【作者】 马克学；

【导师】 陈广文；

【作者基本信息】 河南师范大学 ， 动物学， 2014， 博士

【摘要】 淡水涡虫是一类非常独特的动物，具有极强的耐饥饿能力。一条成体涡虫饥饿数月后仍能存活，除个体大小变化外，机体仍能保持正常的生理功能。关于饥饿对涡虫细胞形态、代谢变化和基因表达的影响国外研究较少，国内未见报道。本文以日本三角涡虫（Dugesia japonica）为材料，研究了持久饥饿对日本三角涡虫细胞形态结构、生化代谢和基因表达的影响，其中，利用HE染色和透射电镜技术观察了持久饥饿过程中日本三角涡虫细胞形态学变化；利用全自动生化分析仪检测了一些酶的活性变化；利用差异显示RT-PCR和差异蛋白质组学技术分析了饥饿诱导上调表达的基因和蛋白质；利用RACE技术克隆了DjHSP90/DjGRP78/DjHSP40cDNA全长序列；利用实时荧光定量PCR技术检测了持久饥饿过程中DjHSP90/DjGRP78/DjHSP40基因的表达变化；利用整体免疫组化技术研究了DjHSP70和DjHSP90的组织分布；利用比色法研究了饥饿诱导的氧化和抗氧化损伤反应。结果报道如下：1.持久饥饿过程中日本三角涡虫组织结构和细胞形态学变化正常涡虫组织结构非常完整，组织间界限清晰。饥饿虫体组织间界限模糊，肠道组织和实质组织中出现大量嗜酸性细胞。非常明显的是，这些嗜酸性细胞经历一个称之为“细胞自溶”的过程，细胞质解体形成无数小泡。超微结构结果表明：一些细胞的细胞核呈空洞状，常染色质被吸收，仅留少量的异染色质；部分细胞仅保留少量的细胞质，细胞质中充满空泡，类似“自噬性细胞死亡”。选择性细胞死亡可能为涡虫在持久饥饿过程中存活提供了营养物质。2.持久饥饿过程中日本三角涡虫体内6种酶活性和PCNA mRNA表达水平的变化结果表明：饥饿过程中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）的活性显著升高，是对照水平的10倍以上，涡虫再喂食后又降低到正常水平。正常个体中肌酸激酶（CK）和乳酸脱氢酶（LDH）的活性很强（分别达到250U/g蛋白质和80U/g蛋白质），饥饿4-6周活性显著下降，涡虫再喂食后又恢复到正常水平。饥饿过程中碱性磷酸酶（ALP）活性降低，而酸性磷酸酶（ACP）的活性升高。受饥饿影响，~140kD和~40kD蛋白水解酶活性显著增强。饥饿过程中PCNA mRNA的表达水平没有明显变化，说明饥饿对涡虫干细胞的影响较小。3.差异RT-PCR和差异蛋白质组学技术筛选饥饿诱导日本三角涡虫上调表达的基因和蛋白质克隆和测序了一些受饥饿诱导上调表达的cDNA片段，发现了一些有价值的EST序列，这些EST序列涉及细胞代谢、细胞死亡、细胞应激、蛋白质水解酶、RNA结合蛋白、转录因子和细胞骨架类蛋白。实时荧光定量PCR结果表明：DjHSP70、DjHSP90、DjHSP40和DjClg3A基因上调表达，而DjGRP78基因转录水平没有明显变化。利用2-D电泳技术分离正常和饥饿涡虫组织差异表达的蛋白质质点，在饥饿涡虫组织中分离出2000个质点，差异质点超过1000个。质谱分析和NCBI数据库检索结果提示：部分上调表达的质点分别归属于细胞应激类、干细胞相关类、蛋白激酶类、膜泡运输类、抗氧化性蛋白类、受体类、转录因子类、细胞代谢类、表观遗传修饰类、细胞死亡相关蛋白类和RNA结合蛋白类。其中，表达水平超过2-倍的蛋白质分别是HSP70/90、Piwi-1蛋白、Vasa相关蛋白、Nanos相关蛋白、非受体丝/苏氨酸激酶、非受体酪氨酸激酶和受体酪氨酸激酶、突触相关蛋白、肌球蛋白重链、抗氧化酶6、细胞色素P450、SOD、G蛋白偶联的受体激酶、HMG蛋白、氨肽酶、U62家族肽酶蛋白、丝氨酸蛋白水解酶、磷酸丙糖异构酶、磷酸甘油酸激酶、Caspase7、ATG11和ATG4B。该工作为进一步研究上调表达基因和蛋白质的功能奠定了良好的基础。4.持久饥饿诱导日本三角涡虫氧化和抗氧化性损伤反应持久饥饿对脂质过氧化的影响采用丙二醛（MDA）水平进行评估。结果表明，涡虫饥饿30天后，MDA水平升高1.7倍，饥饿60天升高2倍，饥饿90天升高4倍。饥饿30天后抗氧化酶SOD活性升高50%，饥饿60天升高76%，饥饿90天升高123%。饥饿30天后CAT活性升高10%，饥饿60-90天后升高20%。结果表明：持久饥饿导致氧化性损伤，抗氧化酶SOD和CAT活性提高能保护细胞免受损伤。5.日本三角涡虫DjHSP90、DjGRP78和DjHSP40基因克隆及生物信息学分析DjHSP90cDNA全长2354bp，包含2148bp的开放阅读框（ORF），编码715个氨基酸，氨基酸序列上含有HSP90蛋白家族的5个标签序列。ORF区DNA测序表明，DjHSP90基因结构区仅含有1个48bp内含子。HSP90系统发生树的基部是单细胞酵母，依次是植物、腔肠动物、扁形动物、软体动物、节肢动物和脊椎动物。HSP90系统发生树所体现的动物之间的亲缘关系与传统动物学分类相一致。DjGRP78cDNA全长2121bp，包含1983bp的开放阅读框（ORF），编码660个氨基酸，氨基酸序列上含有HSP70蛋白家族的3个标签序列。DjGRP78N-端氨基酸含有信号肽，C-端含有KTEL基序，说明该蛋白定位于内质网。ORF区DNA测序表明，DjGRP78基因结构区仅含有1个44bp内含子。GRP78系统发生树显示节肢动物、脊椎动物、腔肠动物和软体动物分别聚在一起，涡虫位于进化树的基部。GRP78系统发生树不能确定物种进化关系，但能反应出种属的特异性。DjHSP40cDNA全长1378bp，包含1236bp的开放阅读框，编码411个氨基酸，分子量46.2kDa，等电点是7.52。氨基酸序列分析表明：DjHSP40含有J、G/F、CR和C-末端4个结构域，说明它归属于Ⅰ型HSP40亚家族。HSP40系统发生树显示脊椎动物和节肢动物聚类在一起，与扁形动物形成姊妹分支。HSP40系统发生树与HSP90系统发生树和GRP78系统发生树显著不同，该现象说明涡虫应激蛋白在进化上产生分歧。6.持久饥饿对日本三角涡虫DjHSP90、DjGRP78和DjHSP40基因表达的影响应用荧光实时定量PCR技术研究持久饥饿对DjHSP90、DjGRP78和DjHSP40基因表达的影响。结果表明，饥饿30天涡虫DjHSP90转录水平升高1.5倍，饥饿45-60天维持在1.6倍，饥饿90天达到2倍，再喂食后没有恢复到正常水平。饥饿30天后DjGRP78的转录水平没有增加，饥饿60-90天甚至略低于正常水平（分别是0.65和0.69倍），再喂食后恢复到正常水平。饥饿30天涡虫DjHSP40转录水平升高1.6倍，饥饿60-90天分别是2倍和2.6倍，饥饿90天略有降低但仍高于对照水平（2倍）。再喂食后DjHSP40的转录水平非但没有降低，却高于对照水平3倍。7.持久饥饿对日本三角涡虫DjHSP90和DjHSP70蛋白表达模式的影响整体免疫组化显示，DjHSP90阳性信号在饥饿1月涡虫的背部两侧平行分布，对照虫体没有检测到阳性信号。DjHSP90的表达模式非常类似于涡虫精巢特异性基因DeY1的表达模式，提示DjHSP90阳性细胞很可能是精巢组织。DjHSP90上调表达可能保护精巢免受饥饿诱导的损伤。除涡虫头部和咽外，DjHSP70阳性细胞通体分布。此外，持久饥饿没有改变DjHSP70表达部位，但显著提高其表达水平。令人感兴趣的是，DjHSP70阳性细胞的特征非常类似于文献描述的涡虫干细胞（neoblasts）。由此推断，DjHSP70上调表达可能在维持涡虫干细胞稳态和保护干细胞免受饥饿诱导的损伤方面发挥重要作用。结论：1.持久饥饿严重损伤涡虫组织和细胞结构，导致选择性细胞死亡，细胞死亡可能为涡虫在持久饥饿过程中存活提供营养物质。2.持久饥饿导致氧化性损伤，氧化性损伤可能是涡虫细胞死亡的主要诱因。3.为抵御饥饿诱导的应激效应，HSP蛋白上调表达能保护涡虫免受伤害。特别是DjHSP70在维持涡虫干细胞稳态和保护干细胞免受饥饿诱导的损伤中发挥着重要作用。更多还原

