CN109512824B - 刃天青在作为牛胰岛素淀粉样纤维抑制剂方面的应用 - Google Patents
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Abstract
刃天青在作为牛胰岛素淀粉样纤维抑制剂方面的应用,属于生物医学技术领域。本发明公开了在制备牛胰岛素淀粉样的过程中加入不同浓度的刃天青后,再加入ThT检测荧光强度,荧光光谱图显示随着刃天青浓度的增加,荧光强度依次降低。圆二色谱图显示加入刃天青的牛胰岛素的二级结构出现β折叠的时间出现延迟。原子力照得的形貌图同样显示刃天青的加入使牛胰岛素停留在成核时期,即刃天青的的加入明显抑制了牛胰岛素淀粉样纤维的形成。
Description
技术领域
本发明属于生物医学技术领域,具体涉及刃天青在作为牛胰岛素淀粉样纤维抑制剂方面的应用。
背景技术
无论是在生物医学领域还是在化学领域,淀粉样纤维的研究都处于至关重要的位置。因为越来越多的疾病的致病原因直指淀粉样纤维,如阿尔兹海默症、海绵状脑病、帕金森亨廷顿病、心律失常、II型糖尿病、动脉粥样硬化、类风湿性关节炎、泌乳素瘤和多发性精神病等。但目前市面上治疗这类疾病的药物基本没有,所以开发有效且对人体副作用小的淀粉样纤维抑制剂,对生物医学有非常重要的意义。
发明内容
刃天青,又名偶氮间苯四酚,为已知结构化合物,其结构式如下所示,刃天青可以与牛胰岛素自发结合,在作为牛胰岛素淀粉样纤维抑制剂方面得到应用。
附图说明
图1:分别加入0mM(曲线1)、0.05mM(曲线2)、0.1mM(曲线3)、 0.2mM(曲线4)刃天青溶液的牛胰岛素在25mM、NaCl/HCl(pH=1.6),65℃条件下孵育240min后加入ThT的荧光强度曲线,用来检测纤维的形成状况;
图2:分别加入0mM(曲线1)、0.05mM(曲线2)、0.1mM(曲线3)、 0.2mM(曲线4)刃天青溶液的牛胰岛素在25mM、NaCl/HCl(pH=1.6),65℃条件下孵育,取不同时间段的样品加入THT检测牛胰岛素淀粉样纤维形成过程的荧光光谱动力学图;
图3:分别加入0mM(图A)、0.05mM(图B)、0.1mM(图C)、0.2mM (图D)刃天青溶液的牛胰岛素在pH=1.6NaCl/HCl(25mM),65℃条件下孵育60min(曲线1)、150min(曲线2)、240min(曲线3)、420min(曲线4) 的圆二色谱图(纵坐标表示椭圆率,横坐标表示波长);
图4:不存在和存在不同浓度刃天青溶液的牛胰岛素在pH=1.6NaCl/HCl(25 mM),65℃条件下孵育120min、240min和360min的原子力图。
具体实施方式
实施例1:
PBS缓冲液配方:19mL、0.2M NaH2PO4水溶液和81mL、0.2M Na2HPO4水溶液的混合。
25mM NaCl/HCl溶液配方:用HCl调节NaCl水溶液的pH=1.6,NaCl水溶液中NaCl的浓度为25mM。
配制不同浓度的刃天青溶液(溶剂为超纯水):0mM、0.05mM、0.1mM、 0.2mM。
将牛胰岛素加入到0.95mL NaCl/HCl(25mM,pH=1.6)溶液中,牛胰岛素的浓度为0.5mM,分别加入不同浓度的刃天青溶液0.05mL,在65℃的条件下孵育240min。各取样品溶液0.05mL,再加入0.05mL的ThT(1mM,thioflavin,硫代磺素T)溶液,最后用0.9mL的PBS缓冲溶液稀释到1mL,将上述稀释后样品在荧光光谱仪中测试其荧光强度(λex=440nm),荧光光谱图显示随着刃天青浓度的增加,荧光强度依次减弱,如图1所示。由于荧光强度的明显性差异,表明刃天青可以抑制牛胰岛素纤维化。
