發(fā)布時間：2018-05-30 13:11

  本文選題：冬眠 + 糖蛋白質組學��； 參考：《西北大學》2016年博士論文

【摘要】：廢用性肌萎縮是臨床上亟待解決的重要問題之一。盡管科研人員已經作了大量的研究,但目前仍未找到阻止和治療廢用性肌萎縮的有效措施,其主要的原因在于我們對廢用性肌萎縮的分子機制仍缺乏了解。冬眠是冬眠動物應對冬季低溫和食物缺乏等不利因素所特有的生存策略,其過程可以長達數(shù)月。在歷經長期的冬眠期不活動后,冬眠動物的骨骼肌并未出現(xiàn)明顯的廢用性肌萎縮。因此,冬眠動物是一個天然的抗廢用性肌萎縮研究模型。闡明冬眠動物骨骼肌這種特殊適應現(xiàn)象的發(fā)生機制是生理生態(tài)學領域亟待研究的重要課題,對航天、臨床、運動及康復醫(yī)學等領域亦有重要借鑒意義。蛋白的糖基化(glycosylation)是指將糖或者寡糖通過酶促反應連接到蛋白質上,這是蛋白質最重要的翻譯后修飾方式之一。據(jù)估計,人類約70%的蛋白質被多種糖結構所修飾。越來越多的證據(jù)表明多種病理狀況引起的肌肉萎縮與蛋白的糖基化密切相關。作為糖基化方式之一,O-乙酰葡萄糖胺(O-acetylglycosamine,O-GlcNAc)可以通過控制肌蛋白合成來調控廢用性肌萎縮；蛋白的唾液酸化(sialylation)水平的降低是導致肌纖維降解、肌肉力量減弱及肌重減輕的重要原因之一；多種導致肌肉萎縮或功能障礙的肌肉疾病如有鑲邊空泡遠端肌病(distal myopathy with rimmed vacuoles, DMRV)和先天性肌營養(yǎng)不良(congenital muscular dystrophy, CMD)均被證實與肌肉中蛋白糖基化的異常緊密相關。我們本次研究以達烏爾黃鼠(Spermophilus dauricus)為實驗對象,選取典型的慢肌比目魚肌(soleus, SOL)為代表,首次對冬眠不同時期黃鼠比目魚肌糖蛋白組質學進行系統(tǒng)研究,從新的角度探討冬眠動物骨骼肌的抗廢用性肌萎縮機制。第一部分：采用凝集素芯片技術檢測冬眠不同時期達烏爾黃鼠比目魚肌中糖蛋白糖基化的變化情況,并使用凝集素組化技術對芯片結果進行驗證。芯片的分析結果顯示,有8種凝集素的熒光強度值在冬眠過程中發(fā)生了顯著性變化,分別為馬鞍樹凝集素-Ⅱ(Maackia Amurensis Lectin Ⅱ, MAL-Ⅱ)、四棱豆凝集素-Ⅰ(Psophocarpus Tetragonolobus Lectin Ⅰ,PTL-Ⅰ)、紫藤花凝集素(Wisteria Floribunda Lectin, WFA)、加納子凝集素-Ⅰ(Griffonia Simplicifolia Lectin I, GSL-Ⅰ)、四棱豆凝集素-Ⅱ(Psophocarpus Tetragonolobus Lectin Ⅱ, PTL-Ⅱ)、曼陀羅凝集素(Datura stramonium, DSA)、歐洲衛(wèi)矛凝集素(Euonymus Europaeus Lectin,EEL)和菜豆凝集素-E+L(Phaseolus vulgaris Agglutinin E+L,PHA-E+L)。我們的結果顯示：1)與冬眠前組相比,冬眠組凝集素MAL-Ⅱ所識別的糖鏈結構唾液酸(Sialic acid, SA)α2-3半乳糖(Galactose, Gal) (SAa2-3Gal)顯著降低,而在激醒組和出眠組顯著增加,恢復到冬眠前水平；2)PTL-Ⅰ所識別的αN-乙酰半乳糖胺(αGalNAc)和Gal結構與SAa2-3Gal的變化趨勢相似,即在冬眠中顯著下降,在激醒和出眠時顯著增加；3)與冬眠組相比,激醒組和出眠組的WFA特異性識別的末端GalNAc (Terminal GalNAc)結構顯著增加；4)與其它三組相比,GSL-Ⅰ和PTL-Ⅱ特異性識別αGalNAc和Gal結構在激醒組中顯著性的增加；5)與冬眠前組相比,DSA識別的GlcNAc結構在冬眠組顯著性增加,而在激醒組和出眠組顯著下降,恢復到冬眠前水平；6)與其它三組相比,凝集素EEL識別的半乳糖α1-3(巖藻糖α1-2)Gal(巖藻糖,Fucose, Fuc)結構在冬眠組顯著增加；7)與冬眠前組相比,冬眠組、激醒組和出眠組SOL的PHA-E+L特異性識別的三天線和四天線復雜型N-糖鏈(Tri-and tetra-antennary complex-type N-glycan)結構顯著性增加。我們選取了MAL-II, PTL-I,DSA和PHA-E+L四種凝集素作了凝集素組化實驗。組化的結果與凝集素芯片的結果相一致,進一步驗證了凝集素芯片的實驗結論�？傊�,這一部分的結果提示,黃鼠周期性陣間激醒過程中,SAa2-3Gal結構的顯著性增加有效地補償了其在冬眠過程中的減少,對于維持冬眠黃鼠骨骼肌正常的結構和功能,防止其廢用性萎縮起到了重要作用；由于PTL-Ⅰ、WFA、GSL-Ⅰ、PTL-Ⅱ四種凝集素特異性識別的糖鏈結構均為O-連接的糖鏈,因此推測,O-連接的糖鏈在激醒過程中的增加可能與冬眠黃鼠特有的保護性機制有關；此外,三天線和四天線型的糖蛋白N-糖鏈在整個冬眠過程中均顯著性增加,可能與黃鼠骨骼肌對冬眠廢用的生理適應機制有關。第二部分：本文第二章的結果表明,黃鼠周期性陣間激醒過程中,SAa2-3Gal結構的顯著性增加有效地補償了其在冬眠過程中的減少,而正常的唾液酸含量對于維持肌肉的形態(tài)、結構、功能有著重要的作用。因此,本部分實驗的研究目的是通過測定冬眠不同時期黃鼠比目魚肌中β-半乳糖-α-2,3-唾液酸糖基轉移酶(sialyltransferasebeta-galactoside alpha-2,3-sialyltransferases)(包括ST3Gall、ST3Gal2、 ST3Gal3、ST3Gal4、T3Gal5、ST3Gal6)與唾液酸糖苷酶(sialidases)(包括NEU1、 NEU2、NEU3、NEU4) mRNA表達的變化情況,從糖相關基因mRNA水平對SAa2-3Ga在冬眠不同時期的變化機制進行分析。我們的結果顯示,與冬眠前組相比,冬眠組ST3Gall的mRNA表達量顯著性降低(-55.00%；P0.01),而激醒組和出眠組ST3Gall的mRNA表達量與冬眠組相比,均顯著性升高(114.30%和126.20%,P0.01),與冬眠前組相比無顯著性的差異(P0.05)。相比于冬眠前組,ST3Gal2的mRNA表達量在冬眠組顯著性降低(-42.70%,P0.01),而與冬眠組相比,激醒組和出眠組mRNA表達量均顯著性升高(77.30%和76.50%,P0.01),冬眠前組、激醒組與出眠組的ST3Gal2的mRNA表達量無顯著性的差異(P0.05)。冬眠組ST3Gal5的mRNA相對表達量相比于冬眠前組顯著性降低(-60.91%,P0.01),而在激醒組和出眠組中ST3Gal5的mRNA表達量分別較冬眠組出現(xiàn)顯著性升高(160.56%和125.48%,P0.01),與冬眠前組相比沒有顯著的差異(P0.05)。NEU1、NEU3和ST3Gal6的mRNA表達量在冬眠四組中沒有顯著的差異(冬眠前組,冬眠組,激醒組,出眠組)(P0.05)。