【Abstract】 Freshwater planarians are unique animals, exhibiting strong tolerance to prolonged starvation. Anadult planarian can still survive after several months of starvation. Except for the reduction in body size, thestarved planarians maintain the normal physiological function. The effects of prolonged starvation oncytomorphology, metablic change and genes expression in planarians Dugesia japonica has been done verylittle in and out of abroad. In this paper, the HE staining and transmission electron microscopy (TEM) wereemployed to observe the cytomorphological changes in planarians Dugesia japonica during prolongedstarvation; The automated biochemistry analyzer was used to detect some enzymes activity; Thedifferential-display RT-PCR and differential proteomics approach were used to investigate the up-regulatedgenes and proteins; The RACE technique was used to clone the full-length ofDjHSP90/DjGRP78/DjHDP40cDNA sequences; The fluorescent real time PCR was employed to detectthe changes of DjHSP90/DjGRP78/DjHDP40gene expression during prolonged starvation; Thewhole-mount immunohistochemistry was used to determine the tissue distribution of DjHSP70andDjHSP90; The colorimetry method was used to investigate the starvation-induced oxidative stress andantioxidant defenses. The results were as follows.1. Tissue structure and cytomorphological changes during prolonged starvation in planarian DugesiajaponicaIn the normal living planarian Dugesia japonica, the tissues structure is very integrity, and theboundary of different tissues is obvious. Whereas, the boundary of different tissues is obscure, and a largenumber of acidophile cells can be seen in the intestinal tissues and parenchymal tissues in starved animals.Notably, these acidophile cells undergo a process called “cell autolysis”, of which the cytoplasms aredisintegrated into numerous vesicles. The ultrastructure observation reveals that the nuclei of some cells areempty, the euchromatins are resorbed and only little heterochromains remain in the nuclei. And partial cellswith little cytoplasm, of which is full of vacuoles, are more similar to what is described “autophagic celldeath”. The selective cell death may provide nutrients for planarian survival during prolonged starvation.2. Changes of six enzymes activity and the expression level of PCNA mRNA during prolonged starvation in planarian Dugesia japonicaThe results show that the activity of alanine aminotransferase (ALT) and aspartate aminotransferase(AST) elevated apparently during starvation,10-fold higher than the control level, but reduced to thenormal level