实施例2:
由于有关淀粉样纤维有关疾病的机理仍在探索中,而淀粉样纤维的动力学过程可能会为机理提供相关信息,因此,用ThT监测牛胰岛素淀粉样纤维的形成过程中加入刃天青。将牛胰岛素溶解在0.95mL NaCl/HCl(25mM,pH=1.6)溶液中,牛胰岛素的浓度为0.5mM,分别加入不同浓度的刃天青溶液0.05mL,在 65℃下纤维化。在不同时间点各取样0.05mL,再加入0.05mL ThT(1mM)溶液,最后用0.9mL的PBS缓冲溶液稀释到1mL,每组样品做3个平行样,将上述所有样品在荧光光谱仪上测定其荧光强度。可以发现,随着刃天青浓度的增加,不仅荧光强度逐渐降低,出现纤维的时间也逐渐推迟,如图2所示。牛胰岛素纤维化的动力学过程,进一步表明刃天青明显的抑制作用。
实施例3:
取牛胰岛素固体溶解在0.95mL pH=1.6、25mM NaCl/HCl溶液中,样品浓度为0.5mM,分别加入0.05mL不同浓度的刃天青溶液,终体积为1mL,在温度为65℃,pH=1.6的条件下进行纤维化。在60min、150min,240min,420min 的时间点各取0.05mL样品,用PBS缓冲溶液稀释至1mL,牛胰岛素的测定浓度为0.025mM。然后用圆二色谱仪测其圆二色谱,在195~260nm的范围内采集光谱,所用石英比色皿的路径长度为1mm,步长为1,并用PBS缓冲溶液扣除背景。结果由摩尔椭圆率表示,由如下公式计算:
[θ]λ=θ(M/10dcn)
[θ]λ(deg.cm2.dmol-1)为蛋白的摩尔椭圆率;θ为直接测得的蛋白椭圆率; M(g.mol-1)为蛋白的摩尔质量;c(g.L-1)为蛋白的浓度;d(cm)为比色皿的厚度;n为蛋白的氨基酸残基数。
如图3所示,随着刃天青浓度增加,牛胰岛素纤维化后二级结构变化的时间延长,并且纤维化后β折叠的程度也减小,再次验证刃天青的加入对牛胰岛素纤维化有抑制作用。
实施例4:
为了更加直观的观察刃天青对牛胰岛素纤维化过程的抑制作用,采用原子力显微镜观察纤维化过程中样品的形貌,在120min、240min、360min各取0.005 mL样品,用超纯水稀释至1mL,在处理过后的硅片(将硅片用过氧化氢和浓硫酸(体积比为3:7)处理,再用超纯水冲洗)上涂满、晾干,在室温空气中采集 AFM图像,其中测量模式为tapping mode,悬臂梁的共振频率为170kHz,典型力常数为7.5n/M。
如图4所示,图A-图C可以看出两小时牛胰岛素已经开始形成纤维,四小时和六小时的纤维已经较为成熟;图D-图F为同等条件下,用50μM刃天青抑制牛胰岛素纤维化后制得的样品,可以看出2-4小时无纤维存在,只有点状物的存在,6小时样品中出现了少量的纤维;图G-图I,图J-图L分别为用100μ M和200μM刃天青抑制牛胰岛素纤维化的样品,在六小时内样品都以点状物的形式存在,无纤维出现。图中的点状物的状态为成纤维前的的成核状态,从图3 的系列原子力显微镜图中可以看出,随着刃天青浓度的增大,样品出现纤维的时间延长,出现纤维的量也大幅度减少,可得出结论:刃天青对牛胰岛素纤维化过程有抑制作用,且作用在了其纤维化的成核期。
Claims (1)
1.刃天青在制备体外抑制牛胰岛素淀粉样纤维的试剂中的应用,所述试剂的pH为1.6。
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