我們的結果提示,ST3Gall、ST3Gal2及ST3Gal5 mRNA相對表達量的改變,即在冬眠中明顯下降,在陣間激醒過程中和出眠后又恢復至冬眠前水平,可能是造成黃鼠SOL的SAa2-3Gal結構在冬眠期間變化的主要因素。第三部分：本文第二章的研究表明,達烏爾黃鼠比目魚肌中凝集素MAL-Ⅱ特異性識別的糖鏈結構SAa2-3Gal在冬眠過程中的顯著變化可能在其抗廢用性肌萎縮機制中起到重要作用。因此,本部分實驗制備制備凝集素MAL-Ⅱ-磁性微粒復合物,分別從冬眠前和冬眠黃鼠比目魚肌中提取末端為SAa2-3Gal的糖蛋白,利用LC-ESI-MS/MS對提取的糖蛋白進行鑒定,通過生物信息學對鑒定的糖蛋白進行進一步分析。從冬眠前組和冬眠組中分別鑒定出糖蛋白227個和185個。其中,只在冬眠前組鑒定出的糖蛋白為141個,只在冬眠組鑒定出的糖蛋白為99個,在兩組中均鑒定出的糖蛋白數(shù)為86個。GO (Gene Ontology)功能注釋分析顯示：根據(jù)細胞成分注釋,鑒定的糖蛋白主要是胞內蛋白,并且很多為大分子蛋白質,主要分布在細胞器中和膜結構上。根據(jù)分子功能注釋,這些糖蛋白具有結合、催化以及結構分子活性等方面的功能。根據(jù)參與的生物過程注釋,這些糖蛋白主要涉及細胞過程、刺激應答、代謝過程以及生物調節(jié)等生物過程。KEGG(Kyoto Encyclopedia of Genes and Genomes)通路富集分析顯示,從冬眠前組和冬眠組SOL分離出的糖蛋白顯著性富集的通路分別為40條和36條,其中,只在冬眠前組顯著性富集的通路有14條,而只在冬眠組顯著性富集的有10條。使用IntAct數(shù)據(jù)庫進行蛋白質-蛋白質相互作用分析,利用Cytoscape欠件分布構建了冬眠前組和冬眠組糖蛋白的蛋白質相互作用網絡圖(The protein-protein interaction network, PPI network),篩選出9個度值(degree)大于30的核心蛋白質。第四部分：本部分實驗選取了2個糖蛋白進行驗證,分別為熱休克同源蛋白(heat shock cognate 70,HSC70)和丙酮酸激酶(pyruvate kinase,PK),檢測冬眠過程中其蛋白表達和SAa2-3Gal結構的變化情況。此外,檢測黃鼠SOL中14-3-3 protein epsilon蛋白表達在冬眠不同時期的變化情況,分析其在抗廢用性肌萎縮機制中的潛在作用。我們的結果顯示,與冬眠前相比,冬眠組的HSC70蛋白的表達量顯著降低了37.39%(P0.01),與冬眠組相比,激醒組的HSC70蛋白的表達量顯著上升了51.39%(P0.01),出眠組顯著上升了41.67%(P0.01),冬眠前組、激醒組和出眠組三組之間無顯著性差異(P0.05)；PK蛋白的表達在四組之間無顯著性差異(冬眠前組,冬眠組,激醒組,出眠組)(P0.05)；與冬眠前組相比,冬眠組的14-3-3 protein epsilon的表達量顯著降低了42.59%(P0.01),與冬眠組相比,激醒組的14-3-3 protein epsilon的表達量顯著性上升了51.61%(P0.01),出眠組顯著性上升了58.06%(P0.01),冬眠前組、激醒組和出眠組三組之間無顯著性差異(P0.05)；與冬眠前組相比,冬眠組糖蛋白HSC70的SAa2-3Gal結構顯著減少30.08%(P0.01),與冬眠組相比,激醒組的SAa2-3Gal結構顯著增加了37.66%(P0.05),而出眠組顯著性增加54.50%(P0.01),冬眠前組、激醒組和出眠組三組之間無顯著性差異(P0.05)；與冬眠前組相比,冬眠組糖蛋白PK末端SAa2-3Gal結構顯著減少29.40%(P0.01),與冬眠組相比,激醒組的SAa2-3Gal結構顯著增加了30.91%(P0.05),出眠組顯著性增加34.41%(P0.05),冬眠前組、激醒組和出眠組三組之間無顯著性差異(P0.05)。結果表明,冬眠不同時期黃鼠比目魚肌中HSC70、PK、14-3-3 protein epsilon三種蛋白的變化(包括蛋白表達和SAa2-3Gal糖結構)可能與其特有的抗廢用性肌萎縮機制相關。
[Abstract]:Wasting muscle atrophy is one of the most important problems to be solved in clinic. Although a lot of researchers have done a lot of research, the effective measures to prevent and treat the disused muscle atrophy are still not found at present. The main reason is that we still lack the understanding of the molecular mechanism of the disused muscle atrophy. Hibernating is a hibernating animal to deal with the low winter. The process of survival specific to adverse factors, such as mild food deficiency, can last several months. After a long hibernation inactivity, the skeletal muscles of hibernating animals do not have obvious disuse muscle atrophy. Therefore, hibernating animals are a natural model of anti waste myatrophy. The mechanism of adaptation is an important subject in the field of physiological ecology. It is also of great significance to the fields of space, clinical, sports and rehabilitation medicine. The glycosylation of protein (glycosylation) refers to the connection of sugar or oligosaccharides to the protein by enzymatic reaction, which is the most important post-translational method of protein. It is estimated that about 70% of human protein is modified by a variety of sugar structures. More and more evidence suggests