【作者】 陈浩；

【导师】 刘剑峰；

【作者基本信息】 华中科技大学 ， 生物物理学， 2020， 硕士

【摘要】 影响衰老的因素多种多样,了解衰老及其机制有重要的科学意义和实用价值。秀丽隐杆线虫是一种土壤中自由生活的线虫,实验室条件下通常以大肠杆菌OP50为食。秀丽隐杆线虫具有生活周期短、后代数量多以及全基因组测序完成等特点,使其成为一种研究衰老及相关机制的经典模式动物。大量的研究数据发现,温度对衰老进程有着非常重要的影响。我们前期的研究数据表明TRPA-1是线虫体内的一类温度敏感型离子通道受体,以温度差异性的方式调控线虫寿命:高温培养条件下TRPA-1对线虫寿命没有明显的调节作用;而低温条件下肠道和神经元中的TRPA-1被激活,进而通过调控下游信号分子延长线虫寿命。在此,我们的研究发现:TRPA-1调控线虫寿命与其摄取的食物有关,低温下喂食线虫活菌才能激活TRPA-1并延长其寿命,而喂食线虫紫外杀死的细菌,TRPA-1不能明显地促进线虫寿命的延长;而在高温下,无论以活菌为食还是死菌为食,TRPA-1对线虫寿命都没有明显的影响。进一步的研究发现,线虫肠道内的TRPA-1而不是神经元中的TRPA-1介导了这种独特的寿命调控机制。接下来,通过RNA干扰技术,我们发现DBL-1和DAF-16是低温条件下TRPA-1调控线虫寿命所必需的,然而敲低它们并不能消除食物的死活性质对线虫寿命的影响。我们进一步的分析发现,敲低PMK-1、MPK-1、KGB-1和JNK-1等经典的调控线虫免疫信号通路的关键分子并不影响TRPA-1对线虫寿命调控。另外,通过实时荧光定量PCR和线虫活体荧光成像技术,我们证实了因喂食食物的死活不同而引起的线虫体内9种免疫信号相关因子,即IRG-1、IRG-2、LYS-1、CLEC-67、MUL-1、K08D8.5、GST-4、F35E12.5和SOD-3,的表达水平变化并不依赖于TRPA-1对食物性质的响应,这表明相关的免疫信号通路极有可能不是TRPA-1响应食物变化调控线虫寿命所必需的。终上所述,我们直接或者间接证实了上述15个免疫信号相关因子均不是较低温度和食物性质通过TRPA-1调控线虫寿命所必需的;而这15个免疫因子几乎参与了线虫体内所有已知的免疫信号通路,因此我们在此推断:较低温度下食物死活性质依赖的TRPA-1寿命调控过程极有可能是不依赖于线虫体内已知的免疫信号通路。更多还原

【Abstract】 There are a variety of factors affecting aging,and it is of great scientific and practical significance to understand aging and its mechanisms.Caenorhabditis elegans is a soil-free nematode.It usually feeds on Escherichia coli OP50 under the laboratory conditions.Its short life cycle,large number of progeny and completed whole genome sequence make C.elegans a classic model animal for studying aging and its mechanisms.It has been reported that temperature has an important effect on aging.Our previous studies have shown that TRPA-1 is a temperature-sensitive receptor in C.elegans,which regulates the lifespan of C.elegans in a temperature dependent manner.TRPA-1 does not have an obvious effect on C.elegans lifespan at high temperatures;while TRPA-1 in the intestine and neurons are activated to extend the lifespan of C.elegans at low temperatures through downstream signaling factors.Here,our research found that TRPA-1 regulated the lifespan of C.elegans,which was related to the food they ingest.Feeding C.elegans with live bacteria at low temperature could activate TRPA-1 and prolong its lifespan,while TRPA-1 could not significantly prolong the lifespan of C.elegans fed with UV-killed bacteria.In addition,we also found that TRPA-1 was not able to change the lifespan of C.elegans on either live bacteria or UV-killed bacteria at high temperature.Further studies found that intestinal TRPA-1,but not neuronal TRPA-1,was involved in this unique longevity regulation process.Next,through RNA interference technology,we found that DBL-1 and DAF-16 participated in the life regulation of TRPA-1 in response to different temperature,but not to different diet(live vs dead diet).Our further analysis found that knocking down some key molecules of classic immune signaling pathways,such as PMK-1,MPK-1,KGB-1 and JNK-1,did not affect the longevity modulation by TRPA-1.In addition,via real-time quantitative PCR and in vivo fluorescent microscopy imaging,we figured out that the expression level changes of 9 immune signal-related factors(IRG-1,IRG-2,LYS-1,CLEC-67,MUL-1,K08D8.5,GST-4,F35E12.5 and SOD-3)in response to different diet(live vs dead diet)did not dependent on TRPA-1,which indicated that the relevant immune signaling pathways were most likely not necessary for longevity modulation in response to different diet by TRPA-1.Thus,we had found that those 15 immune factors were not required for TRPA-1 to modulate the lifespan of C.elegans in response to different diet(live vs dead diet).As those 15 immune factors are involved in almost all known immune signaling pathways in C.elegans,so we could draw a conclusion: known immune response signaling pathways/factors are not required for TRPA-1 to regulate the lifespan of C.elegans in diet(live or dead)dependent manner at low temperature.更多还原