after re-fed; The eznzyme activity of creatinekinase (CK) and the lactate dehydrogenase (LDH)are very strong (250U/g protein and80U/g protein, respectively) in normal palnarians, but decreasedsignificantly after4-6weeks starvation, and returned to the normal after re-fed. The activity of alkalinephosphatase (ALP) decreased during starvation, whereas the activity of acid phosphatase (ACP) increasedapparently; In response to starvation, the activity of~140kD and~40kD proteinases increased drastically.However, no changes of the expression level of PCNA mRNA during starvation were observed, whichindicated that starvation had little influences on planarian stem cells.3. Screening the up-regulated genes and proteins induced by prolonged starvation in planarianDugesia japonica by differential-display RT-PCR and by differential proteomics approachThe up-regulated cDNA fragments induced by starvation were screened and sequenced. The resultsshow that some interesting ESTs are involved in cell metabolism, cell death, cell stress, proteinases, RNAbinding proteins, transcription factors and cytoskeleton proteins. The fluorescent real time RT-PCR wasfurther used to verify the up-regulated genes, and found that the expression levels of DjHSP70, DjHSP90,DjHSP40and DjClg3A increased after1months of starvation, but the expression level of DjGRP78remained stable.Two-dimensional electrophoresis (2-DE) was firstly used to separate the total proteins from thenormal and starved tissues of planarians, and found that over2000protein spots were separated in starvedplanarian tissues, over1000protein spots were differentially expressed. Parts of the up-regulated proteinspots were identified by mass spectrometry, and subsequently the data from mass spectrometry wereblasted in NCBI database and planarians protein database, the results show that these proteins are belongedto the groups of stress related proteins, stem cell related proteins, protein kinases, vesicle transportation,anti-oxidation proteins, receptors, transcription factors, protein degradations, cell metabolism, epigeneticmodification, cell death related protein and RNA binding protein. Of which, the over2-fold expressionlevels of proteins were HSP70/90, Piwi-1liked protein, Vasa-related protein, Nanos-related protein, Nonreceptor serine/threonine kinase, Non receptor tyrosine kinase and Receptor tyrosine kinase, Synaptosomal-associated protein, Myosin heavy chain, Peroxiredoxin-6, Cytochrome P450, Superoxidedismutase, G protein-coupled receptor kinase, HMG protein, Aminopeptidase, Peptidase U62familyprotein, Serine protease, Triosephosphate isomerase, Phosphoglycerate kinase, Caspase7,Autophagy-related protein11（ATG11）and Cysteine protease ATG4B. The above works play a goodfoundation for the further studying the function of the up-regulated genes and proteins.4. Oxidative damage and antioxidant defenses during prolonged starvation in planarian DugesiajaponicaThe effects of prolonged starvation on lipid peroxidation are