that muscle atrophy caused by a variety of pathological conditions is closely related to the glycosylation of proteins. As a glycosylation method, O- acetyl glucosamine (O-acetylglycosamine, O-GlcNAc) can regulate the use of muscle protein synthesis to regulate waste use. Myoatrophy; the decrease of protein's salivary acidification (sialylation) level is one of the important causes of muscle fiber degradation, muscle strength and muscle weight reduction; many muscle diseases that cause muscle atrophy or dysfunction such as distal myopathy with rimmed vacuoles (DMRV) and congenital muscular dystrophy (DMRV). Congenital muscular dystrophy, CMD) were proved to be closely related to the abnormal protein glycosylation in the muscles. In this study, we selected the typical slow muscle soleus (soleus, SOL) as the representative of the Spermophilus dauricus (Spermophilus dauricus) for the first time, and for the first time, the myosin composition of the soleus flounder in different periods of hibernating was carried out. The mechanism of anti waste muscle atrophy in hibernating animal skeletal muscles was explored from a new perspective. The first part: using lectin chip technique to detect the changes of sugar glycosylation in the soleus muscle of dormant of dormans at different periods of hibernating, and to verify the results by using the agglutinin histochemical technique. There were significant changes in the fluorescence intensity of 8 lectins during the hibernation process, namely, saddle tree agglutinin - II (Maackia Amurensis Lectin II, MAL- II), four ribbean lectin - I (Psophocarpus Tetragonolobus Lectin I, PTL- I), wisteria lectin (Wisteria Floribunda Lectin, WFA), and Garner lectin - I Onia Simplicifolia Lectin I, GSL- I), four prismatic lectin - II (Psophocarpus Tetragonolobus Lectin II, PTL- II), Mandala agglutinin (Datura stramonium, DSA), European Euonymus agglutinin and bean agglutinin. Our results show: 1) Compared with the pre hibernating group, the sugar chain structure sialic acid (Sialic acid, SA) 2-3 semi lactose (Galactose, Gal) (SAa2-3Gal) identified by the hibernating group of agglutinin II (Galactose, Gal) (SAa2-3Gal) significantly decreased in the waking and hibernating group and recovered to the pre hibernating level; 2) PTL- I identified the alpha N- acetyl galactoamine (alpha GalNAc) and Gal structure and SAa2-3Gal. The change trend is similar, that is, in hibernation, significant decrease in waking and hibernation; 3) the terminal GalNAc (Terminal GalNAc) structure of WFA specific recognition in the waking and hibernating group increases significantly compared with the hibernating group; 4) GSL- I and PTL- II specific identification of alpha GalNAc and Gal structure are significant in the waking group compared with the other three groups. Increase in sex; 5) the GlcNAc structure identified by DSA increased significantly in hibernating group compared with the pre hibernating group, while in the waking and hibernation group, the structure of galactose alpha 1-3 (fucose alpha 