發(fā)布時(shí)間：2018-06-05 18:16

  本文選題：肺炎支原體 + 分泌蛋白質(zhì)組　； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：背景和目的： 肺炎支原體(Mycoplasma pneumoniae, MP)是引起各年齡段呼吸道感染的重要病原體之一。MP感染一般呈自限性過程,預(yù)后良好,但近年來發(fā)現(xiàn)重癥與難治性MP肺炎呈上升趨勢(shì),部分患兒甚至出現(xiàn)嚴(yán)重并發(fā)癥,因此受到更加廣泛的關(guān)注。但MP感染的致病機(jī)制目前仍不完全明確,主要有呼吸道上皮細(xì)胞粘附、MP直接侵入和免疫學(xué)發(fā)病機(jī)制等學(xué)說。雖然MP粘附于呼吸道上皮細(xì)胞并與上皮細(xì)胞相互作用是MP發(fā)病早期中關(guān)鍵的一個(gè)環(huán)節(jié),但對(duì)呼吸道上皮細(xì)胞被MP感染后所發(fā)生的反應(yīng)知之甚少。目前已知呼吸道上皮細(xì)胞受MP刺激后具有釋放某些蛋白質(zhì)參與免疫炎癥反應(yīng)的能力,但迄今為止所有研究都僅基于對(duì)個(gè)別已知蛋白的分析測(cè)定,缺乏整體性,并且無法對(duì)未知蛋白質(zhì)進(jìn)行分析。 基于上述背景,本研究擬從整體角度采用蛋白質(zhì)組學(xué)技術(shù)對(duì)MP感染后的呼吸道上皮細(xì)胞的分泌蛋白質(zhì)組進(jìn)行分離鑒定,比較正常狀態(tài)與受MP感染后分泌蛋白圖譜的差異,通過生物信息學(xué)手段分析并在功能上驗(yàn)證差異蛋白,尋找具有生物學(xué)意義的上皮細(xì)胞分泌蛋白,為進(jìn)一步闡明MP感染的發(fā)病機(jī)制提供重要的理論依據(jù)；同時(shí)尋找特異蛋白作為MP感染診斷/治療的分子指標(biāo)/靶點(diǎn),為臨床診斷/治療提供新的線索。 方法： 1.以MP感染人肺腺癌細(xì)胞(A549細(xì)胞)作為體外研究的模型系統(tǒng),通過MTT比色法、臺(tái)盼藍(lán)染色、細(xì)胞凋亡檢測(cè)等方法確立合適的無血清培養(yǎng)條件。 2.利用液相色譜-串聯(lián)質(zhì)譜技術(shù)聯(lián)合DeCyder MS無標(biāo)記定量軟件,分析比較MP感染(處理組)與非感染(對(duì)照組)情況下,A549細(xì)胞分泌蛋白質(zhì)組的表達(dá)變化。 3.綜合GO數(shù)據(jù)庫、分泌蛋白預(yù)測(cè)軟件(SingalP、SecretomeP)和ExoCarta數(shù)據(jù)庫對(duì)鑒定的所有蛋白進(jìn)行檢索分析,預(yù)測(cè)所有蛋白的細(xì)胞定位。 4. BiNGO軟件分析差異分泌蛋白的細(xì)胞組分、分子功能及其參與的生物學(xué)過程。 5. KEGG數(shù)據(jù)庫分析差異分泌蛋白涉及的生物學(xué)通路。 6. STRING軟件分析差異分泌蛋白之間的相互作用。 7.挑取6個(gè)差異分泌蛋白(ADAM9、SERPNE1、IL-33、IGFBP4、Gal-1、MIF),采用Western blot和RT-PCR對(duì)其表達(dá)水平進(jìn)行檢測(cè)。 8.設(shè)計(jì)Gal-1的siRNA序列,采用Western blot對(duì)其干擾效果進(jìn)行驗(yàn)證,挑取干擾效率良好的siRNA進(jìn)行進(jìn)一步Gal-1的功能研究。Gal-1的功能研究分別采用流式細(xì)胞術(shù)檢測(cè)活性氧(ROS)水平,免疫熒光觀察γH2AX形成情況,Westernblot檢測(cè)γH2AX表達(dá)情況,以及RT-PCR檢測(cè)細(xì)胞因子表達(dá)改變。 9.采用配對(duì)對(duì)照研究的方式,收集MP肺炎患兒和無合并感染的異物吸入患兒的外周血和肺泡灌洗液,應(yīng)用ELISA方法檢測(cè)其中IL-33的水平。 結(jié)果： 1.無血清培養(yǎng)24h時(shí),A549細(xì)胞存活率均在98.5%以上,其細(xì)胞形態(tài)、增殖速率和存活率與含血清培養(yǎng)均無明顯差異。 2.液相色譜-串聯(lián)質(zhì)譜技術(shù)共鑒定256個(gè)非冗余蛋白質(zhì),其中對(duì)照組233個(gè),處理組237個(gè)。