【作者】 姜莹；

【导师】 朴丰源；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 研究目的与背景：有机磷化合物致迟发性神经病（organophosphorus easter-induced delayedneuropathy，OPIDN）是指某些有机磷化合物经一次性大剂量接触或者反复多次接触，经过7-14天（或更长）的潜伏期出现的，以肢体感觉异常、运动障碍、共济失调、麻痹甚至瘫痪为主要临床特征的一类后果严重的神经退行性疾病。磷酸三邻甲苯酯（tri-o-cresyl phosphate，TOCP）被广泛用作增塑剂，软化剂，阻燃剂及机油添加剂，因为其烷基位于苯环的邻位上，是磷酸三甲苯酯三个异构体中唯一可以诱发OPIDN的化合物。目前，因为OPIDN发生确切机制不清楚而尚无有效地治疗方法，因此OPIDN的发生机制得到了广泛的关注。有机磷化合物染毒早期后除了诱发迟发神经病之外，还会表现出急性胆碱能危象和中间综合征，和以上三种病变发生机制相关的蛋白在TOCP染毒早期均会出现异常表达，很难区分表达改变的蛋白中哪些与OPIDN发生有关。国内外研究已证明在TOCP染毒前皮下注射苯甲基磺酰氟（Phenylmethanesulfonyl fluoride，PMSF）可以有效的预防OPIDN发生，因此我们推测在TOCP染毒组表达发生改变，但同时在PMSF干预组表达没有发生改变的蛋白可能与OPIDN发生机制有关。为了筛选出与OPIDN发生发展机制相关的蛋白从而帮助科学家们进一步研究OPIDN的分子机制，本次实验利用PMSF特性，使用蛋白质双向电泳结合质谱分析技术通过比较TOCP染毒组，PMSF干预组及对照组中蛋白表达筛选OPIDN相关蛋白；在筛选出的差异表达蛋白中，由于谷氨酰胺合成酶（glutamine synthetase，GS）在调控谷氨酸代谢中起到关键作用，进而影响细胞内钙离子浓度，引起我们极大兴趣，我们对GS的表达进行验证，并检测另一关键酶GLS基因和蛋白的表达水平。同时也对GS活性，细胞外谷氨酸和谷氨酰胺浓度及细胞内钙离子浓度进行了测定，以期了解TOCP染毒后，GS的表达是否影响其所调控的谷氨酸/谷氨酰胺代谢，并进一步影响细胞内钙离子浓度。研究方法：1、TOCP染毒诱发鸡OPIDN模型的制备成年罗曼母鸡随机分为TOCP染毒组，PMSF干预组和对照组。其中TOCP染毒组浓度分别为1000mg/kg和750mg/kg，经灌胃一次性给予实验鸡；PMSF干预组先将PMSF按浓度40mg/kg给鸡皮下注射，24小时后，再经灌胃一次性给鸡1000mg/kg的TOCP；而对照组则给予等量安慰剂。2、OPIDN症状观察及评分使用6分制来评价OPIDN临床症状。3、OPIDN期间神经组织超微结构的观察在染毒第5天和第21天分别断头处死鸡（3只/组/时间点），制备电镜切片并电镜下观察，拍照。4、蛋白质双向电泳及质谱分析5、生物信息学分析将质谱分析鉴定出来的与OPIDN发生机制相关的蛋白质按NCBI蛋白质数据库序列号输入到DAVID网站进行Gene Ontology富集分析和pathway分析。6、筛选蛋白基因水平验证及对GLS基因表达测定：Real-time RT-PCR技术对蛋白质双向电泳筛选出来的OPIDN相关的差异表达蛋白在基因水平进行验证并对GLS基因表达进行检测。7、GS和GLS蛋白表达测定ELISA技术对TOCP染毒鸡脑及脊髓组织GS和GLS蛋白表达进行检测。8、谷氨酰胺合成酶活性及谷氨酸与谷氨酰胺浓度测定取鸡脑及脊髓组织制备匀浆，分别应用相应的试剂盒进行测定。9、细胞内钙离子浓度测定取鸡脑及脊髓组织制备细胞悬液，利用Fluo3-AM检测细胞内钙离子浓度。研究结果：1、TOCP染毒后鸡行为学改变：临床症状随着时间的延长而渐进性加重，最先出现轻微步履蹒跚，随后染毒组鸡出现下肢无力，行动迟缓现象，并进一步加重为下肢震颤，活动时肢体关节经常突然打弯和臀部着地。而PMSF干预组鸡与对照组鸡实验期间则无任何OPIDN异常表现。2、OPIDN期间神经组织超微结构的改变TOCP组染毒5天后，发现个别线粒体有轻微肿胀，有轴突内微丝微管排列怀疑发生改变，但是其他细胞器没有明显异常改变。TOCP组染毒21天后，脑和脊髓里神经元细胞退行性改变明显，内质网肿胀，线粒体异常改变，细胞骨架排列紊乱，髓鞘板层分离，疏松不紧密并有气泡样改变。3、蛋白质双向电泳结合质谱分析对OPIDN相关蛋白筛选结果（1）蛋白质双向电泳筛选OPIDN机制相关蛋白质点在染毒5天鸡脑中，表达改变的OPIDN相关蛋白质点为102个，其中上调63个，下调为39个。而染毒21天时，表达改变的OPIDN机制相关蛋白质点为150个，其中上调81个，下调为69个。在染毒5天鸡脊髓中，表达改变的OPIDN相关蛋白质点为43个，其中上调24个，下调为19个。而染毒21天时，表达改变的OPIDN机制相关蛋白质点为86个，其中上调46个，下调为40个。（2）质谱分析鉴定鸡神经系统OPIDN相关蛋白在鸡脑中共鉴定蛋白10个，表达上调的蛋白分别是：EIF5A2，DSTN，PSMA1和CKB。表达下调的蛋白分别是：GS，GST1，LDHB，HOMER1，VDAC2和Hsc70。在鸡脊髓中共鉴定蛋白10个，表达上调的蛋白是NEFM。表达下调的蛋白分别是：HSPB1，LMW-PTP，ETFa，STMN1，MDH1，THAP5，MBP，SNCA和VAT1。（3）Gene Ontology和pathway分析结果通过GO富集分析发现质谱鉴定出的蛋白主要定位在胞浆（GO：0005737）及线粒体（GO：0005739）中，参与的生物过程和分子功能主要有蛋白分解调控（GO：0043244），细胞骨架组合调控（GO：0051493），细胞内氨基酸及衍生物代谢（GO：0006519），actin蛋白解聚调控（GO：0030834）及氧化应激调控（GO：0016491）等。（4）Real-time RT-PCR基因验证结果对蛋白质双向电泳筛选出的蛋白LDHB，HSPA8，CKB，PSMA1，STMN1，THAP5，MBP，NEFM和SNCA在基因水平进行验证，OPIDN相关蛋白在基因水平与蛋白水平变化一致。4、TOCP染毒致OPIDN对GS和GLS表达及谷氨酸/谷氨酰胺循环的影响（1）TOCP染毒诱导成年鸡OPIDN对GS和GLS基因和蛋白表达的影响TOCP染毒5天时，与对照组和PMSF干预组相比较，GS蛋白和基因在TOCP组表达均显著下降（在鸡脑中P<0.01，在鸡脊髓中P<0.05），然而TOCP染毒21天时，与对照组和PMSF干预组相比较，GS蛋白在TOCP组鸡脑和脊髓中表达没有明显变化。TOCP染毒5天和染毒21天两个时间点，与对照组和PMSF干预组相比较，GLS蛋白在TOCP组鸡脑和脊髓中表达都没有明显改变。