1-2) Gal (fucose, Fucose, Fuc) structure in the hibernation group increased significantly compared to the other three groups; and the structure of the Gal (fucose, Fucose, Fuc) in the hibernating group increased significantly; 7) The three antenna identified by the hibernating group, the hibernating group, the PHA-E+L specific identified three antenna and the four antenna complex N- sugar chain (Tri-and tetra-antennary complex-type N-glycan) structure increased significantly in the hibernating group and the hibernating group. We selected the four agglutinin histochemical experiments of MAL-II, PTL-I, DSA and PHA-E+L. The results and agglutination of the histochemistry The results of the prime chip are consistent and further verified the experimental conclusions of the lectin chip. In conclusion, the results of this part suggest that the significant increase of SAa2-3Gal structure in the periodic inter matrix arousal of the rat can effectively compensate for its decrease in hibernating process, and to maintain the normal structure and function of the skeletal muscles of the hibernating rat and prevent it. Disuse atrophy has played an important role; because PTL- I, WFA, GSL- I, PTL- II, four lectins specifically identify the sugar chain structures that are O- connected, it is presumed that the increase of the sugar chain in the O- connection may be related to the specific protective mechanism of the hibernating mice; in addition, the three antenna and the four antenna type of glycoprotein N-. The sugar chain is significantly increased during the whole hibernation process, which may be related to the physiological adaptation mechanism of the skeletal muscle to hibernation. The second part: the results of the second chapter show that the significant increase of SAa2-3Gal structure effectively compensates for its decrease during the hibernation process and normal saliva during the periodic waking process of the rat. Acid content plays an important role in maintaining the shape, structure and function of muscle. Therefore, the aim of this part of this experiment is to determine the beta galactose - alpha -2,3- sialidase (sialyltransferasebeta-galactoside alpha-2,3-sialyltransferases) (including ST3Gall, ST3Gal2, ST3G) in the soleus muscle of the rat at different periods of hibernating. Al3, ST3Gal4, T3Gal5, ST3Gal6) and the changes in the expression of sialidase (sialidases) (including NEU1, NEU2, NEU3, NEU4) mRNA, from the mRNA level of sugar related genes to the mechanism of the variation of SAa2-3Ga in different periods of hibernating. 00%; P0.01), while the mRNA expression of ST3Gall in irritable and hibernating groups was significantly higher than hibernating group (114.30% and 126.20%, P0.01), and there was no significant difference compared with the hibernating group (P0.05). Compared with the hibernating group, the mRNA expression of ST3Gal2 decreased significantly in the hibernating group (-42.70%, P0.01), and compared with the hibernating group, the arousal group and out of the hibernating group were