应用iTRAQ技术对动式染尘矽肺大鼠肺组织差异蛋白的定量分析

朱莹; 段雨霞; 程琦云; 姚婧昕; 徐洪; 袁聚祥

doi:10.16462/j.cnki.zhjbkz.2020.07.015

应用iTRAQ技术对动式染尘矽肺大鼠肺组织差异蛋白的定量分析

doi: 10.16462/j.cnki.zhjbkz.2020.07.015

1.
063210 唐山, 华北理工大学公共卫生学院
2.
063210 唐山, 华北理工大学医学实验研究中心, 河北省器官纤维化重点实验室

基金项目:

国家科技部重点研发计划资助项目 2016YFC0900605

河北省医学科学研究课题 20190108

唐山市科技计划 18130206a

详细信息

通讯作者:
袁聚祥, E-mail:yuanjx@ncst.edu.cn

中图分类号: R181.1
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- 被引次数: 0
出版历程
- 收稿日期: 2019-11-07
- 修回日期: 2020-02-03
- 刊出日期: 2020-07-10

Quantitative analysis of differential proteins in lung tissue of silicosis rats by using iTRAQ profile

1.
School of Public Health, North China University of Science and Technology, Tangshan 063210, China
2.
Medical Research Center, The Hebei Key Laboratory for Organ Fibrosis Research, North China University of Science and Technology, Tangshan, China

Funds:

Research and Development Program Funded by the Ministry of Science and Technology of the People's Republic of China 2016YFC0900605

Medical Science Research Project of Hebei Province 20190108

Science and Technology Project of Tangshan 18130206a

More Information

Corresponding author: YUAN Ju-xiang, E-mail:yuanjx@ncst.edu.cn

摘要

摘要: 目的筛选动式染尘矽肺大鼠肺组织差异蛋白并在动物体内及体外中验证，以期为矽肺早期诊断及相关机制研究提供新思路。方法构建动式染尘矽肺大鼠动物模型，采用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantification，iTRAQ）技术联合液相色谱-串联质谱（liquid chromatography-trandem mass spectrometry，LC-MS/MS）技术筛选肺组织中的差异蛋白。免疫印迹试验检测差异蛋白在动物模型肺组织和SiO₂诱导的小鼠Ⅱ型肺泡上皮细胞中的表达。结果共得到471个差异蛋白，其中252个上调蛋白，219个下调蛋白。差异倍数>1.5，共有28种差异表达蛋白，其中上调差异蛋白20种。在动物模型肺组织及SiO₂诱导的小鼠Ⅱ型肺泡上皮细胞中Factor B、PTPN2、VRK1和MOT4蛋白表达上调，差异均具有统计学意义（均有P < 0.05）。结论差异蛋白Factor B、PTPN2、VRK1和MOT4蛋白可能是矽肺纤维化进程中的关键蛋白，为矽肺纤维化的研究提供了新靶点。
- 蛋白质组学 /
- 小鼠Ⅱ型肺泡上皮细胞 /
- 矽肺 /
- 差异蛋白
Abstract: Objective We aimed to screened the differential protein of silicosis in the lung tissue of rats exposed to movable dust and verified it in vivo and in vitro, so as to provide new targeted for the early diagnosis of silicosis and the related molecular mechanism. Methods The animal model of silicosis was established by rats exposed to movable dust. The differential proteins in lung tissue were screened by isobaric tags for relative and absolute quantification(iTRAQ) and liquid chromatography-trandem mass spectrometry(LC-MS/MS). Western blot was used to detect the expression of differential protein in lung tissue of animal model and type Ⅱ alveolar epithelial cells induced by SiO₂. Results Our results identified a total of 471 differential proteins, 252 of which were up regulated and 219 down regulated. There were 28 differentially expressed proteins, twenty of which were up-regulated. The expression of Factor B, PTPN2, VRK1 and MOT4 protein were up-regulated in lung tissue of animal model and type Ⅱ alveolar epithelial cells induced by SiO₂ (all P < 0.05). Conclusions Differential proteins Factor B, PTPN2, VRK1 and MOT4 proteins may the novel key proteins in the process of silicosis fibrosis, which provides a new target for silicosis fibrosis.
- Proteomic /
- Mouse type Ⅱ alveolar epithelial cell /
- Silicosis /
- Differential protein

HTML全文

图 1 矽肺动物模型肺组织差异蛋白GO分析结果

注：A：细胞组分分析；B：分子功能分析；C：生物过程分析。

Figure 1. Go analysis of the difference protein in lung tissue of silicosis animal models

下载: 全尺寸图片幻灯片

图 2 尘肺动物模型肺组织差异蛋白KEGG分析

注：A：KEGG分析；B：相互作用网络分析。

Figure 2. KEGG analysis of differential protein in lung tissue of pneumoconiosis animal models