measured as malondialdehyde (MDA)levels. The results show that MDA levels increased1.7-fold after30days of starvation,2-fold after60daysof starvation, and4-fold after90days of starvation. The activity of the antioxidant enzyme superoxidedismutase (SOD) increases by50%after30days of starvation,76%after60days of starvation, and123%after90days of starvation with respect to controls. The activity of CAT increases by10%after30days ofstarvation,20%after60-90days of starvation. The above results indicate that prolonged starvation leads tooxidative stress, and the activation of the antioxidant enzyme SOD and CAT can protect cells fromdamage.5. Clone and bioinformatics analysis of DjHSP90, DjGRP78and DjHSP40gene from planarianDugesia japonica.The full-length cDNA of planarian Dugesia japonica HSP90(designated DjHSP90) is2354bp,including an open reading frame (ORF) of2148bp encoding a polypeptide of715amino acids with all fiveHSP90family signatures. The ORF sequences from genomic DNA was sequenced, and found only oneintron (48bp) existed in DjHSP90gene structure. In the root of HSP90phylogenetic tree is unicellularyeast, next to plants, coelenteratas, platyhelminthes, molluscs, arthropodas, and vertebrates. Therelationships displayed in the phylogenic tree are in agreement with the concept of traditional taxonomy.The full-length cDNA of planarian Dugesia japonica GRP78(designated DjGRP78) is2121bp,including an open reading frame (ORF) of1983bp encoding a polypeptide of660amino acids with threeHSP70family signatures. DjGRP78contains signal peptides at the N-terminus and a KTEL peptide motifat the C-terminus, which suggests that it localizes in the endoplasmic reticulum (ER). In addition, the ORFsequences from genomic DNA was sequenced, and found only one intron (44bp) existed in the DjGRP78 gene structure. The GRP78phylogenetic tree shows that animals of Arthropods, Vertebrates, Cnidariansand Mollusks are clustered together, with planarians located at the root of the tree. The GRP78phylogenetic tree does not define the evolutionary relationship of species, rather it shows a current look atthe similarities between species based on the similarity of GRP78homolog sequences.The full-length cDNA of planarian Dugesia japonica HSP40(designated DjHSP40) is1378bp,including an open reading frame (ORF) of1236bp encoding a polypeptide of411amino acids with apredicted molecular mass of46.2kDa and theoretical isoelectric point of7.52. A database search with thededuced amino acid sequence indicates that DjHSP40contains four domains structure: J, G/F, CR, andC-terminal, which characteristics of DjHSP40suggestes that it belongs to the TypeⅠ HSP40subfamily.The HSP40phylogenetic tree shows that animals from Vertebrates and Arthropods are clusted together,which forms a sister branch with the animals from Platyhelminthes. The relationships displayed in theHSP40phylogenic tree are very not in agreement with that displayed in HSP90and GRP78phylogenic tree.This phenomenon suggests that