compared with hibernating group. The expression of mRNA in the sleeping group increased significantly (77.30% and 76.50%, P0.01). Before hibernating group, there was no significant difference in the mRNA expression of ST3Gal2 in the waking and sleeping group (P0.05). The mRNA relative expression of ST3Gal5 in hibernating group was significantly lower than that in the pre hibernating group (-60.91%, P0.01), while the mRNA expression of ST3Gal5 in the waking and hibernation group was respectively respectively. There was a significant increase in the hibernating group (160.56% and 125.48%, P0.01), and there was no significant difference between the hibernating group (P0.05).NEU1, the mRNA expression of NEU3 and ST3Gal6 was not significantly different in the hibernating group (hibernating group, hibernating group, waking group, and dormancy group) (P0.05). Our results suggest that ST3Gall, ST3Gal2 and ST3Gal5 mRNA are expressed in relative terms. The change of the quantity, that is, in hibernation, a significant decrease in the wake-up process and after the hibernation to the pre hibernation level, may be the main factor causing the changes in the SAa2-3Gal structure of the SOL in the rat. The third part: the study in the second chapter of this article shows that the sugar of the lectin MAL- II in the soleus muscle of the squirrel's soleus is specific. The significant changes of chain structure SAa2-3Gal in hibernating process may play an important role in the mechanism of anti waste muscle atrophy. Therefore, the preparation of lectin MAL- II magnetic particle complex is prepared by this experiment, and the glycoprotein of SAa2-3Gal is extracted from the soleus muscle of hibernating and hibernating squirrel, respectively, and extracted by LC-ESI-MS/MS The glycoprotein was identified by bioinformatics. 227 glycoproteins and 185 glycoproteins were identified from the pre hibernation group and the hibernation group. Among them, only 141 glycoproteins were identified before hibernating group, and only 99 glycoproteins identified in hibernation group were identified, and the number of glycoproteins identified in the two groups was identified. The annotation analysis for 86.GO (Gene Ontology) functional annotation shows that the glycoproteins identified according to the cell composition are mainly intracellular proteins, and many are macromolecular proteins, mainly distributed in the organelles and membrane structures. According to the molecular functions, these glycoproteins have functions of binding, catalysis and structural molecular activity. .KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that the glycoproteins isolated from pre hibernating group and hibernating group SOL were 40 and 36 respectively, according to the participating biologic process annotation. There are 14 significant enrichment pathways in the pre hibernating group, but only 10 in the hibernating group, and the protein protein interaction analysis using the IntAct database, and the network diagram of the egg white interaction of