【作者】 周琳；

【导师】 付学奇；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2019， 博士

【摘要】 低分子量蛋白质酪氨酸磷酸酶(low molecular weight protein tyrosine phosphatase,LMW-PTP)是一种结构不典型的蛋白质酪氨酸磷酸酶(protein tyrosine phosphatases,PTPs),在PTPs家族中被单独分成一类,进化上高度保守,广泛表达于多种生物中,包括植物、原核生物和古细菌等。天然产物在自然界中含量丰富,是重要的药物宝库,在药物开发领域得到了广泛的研究。秀丽线虫(Caenorhabditis elegans,C.elegans)是一种优秀的模式生物,具有繁殖能力强、生命周期短、虫体小易操作、通体透明便于观察、可在液氮中长期冻存、细胞谱系已完全确定等优点。同时,线虫有许多在进化上高度保守的信号通路,与人类基因同源性达60%~80%。线虫lmwptp基因与人类acp1基因同源,经计算机结构模拟得知两者蛋白质三级结构极为相似,因此推测两者生物学功能及作用机制具有相似之处。应激反应是生物体对抗外界刺激所发生反应的总称,随着自然环境的恶化及生活节奏的加快,人们受到体内外刺激的种类和频率逐渐增多,这些刺激会破坏机体氧化和抗氧化系统之间的平衡,影响生物体的日常行为和健康,导致多种疾病的发生,因此生物抗应激的预防及治疗日益受到重视。本文应用线虫模型研究了LMW-PTP在应激反应中的作用机制,并对抗应激天然产物进行了筛选。首先,本文成功构建了用于线虫lmwptp RNAi的pPD129.36-lmwptp质粒并诱导表达,通过免疫家兔成功制备出线虫LMW-PTP多克隆抗体,通过qPCR和Western Blot两种方法证明了lmwptp RNAi的有效性。其次,本文检测了lmwptp RNAi对线虫一般生理指标的影响,发现lmwptp RNAi不影响线虫的寿命、产卵数、吞咽频率、体长,但lmwptp RNAi后第7天的线虫出现了一种特殊运动状态,线虫除了在培养基平面上做正弦曲线运动外,还在垂直方向“抬头”和/或“翘尾”;lmwptp RNAi后线虫体内脂肪含量明显增多,这一现象的产生与lmwptp RNAi后降低一些脂肪分解基因如acdh-1、acs-2的表达量和升高一些脂肪合成基因如fat-6、mdt-15、pod-2、elo-5的表达量有关;并且lmwptp RNAi明显提高了线虫的应激抵抗性,包括热应激、紫外辐射应激和氧化应激抵抗性。然后,本文进一步探究了LMW-PTP在热应激反应中的作用机制,发现lmwptp RNAi可降低线虫体内活性氧簇(reactive oxygen specie,ROS)水平,提高线虫体内超氧化物歧化酶(superoxide dismutase,SOD)活性,减少线虫体内脂褐素累积量,LMW-PTP负调控线虫应激抵抗性的作用与类胰岛素信号通路有关,LMW-PTP可能作用于DAF-2,进而改变类胰岛素信号通路下游转录因子DAF-16和HSF-1的细胞核转位及两者下游靶基因的表达量。接下来,本文应用线虫模型对50种天然产物的抗紫外辐射功能进行了筛选,由于薄荷水提物抗紫外辐射的作用效果稳定,因此本文进一步探究了薄荷水提物提高线虫紫外辐射抵抗性的原因,发现薄荷水提物可以降低紫外辐射后线虫体内ROS含量和提高SOD活性,并且这种作用与类胰岛素信号通路有关,薄荷水提物可以促进DAF-16细胞核转位并增加其下游靶基因sod-3、hsp-12.6、hsp-16.1、hsp-16.49的表达量。最后,本文探究了薄荷中的水溶性成分香蜂草苷在线虫抵抗紫外辐射应激过程中的作用和机制,发现了0.1 mM香蜂草苷可以延长线虫寿命,提高线虫抵抗紫外辐射应激、35°C热应激、胡桃醌氧化应激的能力,并且可以降低紫外辐射后线虫体内ROS水平并且提高SOD活性,其提高线虫紫外辐射应激抵抗性的作用与类胰岛素信号通路有关,香蜂草苷可能作用于该通路上游的DAF-2来发挥作用,进而促进DAF-16的细胞核转位,增加下游基因sod-3和hsp-16.2的表达量。综上所述,本文应用线虫模型研究了低分子量蛋白质酪氨酸磷酸酶的生物学功能,着重研究了其在应激反应中的作用机制,并对50种天然产物水提物的抗紫外辐射功能进行了筛选,着重研究了薄荷水提物及其水溶性成分香蜂草苷提高线虫紫外辐射抵抗性的作用机制。本文不仅发现了PTPs家族的新功能,还为抗应激药物的设计和开发提供了理论依据,具有一定的市场前景和社会意义。更多还原

