的药物中的应用。
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