在C.elegans中rBTI延缓衰老及对衰老相关疾病的影响
发布时间：2018-07-17 07:42
【摘要】：荞麦是一种药食同源的小杂粮作物,含有丰富的营养物质及生物活性成分,常食荞麦可降低罹患高血压、高血脂、糖尿病、癌症等风险。荞麦胰蛋白酶抑制剂(buckwheat typsin inhibitor,BTI)是一种提取自荞麦种子的含有69个氨基酸的小分子多肽,分子量为7.9 k D,具有很好的水溶性,属于Potato I型蛋白酶抑制剂家族,特异性抑制胰蛋白酶活性。为了方便BTI的研究和应用,本课题组利用基因克隆技术构建了含有BTI基因的表达质粒p Ex Sec I-BTI经大肠杆菌原核表达和离子交换层析,获得了重组荞麦胰蛋白酶抑制剂(recombinant buckwheat typsin inhibitor,r BTI),r BTI和天然提取BTI具有相同的氨基酸序列及生物活性。课题组先前的研究表明,r BTI对Hep G2,EC9706,K562,HL60等实体瘤细胞具有很好的抑制增殖作用,在Hep G2中r BTI可以通过诱导活性氧(reactive oxygen species,ROS)水平的增加促进线粒体自噬,并且可以将细胞周期阻滞在G0/G1期,使细胞单次分裂周期延长;近期在黑腹果蝇中的实验证明,r BTI可以通过增强黑腹果蝇抗氧化能力延长其寿命,但其具体机制尚不明确。本研究利用经典模式生物秀丽隐杆线虫(caenorhabditis elegans,C.elegans),探究了r BTI对衰老及衰老相关疾病的影响及其作用机制,主要研究内容和结果如下:1、r BTI对C.elegans健康寿命、摄食及运动能力的影响利用五氟尿嘧啶(5-fluoro-2′-deoxyuridine,FUd R)寿命检测法检测r BTI对N2野生型C.elegans寿命的影响,结果显示,和对照组相比,在2.5-10μM的范围内r BTI能明显延长虫体寿命,且具有浓度依赖性;对衰老相关指标检测表明,r BTI明显降低了衰老相关的β-半乳糖苷酶活性及线虫肠道褐脂质积累;同时,显微观察统计结果显示r BTI不会明显影响年轻虫体运动及咽抽速率,但改善了老年虫体的运动能力,表明r BTI延长了C.elegans的健康寿命。2、r BTI延长C.elegans寿命的作用方式采用反相高效液相色谱法、耗氧量检测、荧光探针、酶活检测及胡桃醌诱导氧化应激等方法检测显示,r BTI可以增强线虫体内AMP与ATP的比率,诱导其呼吸作用和ROS产生的增强。增强的ROS可激活虫体内源性抗氧化防御系统的活性,产生反馈保护作用,降低ROS的含量,进而增强虫体抗氧化应激能力。利用ROS抑制剂N-乙酰-L-半胱氨酸(N-Acetyl-L-cysteine,NAC)的检测显示,当ROS信号被抑制后,r BTI失去了延长虫体寿命的能力,表明r BTI延长寿命的作用依赖于瞬时ROS信号产生,该机制和热量限制(calorie restriction,CR)延长寿命的机制非常相似,据此我们认为:r BTI延长寿命的功效可能通过一种热量限制模拟(calorie restriction mimetic,CRM)的方式发挥作用。利用q RT-PCR方法检测显示,r BTI下调了胰岛素/类胰岛素生长因子信号(insulin/IGF-1 signaling,IIS)通路各组分的转录水平;同时转基因C.elegans及q RT-PCR等方法检测显示,r BTI可以增强IIS通路主要转录因子DAF-16的转录活性,增强其靶基因的表达,但不影响IIS通路中另外两个转录因子HSF-1和SKN-1的转录活性。突变体线虫品系寿命检测显示,r BTI不能延长daf-2和daf-16突变体线虫寿命,表明r BTI介导的寿命延长依赖于daf-2和daf-16,即依赖于IIS通路;同时,荧光探针检测显示,在daf-2、daf-16突变体中,r BTI不能诱导瞬时ROS信号产生,但是当用NAC抑制ROS信号产生时,r BTI仍能够下调IIS通路,表明r BTI介导的ROS信号诱导同样依赖于IIS通路。3、r BTI对C.elegans相关代谢的影响与CR对代谢影响相似,q RT-PCR及酶活检测显示,r BTI降低了C.elegans中参与糖酵解过程相关酶活性,增强了虫体脂肪酶活性,同时油红染色结果显示,和对照组相比,r BTI处理组虫体中脂肪积累明显减少,表明r BTI可以诱导C.elegans体内糖代谢产能向脂代谢产能的转变;此外,转基因线虫及q RT-PCR显示,r BTI能够增强C.elegans自噬过程。4、r BTI延长寿命的作用与其本身分子结构的关系根据r BTI的结构特点和活性部位,通过定点突变技术,分别对r BTI的45位、53位和44位氨基酸活性位点进行突变,获得了4种r BTI突变体,分别命名为r BTI-R45A、r BTI-R45F、r BTI-W53R和r BTI-P44T。C.elegans寿命检测实验表明失去胰蛋白酶抑制活性的r BTI-R45A、r BTI-R45F及胰蛋白酶抑制活性显著升高的r BTI-P44T均不同程度失去了延长寿命的功能。同时,转基因线虫荧光显微观察及q RT-PCR显示,四种突变体均不能增强DAF-16的转录活性,表明r BTI延长寿命及增强DAF-16转录活性的功能均依赖于其适当的胰蛋白酶抑制活性,活性位点突变会导致其相应的延长寿命功能减弱或丧失。5、rBTI对衰老相关疾病的影响及其机制阿尔兹海默症(Alzheimer's disease,AD)是最常见的一种神经退行性疾病,其发生发展与衰老密切相关。虽然AD的发病机制还不太清楚,但普遍认为β样淀粉蛋白(β-amyloid,Aβ)诱导的毒性是AD发病的主要原因。研究表明具有延寿效应的物质能够不同程度的保护对抗AD,本研究中利用一种能够表达人类Aβ3-42的AD模型线虫CL4176检测了r BTI对AD的作用,并用western blot、免疫印迹、荧光染色、RNA干扰(RNA interference,RNAi)等方法初步探究了其作用机制,结果显示r BTI能够很好的减缓Aβ毒性诱导的AD线虫僵化速率,促进AD线虫自噬-溶酶体降解途径,降低其体内Aβ积累,但是对蛋白酶体降解途径没有明显影响,且和在N2野生型线虫中作用相似,r BTI保护效应的发挥依赖于转录因子DAF-16。这些数据表明r BTI不仅具有延缓衰老的效应,还是一种潜在的有效治疗AD的天然化合物。此外,在亨廷顿症(Huntington's disease,HD)模型线虫中的实验显示,r BTI同样能够减缓HD模型中多聚谷氨酰胺聚体及线虫的僵化表型,表明除了AD外,r BTI可能对另一衰老相关疾病HD也有有益作用。综上所述,本研究证明,r BTI可以改善老年虫体的运动能力,延长C.elegans的健康寿命;类似于CR延长寿命效应,r BTI可通过下调IIS通路,诱导ROS短暂升高,进而激活DAF-16的转录活性,增强其下游靶基因的表达和抗氧化防御能力,最终使虫体寿命延长;r BTI可诱导线虫体内糖代谢产能向脂代谢产能转变;分别利用转基因线虫和AD模型线虫实验显示,r BTI不仅能增强C.elegans自噬过程,而且能通过促进自噬-溶酶体降解途径,适当对抗AD。这些结果为进一步开展生物活性物质在延缓衰老,对抗老年痴呆等疾病中的作用机理和应用研究奠定了良好的基础。