【Abstract】 Low molecular weight protein tyrosine phosphatase(LMW-PTP)is a structural dysfunctionally protein tyrosine phosphatases(PTPs),which is separately divided into one type of PTPs family.LMW-PTP is highly conserved in evolution and exists in almost all organisms,including plants,prokaryotes,and archaea.Natural products are abundant in nature.They are important drug storehouses and have been extensively studied in the field of drug development.Nematode is an excellent model organism with strong reproductive ability,short life cycle,small body and easy to operate,transparent body and easy to observe,long-term cryopreservation in liquid nitrogen,and complete determination of cell lineage.At the same time,C.elegans are highly conserved in many important signaling pathways,with 60% to 80% homology with human genes.The C.elegans’ lmwptp gene is homologous to human acp1 gene.The computer structure simulation shows that the tertiary structure of the two proteins is very similar,so it is speculated that the biological functions and action mechanisms of the two have similarities.Stress response is a general term for the response of organisms to external stimuli.With the deterioration of natural environment and the acceleration of life rhythm,the types and frequencies of stimuli in vivo and in vitro are gradually increasing.These stimuli will destroy the balance between oxidation and antioxidant systems,affect the daily behavior and health of organisms,and lead to the occurrence of many diseases.Therefore,the prevention and treatment of biological anti-stress has been paid more and more attention.This paper used C.elegans model to study the action mechanism of LMW-PTP on stress response,and the anti-stress natural products were screened.Firstly,the pPD129.36-lmwptp plasmid for C.elegans’ lmwptp RNAi was successfully constructed and induced to express,and the LMW-PTP polyclonal antibody was successfully prepared by immunizing rabbits.The validity of lmwptp RNAi was confirmed by qPCR and Western Blot.Secondly,we examined the effect of lmwptp RNAi on the general physiological indexes of C.elegans.It was found that lmwptp RNAi did not affect the lifespan,spawning number,swallowing rate,body length of C.elegans,but the C.elegans on the 7th day after lmwptp RNAi showed a special state of motion.In addition to sinusoidal motion on the medium plane,C.elegans also exhibited “head-up” and/or “tail-up” motion in the vertical direction.We also found that the fat content of C.elegans increased significantly after lmwptp RNAi.This phenomenon was related to reducing the expression of some lipolytic genes,such as acdh-1,acs-2 and increasing the expression of some fat synthesis genes,such as fat-6,mdt-15,pod-2,elo-5 in the C.elegans after lmwptp RNAi.We further found that lmwptp RNAi significantly increased the stress resistance of C.elegans,including 35°C heat shock stress resistance,UV irradiation stress resistance,oxidation stress resistance.Then,we further explored action mechanism of LMW-PTP on 35°C heat stress.lmwptp RNAi was found to reduce ROS levels,increase SOD activity and reduce the accumulation of lipofuscin in C.elegans.LMW-PTP negatively regulated the stress resistance of C.elegans through insulin/IGF-1-like signaling pathway.LMW-PTP might act on DAF-2 to further change the nuclear translocalization of transcription factors DAF-16 and HSF-1 and the expression of their downstream target genes.Next,we selected 50 kinds of natural products to screen for anti-UV irradiation using C.elegans model.Because the effect of mint aqueous extract on UV irradiation resistance was stable,we further explored the reason why mint aqueous extract enhanced the UV irradiation resistance of C.elegans.We found that mint aqueous extract could reduce the ROS levels and increase SOD activity in C.elegans after UV irradiation,and this effect was related to the insulin/IGF-1-like signaling pathway.Mint aqueous extract could promote the nuclear translocalization of DAF-16 and promote the expression levels of its downstream target genes sod-3,hsp-12.6,hsp-16.1,hsp-16.49.Finally,we explored the role and mechanism of the aqueous component of mint,didymin,in the process of UV irradiation stress resistance.We found that 0.1 mM didymin could prolong the lifespan of C.elegans and improve the resistance of C.elegans to UV irradiation,35°C heat shock and juglone oxidative stress.Didymin could reduce ROS levels and increase SOD activity after UV irradiation.The effect of anti-UV irradiation of didymin in C.elegans was related to the insulin/IGF-1-like signaling pathway and might act on DAF-2 upstream of the pathway.Thereby,the nuclear translocalization of DAF-16 was promoted and the expression levels of its downstream genes sod-3 and hsp-16.2 were increased.In summary,we studied the biological function of LMW-PTP by using C.elegans model and explored its action mechanism in stress response.We also screened the anti-UV irradiation function of 50 kinds of natural products aqueous extract by using C.elegans model and focused on the action mechanism of mint aqueous extract and its aqueous component didymin to improve UV irradiation resistance of C.elegans.We not only discovered new functions of the PTPs family,but also provided a theoretical basis for the development of drugs for stress-related diseases.It had certain market prospects and social significance.更多还原