經(jīng)DeCyder MS無標(biāo)記定量分析共發(fā)現(xiàn)表達(dá)差異超過1.5倍(p0.05)的蛋白質(zhì)有113個(gè),其中65個(gè)在處理組表達(dá)上調(diào),48個(gè)在處理組表達(dá)下調(diào)。 3.SingalP和SecretomeP軟件預(yù)測(cè)顯示,在鑒定的256個(gè)蛋白中,83個(gè)屬于經(jīng)典途徑分泌的蛋白,69個(gè)屬于非經(jīng)典途徑分泌蛋白。同時(shí),ExoCarta數(shù)據(jù)庫分析發(fā)現(xiàn),在鑒定的256個(gè)蛋白中,190個(gè)蛋白能通過外泌小體到達(dá)胞外。GO數(shù)據(jù)庫資源檢索發(fā)現(xiàn)鑒定的256個(gè)蛋白中的大部分蛋白對(duì)應(yīng)一種以上的細(xì)胞成分分類注釋,可同時(shí)涉及細(xì)胞核、細(xì)胞膜、細(xì)胞胞漿等多種細(xì)胞組分。 4. BiNGO軟件分析顯示,細(xì)胞組分方面：113個(gè)差異分泌蛋白并不是來自某一特定的細(xì)胞器,而是來自多個(gè)不同的細(xì)胞器,比如線粒體、溶酶體等；分子功能方面：上調(diào)的65個(gè)蛋白主要與氧化還原活性、蛋白結(jié)合活性、酶調(diào)節(jié)功能有關(guān),而下調(diào)的48個(gè)蛋白主要與酶活性抑制和水解酶活性有關(guān)；生物學(xué)過程方面：上調(diào)的蛋白主要涉及糖代謝、炎癥反應(yīng)、氧化還原和防御反應(yīng)這四個(gè)過程,而下調(diào)的蛋白主要涉及系統(tǒng)發(fā)育過程。 5. KEGG通路數(shù)據(jù)庫的檢索發(fā)現(xiàn)113個(gè)差異蛋白共涉及85條生物學(xué)通路,經(jīng)聚類分析發(fā)現(xiàn)其主要富集于丙酮酸代謝、糖酵解/糖原合成、病原體感染等11條通路。 6. STRING在線分析發(fā)現(xiàn)113個(gè)差異蛋白中,有88個(gè)蛋白可與其他蛋白存在相互作用。其中,上調(diào)的65個(gè)蛋白主要形成3個(gè)小的蛋白與蛋白相互作用網(wǎng)絡(luò),分別為應(yīng)激反應(yīng)相關(guān)蛋白網(wǎng)絡(luò)、信號(hào)通路相關(guān)蛋白網(wǎng)絡(luò)和細(xì)胞代謝相關(guān)網(wǎng)絡(luò)；下調(diào)的48個(gè)蛋白,主要形成2個(gè)小的蛋白與蛋白相互作用網(wǎng)絡(luò),分別為糖代謝相關(guān)蛋白網(wǎng)絡(luò)和生物學(xué)過程負(fù)性調(diào)節(jié)相關(guān)蛋白網(wǎng)絡(luò)。 7. RT-PCR和Western blot結(jié)果顯示ADAM9、SERPNE1、IL-33、IGFBP4、 Gal-1、MIF這6個(gè)蛋白的表達(dá)水平與質(zhì)譜鑒定結(jié)果一致。 8. siRNA干擾Gal-1表達(dá)后,A549細(xì)胞內(nèi)ROS水平明顯降低、細(xì)胞核內(nèi)yH2AX焦點(diǎn)形成明顯減少、yH2AX表達(dá)水平亦降低,同時(shí)細(xì)胞因子IL-8和IL-18在24h時(shí)表達(dá)水平明顯升高。 9.IL-33在MP肺炎患兒的血清和肺泡灌洗液中的水平顯著高于無感染的支氣管異物患兒。 結(jié)論： 1.MP感染可引起A549細(xì)胞分泌蛋白表達(dá)的改變；運(yùn)用非標(biāo)記定量鳥槍法蛋白質(zhì)組學(xué)技術(shù)分析A549細(xì)胞在MP感染情況下的差異分泌蛋白質(zhì)組,是探求MP發(fā)病機(jī)制高效、可行的研究策略。 2.非經(jīng)典分泌途徑,特別是外泌小體運(yùn)輸是MP感染后分泌蛋白重要的分泌途徑之一。 3.MP感染后MIF、HSPB1、Gal-1等應(yīng)激相關(guān)蛋白,YWHAZ、ADAM9、 PRDX1等免疫炎癥相關(guān)蛋白,以及GPI、LDHB、ENO1等代謝相關(guān)蛋白的表達(dá)均上調(diào),提示MP感染可引起宿主細(xì)胞應(yīng)激反應(yīng)、免疫反應(yīng)、細(xì)胞代謝等多方面的功能改變。 4.Gal-1和IL-33可能參與了MP對(duì)細(xì)胞的損傷效應(yīng)。
[Abstract]:Background and Purpose :