the pre hibernating group and the hibernating group of the hibernating group and the hibernating group of the hibernating group (The protein-protein inte). Raction network, PPI network), screening out 9 degree values (degree) more than 30 of the core protein. Fourth part: this part of the experiment selected 2 glycoproteins to verify, respectively, heat shock homologous protein (heat shock cognate 70, HSC70) and pyruvate kinase (pyruvate kinase, PK), detection of hibernation in the process of protein expression and knot In addition, the changes in the expression of 14-3-3 protein epsilon protein in the SOL of the rat were detected at different periods of hibernation, and the potential role of the protein in the mechanism of anti waste muscle atrophy was analyzed. Our results showed that the expression of HSC70 protein in hibernating group was significantly reduced by 37.39% (P0.01) compared with hibernating group, compared with hibernating group. The expression of HSC70 protein increased by 51.39% (P0.01) and 41.67% (P0.01) in the hibernating group. There was no significant difference between the three groups in the waking and hibernation group (P0.05), and there was no significant difference between the four groups (hibernating group, hibernating group, waking group, hibernating group) (P0.05), and pre hibernating group. The expression of 14-3-3 protein epsilon in hibernating group was significantly reduced by 42.59% (P0.01). Compared with the hibernating group, the 14-3-3 protein epsilon expression increased by 51.61% (P0.01) and 58.06% (P0.01) in the hibernating group. There was no significant difference between the waking and hibernating groups (P0.05) between the hibernating group and the hibernating group (P0.05). Compared with the pre hibernating group, the SAa2-3Gal structure of the hibernating group glycoprotein HSC70 was significantly reduced by 30.08% (P0.01). Compared with the hibernating group, the SAa2-3Gal structure of the waking group increased by 37.66% (P0.05), and the saliency group increased by 54.50% (P0.01). There was no significant difference between the pre hibernating group and the waking and hibernating group (P0.05). Compared with the hibernating group, the winter group had no significant difference (P0.05). The structure of PK terminal SAa2-3Gal in the sleeping group decreased by 29.40% (P0.01). Compared with the hibernating group, the structure of SAa2-3Gal increased by 30.91% (P0.05) and 34.41% (P0.05) in the hibernating group. There was no significant difference between the waking group and the three groups (P0.05) before hibernating group (P0.05). The results showed that the soleus muscle of the hibernation at different stages of the dormancy Changes in the three proteins of HSC70, PK, 14-3-3 protein epsilon (including protein expression and SAa2-3Gal sugar structure) may be related to its unique mechanism of anti waste muscle atrophy.
【學位授予單位】：西北大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q445
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本文編號：1955417