stress proteins in planarians display a divergent evolution.6. Expression levels of DjHSP90, DjGRP78and DjHSP40during prolonged starvation in planarianDugesia japonicaThe fluoresent real-time quantitative RT-PCR was employed to measure the temporal expression ofDjHSP90, DjGRP78and DjHSP40during prolonged starvation in planarian Dugesia japonica. The resultsshowed that DjHSP90increased1.5-fold after30days of starvation, maintained1.6-fold after45-60daysof starvation, and reached2-fold after90days of starvation, but not restored to the normal level in there-fed (RF) animals. The transcriptional level of DjGRP78did not increase after30days of starvation, evenwas slighly lower (0.65and0.69-fold, respectively) than the control level after60-90days of starvation,and restored to the normal level in the re-fed (RF) animals. The expression level of DjHSP40mRNAincreased1.6-fold after30days of starvation, reached2-fold and2.6-fold after45-60days of starvationrespectively. After90days of starvation, DjHSP40mRNA level decreased slightly, but still higher than thecontrol level (2-fold). Other than reducing the DjHSP40mRNA level in the re-fed animals, it increased to3-fold higher than the control level.7. Expression pattern of DjHSP90and DjHSP70during prolonged starvation in planarian Dugesiajaponica The results by whole-mount immunohistochemistry reveal that the distribution of DjHSP90positivesignal is located in long lateral and parallel lines in the dorsal part of the body in1months starved animals,no positive signal is detected in control samples. The expression pattern of DjHSP90is more similar to thatof the testes-specific expression gene DeY1. The results suggest that DjHSP90-positive tissues may be thetestes tissues, and the up-regulation expression of DjHSP90may be involved in protecting testes tissuesfrom damage induced by prolonged starvation. The DjHSP70-positive cells are present throughout theplanarian body, except for the head and the pharynx. In addition, prolonged starvation does not affect thetissue distribution of DjHSP70, but can elevate its expression level evidently. Interestingly, thecharacteristics of DjHSP70-positive cells is more similar to what is described the planarian stem cells(neoblasts). It is inferred that the up-regulation of DjHSP70may have an important role in maintainingplanarian stem cell homeostasis and protecting them from starvation-induced damage.Conclusion:(1) Prolonged starvation can severely damage the tissue and cell structures in planarian, and leads toselective cell death, which may provide nutrients for planarian survival during prolonged starvation.(2) Prolonged starvation can lead to oxidative damage, which may be the major cell death inducer inplanarians.(3) To defend against starvation-induced stress in planarians, the up-regulation of HSP proteins can protectthem from damage. Especially, DjHSP70may have an important role in maintaining planarian stem cellhomeostasis and protecting them from starvation-induced death.更多还原