（2）谷氨酰胺合成酶活性测定结果TOCP染毒5天时，谷氨酰胺合成酶活性在TOCP组与对照组比较表达显著下降，（在鸡脑和脊髓中P<0.05）；然而TOCP染毒21天时，与对照组和PMSF干预组相比较，谷氨酰胺合成酶活性在TOCP组鸡脑和脊髓中表达没有明显变化。（3）谷氨酸和谷氨酰胺含量测定结果TOCP染毒5天时，与对照组和PMSF干预组相比较，谷氨酸含量在TOCP组表达显著上升（在鸡脑中P<0.01，在鸡脊髓中P<0.05），相反，谷氨酰胺含量在TOCP组表达显著下降（在鸡脑中P<0.01，在鸡脊髓中P<0.05），然而TOCP染毒21天时，与对照组和PMSF干预组相比较，谷氨酸与谷氨酰胺含量在TOCP组鸡脑和脊髓中表达没有明显变化。（4）细胞内钙离子含量测定结果TOCP染毒5天时，细胞内钙离子含量在TOCP组与对照组比较表达显著上升（在鸡脑中和鸡脊髓中P<0.05），然而TOCP染毒21天时，与对照组和PMSF干预组相比较，细胞内钙离子含量在TOCP组鸡脑和脊髓中表达没有明显变化。结论：1、TOCP暴露可上调鸡脑组织EIF5A2、DSTN、PSMA1和CKB及下调GST1、LDHB、HOMER1、VDAC2和Hsc70。2、TOCP暴露可上调鸡脊髓组织NEFM及下调HSPB1，LMW-PTP，ETFa、STMN1、MDH1、THAP5、MBP、SNCA和VAT1。3、些OPIDN相关蛋白主要定位在胞浆及线粒体中，主要参与蛋白分解调控，细胞骨架组合调控，细胞内氨基酸及衍生物代谢，actin蛋白解聚调控及氧化应激调控等生物过程。4、TOCP在暴露早期可诱导鸡神经组织GS表达水平和活性显著下降。5、TOCP在暴露早期可诱导鸡神经组织谷氨酸水平显著升高。6、TOCP在暴露早期可诱导鸡神经组织谷氨酰胺水平显著下降。7、TOCP在暴露早期可诱导鸡神经组织细胞钙离子浓度显著升高。8、TOCP暴露早期鸡神经组细胞钙离子浓度显著升高可能与GS活性下降所导致的谷氨酸水平显著升高有关。9、GS活性抑制所导致的谷氨酸/谷氨酰胺循环障碍和钙离子浓度显著升高可能与TOCP暴露鸡诱发OPIDN机制密切相关。更多还原

【Abstract】 Background and ObjectiveOrganophosphate-induced delayed neuropathy （OPIDN） is a well-recognizedserious neuropathy induced by organophosphorus compounds （OPs） during2–3weeksafter acute exposure. Tri-ortho-cresyl-phosphate （TOCP） is widely used as additive orsoftener and has been believed to be associated with OPIDN. The mechanism ofOPIDN is still unclear, so OPIDN has been increasingly concerned.Because enoughdoses of TOCP administered to the laboratory animals not only promoted the initiationof OPIDN but also induced immediate neurotoxic action at the same time, it is difficultto identify the biochemical changes resulted from acute toxicity with those from OPIDN.It has been documented that pretreatment of Phenylmethylsulfonyl fluoride （PMSF）prior to OPs administration could prevent the signs and symptoms of the delayedneuropathy in hens. The biochemical changes both related to OPIDN and acute toxicityoccur when only TOCP is administered to hens, but the biochemical changes related toacute toxicity occur alone when hens exposed to TOCP followed by PMSF. In thepresent study, to screen the changed protein related to OPIDN and help researchersfurther explore the molecule mechanism of OPIDN, Two-dimensional polyacrylamidegel electrophoresis （2D-PAGE） and mass spectrometry （MS） were used to investigatethe disturbance of proteins in the brain of hens in OPIDN. As the result of ourproteomic analysis, we found that glutamine synthetase （GS） was showed dramaticallydecreased expression in OPIDN. Because GS play an important role in intracellularCa²⁺homeostasis, we further studied the expression of GS by real time RT-PCR andELISA, and the expression of another protein glutaminase （GLS）, which were notidentified by our2D-PAGE but was thought to be closely related to the physiologicalfunction of GS. At last, we also examined the levels of glutamate （Glu） and glutamine （Gln）, and the concentration of intracellular Ca²⁺.Methods1. Animal model of OPIDN: The hens were randomly divided into four groups.TOCP group was treated with TOCP by gavage