肺炎支原体感染呼吸道上皮细胞分泌蛋白质组学研究

发布时间：2018-06-05 18:16

本文选题：肺炎支原体 + 分泌蛋白质组　；参考：《浙江大学》2014年博士论文

【摘要】：背景和目的：肺炎支原体(Mycoplasma pneumoniae, MP)是引起各年龄段呼吸道感染的重要病原体之一。MP感染一般呈自限性过程,预后良好,但近年来发现重症与难治性MP肺炎呈上升趋势,部分患儿甚至出现严重并发症,因此受到更加广泛的关注。但MP感染的致病机制目前仍不完全明确,主要有呼吸道上皮细胞粘附、MP直接侵入和免疫学发病机制等学说。虽然MP粘附于呼吸道上皮细胞并与上皮细胞相互作用是MP发病早期中关键的一个环节,但对呼吸道上皮细胞被MP感染后所发生的反应知之甚少。目前已知呼吸道上皮细胞受MP刺激后具有释放某些蛋白质参与免疫炎症反应的能力,但迄今为止所有研究都仅基于对个别已知蛋白的分析测定,缺乏整体性,并且无法对未知蛋白质进行分析。基于上述背景,本研究拟从整体角度采用蛋白质组学技术对MP感染后的呼吸道上皮细胞的分泌蛋白质组进行分离鉴定,比较正常状态与受MP感染后分泌蛋白图谱的差异,通过生物信息学手段分析并在功能上验证差异蛋白,寻找具有生物学意义的上皮细胞分泌蛋白,为进一步阐明MP感染的发病机制提供重要的理论依据；同时寻找特异蛋白作为MP感染诊断/治疗的分子指标/靶点,为临床诊断/治疗提供新的线索。方法： 1.以MP感染人肺腺癌细胞(A549细胞)作为体外研究的模型系统,通过MTT比色法、台盼蓝染色、细胞凋亡检测等方法确立合适的无血清培养条件。 2.利用液相色谱-串联质谱技术联合DeCyder MS无标记定量软件,分析比较MP感染(处理组)与非感染(对照组)情况下,A549细胞分泌蛋白质组的表达变化。 3.综合GO数据库、分泌蛋白预测软件(SingalP、SecretomeP)和ExoCarta数据库对鉴定的所有蛋白进行检索分析,预测所有蛋白的细胞定位。 4. BiNGO软件分析差异分泌蛋白的细胞组分、分子功能及其参与的生物学过程。 5. KEGG数据库分析差异分泌蛋白涉及的生物学通路。 6. STRING软件分析差异分泌蛋白之间的相互作用。 7.挑取6个差异分泌蛋白(ADAM9、SERPNE1、IL-33、IGFBP4、Gal-1、MIF),采用Western blot和RT-PCR对其表达水平进行检测。 8.设计Gal-1的siRNA序列,采用Western blot对其干扰效果进行验证,挑取干扰效率良好的siRNA进行进一步Gal-1的功能研究。Gal-1的功能研究分别采用流式细胞术检测活性氧(ROS)水平,免疫荧光观察γH2AX形成情况,Westernblot检测γH2AX表达情况,以及RT-PCR检测细胞因子表达改变。 9.采用配对对照研究的方式,收集MP肺炎患儿和无合并感染的异物吸入患儿的外周血和肺泡灌洗液,应用ELISA方法检测其中IL-33的水平。结果： 1.无血清培养24h时,A549细胞存活率均在98.5%以上,其细胞形态、增殖速率和存活率与含血清培养均无明显差异。 2.液相色谱-串联质谱技术共鉴定256个非冗余蛋白质,其中对照组233个,处理组237个。经DeCyder MS无标记定量分析共发现表达差异超过1.5倍(p0.05)的蛋白质有113个,其中65个在处理组表达上调,48个在处理组表达下调。 3.SingalP和SecretomeP软件预测显示,在鉴定的256个蛋白中,83个属于经典途径分泌的蛋白,69个属于非经典途径分泌蛋白。同时,ExoCarta数据库分析发现,在鉴定的256个蛋白中,190个蛋白能通过外泌小体到达胞外。GO数据库资源检索发现鉴定的256个蛋白中的大部分蛋白对应一种以上的细胞成分分类注释,可同时涉及细胞核、细胞膜、细胞胞浆等多种细胞组分。 4. BiNGO软件分析显示,细胞组分方面：113个差异分泌蛋白并不是来自某一特定的细胞器,而是来自多个不同的细胞器,比如线粒体、溶酶体等；分子功能方面：上调的65个蛋白主要与氧化还原活性、蛋白结合活性、酶调节功能有关,而下调的48个蛋白主要与酶活性抑制和水解酶活性有关；生物学过程方面：上调的蛋白主要涉及糖代谢、炎症反应、氧化还原和防御反应这四个过程,而下调的蛋白主要涉及系统发育过程。 5. KEGG通路数据库的检索发现113个差异蛋白共涉及85条生物学通路,经聚类分析发现其主要富集于丙酮酸代谢、糖酵解/糖原合成、病原体感染等11条通路。 6. STRING在线分析发现113个差异蛋白中,有88个蛋白可与其他蛋白存在相互作用。其中,上调的65个蛋白主要形成3个小的蛋白与蛋白相互作用网络,分别为应激反应相关蛋白网络、信号通路相关蛋白网络和细胞代谢相关网络；下调的48个蛋白,主要形成2个小的蛋白与蛋白相互作用网络,分别为糖代谢相关蛋白网络和生物学过程负性调节相关蛋白网络。 7. RT-PCR和Western blot结果显示ADAM9、SERPNE1、IL-33、IGFBP4、 Gal-1、MIF这6个蛋白的表达水平与质谱鉴定结果一致。 8. siRNA干扰Gal-1表达后,A549细胞内ROS水平明显降低、细胞核内yH2AX焦点形成明显减少、yH2AX表达水平亦降低,同时细胞因子IL-8和IL-18在24h时表达水平明显升高。 9.IL-33在MP肺炎患儿的血清和肺泡灌洗液中的水平显著高于无感染的支气管异物患儿。结论： 1.MP感染可引起A549细胞分泌蛋白表达的改变；运用非标记定量鸟枪法蛋白质组学技术分析A549细胞在MP感染情况下的差异分泌蛋白质组,是探求MP发病机制高效、可行的研究策略。 2.非经典分泌途径,特别是外泌小体运输是MP感染后分泌蛋白重要的分泌途径之一。 3.MP感染后MIF、HSPB1、Gal-1等应激相关蛋白,YWHAZ、ADAM9、 PRDX1等免疫炎症相关蛋白,以及GPI、LDHB、ENO1等代谢相关蛋白的表达均上调,提示MP感染可引起宿主细胞应激反应、免疫反应、细胞代谢等多方面的功能改变。 4.Gal-1和IL-33可能参与了MP对细胞的损伤效应。
[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 纬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 .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.6

【共引文献】