下载: 全尺寸图片幻灯片

图 3 矽肺动物模型肺组织验证结果

注：A：体内验证Western条带图；B：差异蛋白统计图。

Figure 3. Validation results of lung tissue in silicosis animal models

下载: 全尺寸图片幻灯片

图 4 SiO₂诱导的小鼠Ⅱ型肺泡上皮细胞验证结果

注：A：体外验证Western条带图；B：差异蛋白统计图。

Figure 4. Validation results of SiO₂ induced typeⅡ alveolar epithelial cells in mice

下载: 全尺寸图片幻灯片

参考文献(27)

[1]	中华人民共和国国家卫生健康委员会. 2017年我国卫生健康事业发展统计公报[EB/OL]. (2018-06-12)[2019-12-12]. http://www.nhfpc.gov.cn/guihuaxxs/s10743/201806/44e3cdfe11fa4c7f928c879d435b6a18.shtml. National Health and Health Commission of the People's Republic of China. 2017 statistical bulletin of China's health development[EB/OL]. (2018-06-12)[2019-12-12]. http://www.nhfpc.gov.cn/guihuaxxs/s10743/201806/44e3cdfe11fa4c7f928c879d435b6a18.shtml.
[2]	吴二丰, 张勤丽, 李海学.尘肺病与现代影像学技术[J].中国工业医学杂志, 2013, 16(12):2026-2028. DOI:10.11723/mtgyyx 1007-9564201312044. Wu EF, Zhang QL, Li HX. Pneumoconiosis and modern imaging technology[J]. Chin J Industrial Med, 2013, 16(12):2026-2028. DOI:10.11723/mtgyyx 1007-9564201312044.
[3]	Chu C, Yan N, Du Y, et al. iTRAQ-based proteomic analysis reveals the accumulation of bioactive compounds in Chinese wild rice (Zizania latifolia) during germination[J]. Food chemistry, 2019, 289:635-644. DOI: 10.1016/j.foodchem.2019.03.092.
[4]	Martyniuk CJ, Alvarez S, Denslow ND. DIGE and iTRAQ as biomarker discovery tools in aquatic toxicology[J]. Ecotoxicol Environ Saf, 2012, 76(2):3-10. DOI: 10.1016/j.ecoenv.2011.09.020.
[5]	Jiang Z, Zhang CH, Gan L, et al. iTRAQ-based quantitative proteomics approach identifies novel diagnostic biomarkers that were essential for glutamine metabolism and redox homeostasis for gastric cancer[J]. Proteom Clin Appl, 2019, 13(4):1-8. DOI: 10.1002/prca.201800038.
[6]	Du C, Weng Y, Lou J, et al. Isobaric tags for relative and absolute quantitationbased proteomics reveals potential novel biomarkers for the early diagnosis of acute myocardial infarction within 3 h[J]. Int J Mol Med, 2019, 43(5):1991-2004. DOI: 10.3892/ijmm.2019.4137.
[7]	Zhou FY, Chen XQ, Chen GX, et al. Identification of SAA and ACTB as potential biomarker of patients with severe HFMD using iTRAQ quantitative proteomics[J]. Clin Biochem, 2019, 67:1-6. DOI: 10.1016/j.clinbiochem.2019.02.011.
[8]	Arora A, Patil V, Kundu P, et al. Serum biomarkers identification by iTRAQ and verification by MRM:S100A8/S100A9 levels predict tumor-stroma involvement and prognosis in Glioblastoma[J]. Sci Rep, 2019, 9(1):2749. DOI: 10.1038/s41598-019-39067-8.
[9]	Fu R, Li Q, Fan R, et al. iTRAQ-based secretome reveals that SiO₂ induces the polarization of RAW264.7 macrophages by activation of the NOD-RIP2-NF-kappaB signaling pathway[J]. Environ Toxicol Pharmacol, 2018, 63:92-102. DOI: 10.1016/j.etap.2018.08.010.
[10]	Na M, Hong X, Fuyu J, et al. Proteomic profile of TGF-beta treated lung fibroblasts identifies novel markers of activated fibroblasts in the silica exposed rat lung[J]. Exp Cell Res, 2019, 375(2):1-9. DOI: 10.1016/j.yexcr.2019.01.010.
[11]	高学敏, 耿玉聪, 李世峰, 等.动式染尘构建矽肺大鼠模型及ACE2-Ang(1-7)-Mas轴动态变化规律[J].医学研究生学报, 2016, 29(12):1276-1280. DOI: 10.16571/j.cnki.1008-8199.2016.12.010. Gao XM, Geng YC, Li SF, et al. Establishment of a rat silicosis model by dynamic silica dust exposure and its dynamic variation rule of ACE2-Ang(1-7)-Mas axis[J]. J Med Postgra, 2016, 29(12):1276-1280. DOI: 10.16571/j.cnki.1008-8199.2016.12.010.
[12]	Mi H, Muruganujan A, Casagrande JT, et al. Large-scale gene function analysis with the PANTHER classification system[J]. Nat Protoc, 2013, 8(8):1551-1566. DOI: 10.1038/nprot.2013.092.