at a single dosage of1000mg/kg and750mg/kg respectively, and control group was given an equivalent volume vehicle bygavage while hens in the PMSF+TOCP group were subcutaneously injected with40mg/kg PMSF followed by1000mg/kg TOCP24h later.2. Neurological behavior scores: OPIDN neurological signs were assessed by asix-point graded scale.3. The ultrastructural alterations in OPIDN: The samples cut into50nm thicksections and examined by transmission electron microscopy.4. Two-dimensional electrophoresis and image analysis and Mass SpectrometryAnalysis.5. Bioinformatics analysis: The GO category and pathway analysis were carriedout statistical analysis tools of DAVID.6. Real-time RT-PCR was used to determination the expression of the proteinsrelated OPIDN and GLS.7. ELISA was used to determination the expression of GS and GLS.8. Measurement of the activity of GS and the level of Glu and Gln.9. Measurement of intracellular Ca²⁺level.Results1. Delayed neurotoxic symptoms: Abnormal gaits progressed in severity with timeand hens were nearly or completely paralyzed by the end of21-day experimental period.However, no clinical signs of delayed neuropathy were observed in hens of the othertwo groups during the whole experiment period.2. Effects of TOCP on morphological changes in OPIDN: The changes includedswelling and vacuolation of mitochondria, the fragmentation of microtubule andneurofilaments in axon, etc.3.2D-PAGE combined MS analysis of protein expression in hens:（1）2D-PAGE analysis of protein expression in hensIn the brain:102spots of the differentially expressed proteins closely related toOPIDN were found in the TOCP group on day5, which63spots were upregulated and39spots were downregulated. On day21,150protein spots were found, which81spotswere upregulated and69spots were downregulated. In the spinal cord:43spots of the differentially expressed proteins closely relatedto OPIDN were found in the TOCP group on day5, which24spots were upregulatedand19spots were downregulated. On day21,86protein spots were found, which46spots were upregulated and40spots were downregulated.（2） Identification of proteins closely related to the OPIDN in brains of hensIn the brain: The upregulated proteins were EIF5A2, DSTN, PSMA1and CKB.And the dowregulated proteins were GS, LDHB, HOMER1, VDAC2, Hsc70andGSTA.In the spinal cord: The upregulated protein was NEFM. And the dowregulatedproteins were HSPB1，LMW-PTP, ETFa, STMN1, MDH1, THAP5, MBP, SNCA andVAT1.（3） The results of GO category: The main GO categories are GO:0005737^cytoplasm, GO:0005739^mitochondrion, GO:0043244^regulation of proteincomplex disassembly, GO:0051493^regulation of cytoskeleton organization, GO:0006519^cellular amino acid and derivative metabolic process, GO:0030834^regulation of actin filament depolymerization and GO:0016491^oxidoreductase activity.