[Abstract]:In order to facilitate the study and application of BTI , the expression plasmid pEx Sec I - BTI , which contains 69 amino acids from buckwheat seeds , has the same amino acid sequence and bioactivity . The results showed that r BTI could increase the activity of endogenous antioxidant defense system . The results showed that r BTI could increase the activity of the organism ' s endogenous antioxidant defense system . The results showed that r BTI could increase the activity of the organism ' s endogenous antioxidant defense system . The results showed that r BTI could increase the activity of the organism ' s endogenous antioxidant defense system . The results showed that r BTI could prolong the life of the body . The results showed that r BTI could prolong the life of the body . The results showed that r BTI could prolong the life of the worm . The results showed that r BTI could prolong the life of the worm . The results showed that r BTI could enhance the activity of the main transcription factors of the IIS pathway . The results showed that r BTI could not extend the activity of the other two transcription factors HSF - 1 and SKN - 1 . The results showed that r BTI could not prolong the activity of the other two transcription factors HSF - 1 and SKN - 1 . r BTI-W53R鍜宺 BTI-P44T.C.elegans瀵垮懡妫，
本文编号：2129642
[Abstract]:In order to facilitate the study and application of BTI , the expression plasmid pEx Sec I - BTI , which contains 69 amino acids from buckwheat seeds , has the same amino acid sequence and bioactivity . The results showed that r BTI could increase the activity of endogenous antioxidant defense system . The results showed that r BTI could increase the activity of the organism ' s endogenous antioxidant defense system . The results showed that r BTI could increase the activity of the organism ' s endogenous antioxidant defense system . The results showed that r BTI could increase the activity of the organism ' s endogenous antioxidant defense system . The results showed that r BTI could prolong the life of the body . The results showed that r BTI could prolong the life of the body . The results showed that r BTI could prolong the life of the worm . The results showed that r BTI could prolong the life of the worm . The results showed that r BTI could enhance the activity of the main transcription factors of the IIS pathway . The results showed that r BTI could not extend the activity of the other two transcription factors HSF - 1 and SKN - 1 . The results showed that r BTI could not prolong the activity of the other two transcription factors HSF - 1 and SKN - 1 . r BTI-W53R鍜宺 BTI-P44T.C.elegans瀵垮懡妫，
本文编号：2129642
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2129642.html
最近更新
教材专著