[13]	Zhang Y, Wu XF, Tao WA. Characterization and applications of extracellular vesicle proteome with post-translational modifications[J]. Trends Analyt Chem, 2018, 107:21-30.DOI: 10.1016/j.trac.2018.07.014.
[14]	Viczian A, Adam E, Staudt AM, et al. Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling[J]. New Phytologist, 2020, 225(4):1635-1650. DOI: 10.1111/nph.16243.
[15]	Elagamey E, Narula K, Chakraborty N, et al. Extracellular matrix proteome:isolation of ECM proteins for proteomics studies[J]. Methods in Molecular Biology, 2019;2057:155-172. DOI: 10.1007/978-1-4939-9790-9_14.
[16]	Li G, Eckert MA, Chang JW, et al. Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation[J]. Proc Natl Acad Sci USA, 2019, 116(43):21493-21500. DOI: 10.1073/pnas.1912934116.
[17]	朱琰琰, 罗执芬, 崔永霞, 等.人类蛋白组学草图的肺癌分子标记物初探[J].生物信息学, 2016, 14(1):43-48. DOI:10. 3969/j.issn.1672-5565.2016.01.08. Zhu YY, Luo ZF, Cui YX, et al. Pilot study on biomarkers for lung cancer based on the draft of human proteome[J]. Chinese Journal of Bioinformatics, 2016, 14(1):43-48. DOI: 10.3969/j.issn.1672-5565.2016.01.08.
[18]	Ye H, Sun L, Huang X, et al. A proteomic approach for plasma biomarker discovery with 8-plex iTRAQ labeling and SCX-LC-MS/MS[J]. Mol Cell Biochem, 2010, 343(1-2):91-99. DOI: 10.1007/s11010-010-0502-x.
[19]	Akiyama N, Hozumi H, Isayama T, et al. Clinical significance of serum S100 calcium-binding protein A4 in idiopathic pulmonary fibrosis[J]. Respirology, 2020, 25(7):743-749. DOI: 10.1111/resp.13707.
[20]	Liu H, Yu H, Cao Z, et al. Kaempferol modulates autophagy and alleviates silica-induced pulmonary fibrosis[J]. DNA Cell Biol, 2019, 38(12):1418-1426. DOI: 10.1089/dna.2019.4941.
[21]	Li N, Feng F, Wu K, et al. Inhibitory effects of astragaloside IV on silica-induced pulmonary fibrosis via inactivating TGF-beta1/Smad3 signaling[J]. Biomed Pharmacother, 2019, 119:109387. DOI: 10.1016/j.biopha.2019.109387.
[22]	刘燕, 耿玉聪, 徐洪, 等. Ac-SDKP调控Gαs/Gαi信号抑制大鼠矽肺纤维化的作用[J].西安交通大学学报(医学版), 2017, 38(1):24-28. DOI: 10.7652/jdyxb201701005. Liu Y, Geng YC, Xu H, et al.Ac-SDKP inhibits silicosis fibrosis in rats by regulating Gαs/Gαi signaling[J]. J Xi'an Med Univ, 2017, 38(1):24-28. DOI: 10.7652/jdyxb201701005.
[23]	Wu M, Chen MH, Jing Y, et al. The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1[J]. Am J Physiol Renal Physiol, 2016, 310(11):1284-1294. DOI: 10.1152/ajprenal.00428.2015.
[24]	Tong H, Liu XH, Li T, et al. INTS8 accelerates the epithelial-to-mesenchymal transition in hepatocellular carcinoma by upregulating the TGF-beta signaling pathway[J]. Cancer Manag Res, 2019, 11:1869-1879. DOI: 10.2147/cmar.s184392.
[25]	Mon AM, MacKinnon AC JR, Traktman P. Overexpression of the VRK1 kinase, which is associated with breast cancer, induces a mesenchymal to epithelial transition in mammary epithelial cells[J]. PLoS One, 2018, 13(9):e0203397. DOI: 10.1371/journal.pone.0203397.
[26]	Zhang S, Xu W, Wang H, et al. Inhibition of CREB-mediated ZO-1 and activation of NF-κB induced IL-6 by colonic epithelial MCT4 destroys intestinal barrier function[J]. Cell Prolif, 2019, 52(6):e12673. DOI: 10.1111/cpr.12673.
[27]	杨萌, 姚三巧.胶原样结构巨噬细胞受体和上皮间质转化在矽肺发病机制中作用研究进展[J].中国职业医学, 2017, 44(6):371-375. DOI: 10.11763/j.issn.2095-2619.2017.03.027. Yang M, Yao SQ. Study advance in the role of collagen-like macrophage receptors andepithelial-mesenchymal transition in the pathogenesis of silicosis[J]. Chin Occup Med, 2017, 44(6):371-375. DOI: 10.11763/j.issn.2095-2619.2017.03.027.