（4） Gene expression of the identified proteinLDHB，HSPA8，CKB，PSMA1，STMN1，THAP5，MBP，NEFM，SNCA werechosen to study whether the differential level of proteins was related to the differentmRNA level and the results were consistent with those from2-DE.4. Impact of TOCP exposure on the expression of GS and GLS, and theglutamate–glutamine cycle:（1）Expressions of GS and GLS after TOCP exposed: the gene and proteinexpression of GS in the CNS of hens decreased significantly in the TOCP groupcompared to control group or to PMSF+TOCP group on day5. However, there was nosignificant difference in the expression of GS between control group and PMSF+TOCPgroup on day21, and there was no significant difference in the expression of GLSbetween control group and PMSF+TOCP group on day5or day21.（2） The activity of GS after TOCP exposed: The activity of GS in the CNS of hensdecreased significantly in the TOCP group compared to control group on day5.However, there was no significant difference in the expression of GS between controlgroup and PMSF+TOCP group on day21.（3） The levels of Glu and Gln after TOCP exposed: On day5, Glu level in CNS of hens was significantly increased in TOCP group compared to control and PMSF+TOCPgroups. There were no significant differences in the Glu level among the three groupson day21. In contrast, Gln level in CNS of hens was significantly in TOCP groupcompared to control and PMSF+TOCP groups. However, there was no significantdifference in the Gln level between control group and PMSF+TOCP group on day5orday21.（4） The level of the intracellular Ca²⁺in brains of hens: Intracellular level of Ca²⁺in the CNS of hens was significantly increased in TOCP group compared to controlgroup. However, there was no significant difference in the intracellular level of Ca²⁺inthe brains of hens among the three groups on day21.Conclusions1. Ten proteins were identified in the brain, which were EIF5A2, DSTN, PSMA1,CKB, GS, LDHB, HOMER1, VDAC2, Hsc70and GSTA.2. Ten proteins were identified in the spinal cord, which were NEFM, HSPB1，LMW-PTP, ETFa, STMN1, MDH1, THAP5, MBP, SNCA and VAT1.3. The results of GO category indicated that proteins related OPIDN were mainlyin cytoplasm and mitochondrion, and involved in regulation of protein complexdisassembly, regulation of cytoskeleton organization, regulation of actin filamentdepolymerization and oxidoreductase activity;4. The expression and the activity of GS were down regulation in the early stageafter TOCP exposure;5. The level of Glu was up regulation in the early stage after TOCP exposure;6. The level of Gln was down regulation in the early stage after TOCP exposure;7. The intracellular Ca²⁺homeostasis was up regulation in the early stage afterTOCP exposure.8. TOCP exposure might induce downregulation of GS and increase in glutamatelevels in the brains of hens in the early stage after administration.9. The increased extracellular glutamate level in brain of hens exposed to TOCPmay be associated with the downregulated expression of GS.更多还原