Mycoplasma pneumoniae ( MP ) is one of the important pathogens causing respiratory tract infection in all ages . MP infection is generally self - limiting and has a good prognosis . However , the pathogenesis of MP infection is not completely clear .

Based on the above background , this study intends to isolate and identify the secretory protein group of respiratory epithelial cells infected with MP from the whole angle , compare the difference between the normal state and the secretion protein map after MP infection , analyze and functionally verify the difference protein by means of bioinformatics , search for the epithelial cell secretory protein with biological significance , and provide an important theoretical basis for further clarifying the pathogenesis of MP infection ;
meanwhile , finding specific protein as the molecular index / target of MP infection diagnosis / treatment , and providing a new clue for clinical diagnosis / treatment .

Method :

1 . Using MP - infected human lung adenocarcinoma cells ( A549 cells ) as the model system of in vitro study , the appropriate serum - free culture conditions were established by MTT assay , trypan blue staining and apoptosis detection .

2 . Using liquid chromatography - tandem mass spectrometry combined with DeCyder MS without labeling quantitative software , the expression of protein secretion in A549 cells was analyzed by comparing MP infection ( treatment group ) with non - infection ( control group ) .

3 . The comprehensive GO database , the secreted protein prediction software ( SingalP ) , the ExotomeP ) and the ExoCarta database were used for the search and analysis of all the proteins identified , and the cellular localization of all proteins was predicted .

4 . BiNGO software analyzes the cellular components , molecular functions and the biological processes involved in the secretion of proteins .

5 . The KEGGS database analyzes the biological pathways involved in the differential secretion protein .

6.0 software analyzes the interaction between the differentially secreted proteins .

7 . The expression levels of 6 differentially expressed proteins were detected by Western blot and RT - PCR .

8 . The siRNA sequence of Gal - 1 was designed . Western blot was used to verify the interference effect . The function of Gal - 1 was studied . The function of Gal - 1 was studied by flow cytometry to detect reactive oxygen ( ROS ) level . The expression of 緯H2AX was detected by Western blot , and the expression of cytokines was detected by RT - PCR .

9 . Peripheral blood and alveolar lavage fluid were collected from children with MP pneumonia and without complicated infection by paired control , and the level of IL - 33 was detected by ELISA .

Results :

1 . The survival rate of A549 cells was more than 98.5 % at 24h without serum culture , and the cell morphology , proliferation rate and survival rate were not significantly different from those in serum - containing culture .

2 . A total of 256 non - redundant proteins were identified by liquid chromatography - tandem mass spectrometry , 233 in the control group and 237 in the treatment group .

The results showed that , among the 256 proteins identified , 83 belonged to the classical pathway secreted proteins , 69 belonged to non - classical pathway secretory proteins . At the same time , the ExoCarta database analysis showed that 190 of the 256 proteins identified were able to reach the extracellular domain through the exosomes . Most of the 256 proteins identified by GO database resource search correspond to more than one cell component classification note , which can also relate to cell components such as cell nuclei , cell membranes , cell cytoplasm , and the like .

4 . BiNGO software analysis showed that 113 differentially secreted proteins were not from a particular organelle but from a number of different organelle , such as mitochondria , lysosomes , etc .
Molecular function : The 65 proteins up - regulated are mainly related to redox activity , protein binding activity , enzyme regulation function , while the down - regulated 48 proteins are mainly related to enzyme activity inhibition and hydrolytic enzyme activity ;
The biological process aspect : up - regulated protein mainly involves four processes of sugar metabolism , inflammatory reaction , redox and defensive reaction , while the down - regulated protein mainly involves the development of the system .

5 . It was found that 113 differentially expressed proteins involved 85 biological pathways , which were mainly enriched in 11 pathways , such as pyruvate metabolism , glycolytic / glycogen synthesis , pathogen infection , etc .

6 . On - line analysis found that 88 of 113 differentially expressed proteins could interact with other proteins . Among them , the up - regulated 65 proteins mainly formed three small proteins interacting with proteins , which were the related protein networks , signal pathway related protein networks and cellular metabolism related networks , respectively .
The down - regulated 48 proteins mainly form two small proteins interacting with proteins , which are the related protein network of sugar metabolism - related protein network and biological process negative regulation , respectively .

7 . The results of RT - PCR and Western blot showed that the expression levels of these six proteins were consistent with the results of mass spectrometry .

8 . After interfering with the expression of Gal - 1 , the level of ROS in A549 cells was significantly decreased , the focal formation of yH2AX in the nucleus decreased significantly , and the expression level of yH2AX was also decreased , while the level of IL - 8 and IL - 18 increased significantly at 24 h .

9 . The levels of IL - 33 in serum and alveolar lavage fluid of children with MP pneumonia were significantly higher than those in children without infection .

Conclusion :

1 . MP infection can cause the change of the expression of secretory protein in A549 cells .
By using non - labeled quantitative shotgun protein group technique to analyze the differentially secreted protein group of A549 cells under MP infection , it is an effective and feasible research strategy for investigating the pathogenesis of MP .

2 . Non - classical secretory pathway , especially exosome transport is one of the important secretory pathways of secretory protein after MP infection .

3 . After MP infection , the related proteins , such as MIF , HSPB1 , Gal - 1 , etc . , were up - regulated by immune inflammatory related protein , such as YWHAZ , DHB , PRDX1 , and so on , suggesting that MP infection could cause various functions of host cell response , immune response , cell metabolism and so on .

4 . Gal - 1 and IL - 33 may participate in the effect of MP on cell damage .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.6

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