煤工尘肺患者肺灌洗回收液差异代谢物研究

Differential metabolites of bronchoalveolar lavage fluid from coal worker's pneumoconiosis patients

[背景]通过代谢组学技术研究煤工尘肺发生发展过程中代谢物和代谢通路的变化,探索其发病机制是目前的研究热点。[目的]研究煤工尘肺患者肺灌洗回收液中代谢物的变化,探究煤工尘肺疾病的代谢调节机制。[方法]以符合GBZ 70-2015《职业性尘肺病的诊断》中煤工尘肺诊断标准,进行大容量肺灌洗手术治疗的煤工尘肺患者作为病例组,以进行气管镜检查的气道狭窄患者为对照组。采集病例组肺灌洗回收液样本及对照组正常肺组织的肺灌洗回收液样本,过滤掉大颗粒杂质与黏液后留取上清液于−80℃冰箱冷冻保存备用。经过添加提取液、冷浴超声、高速离心等处理后,运用液相色谱质谱检测技术检测分析病例组与对照组的样本,得到煤工尘肺患者的代谢谱图及相关数据。通过多元统计分析方法筛选出与煤工尘肺疾病发生发展相关的差异代谢产物,并进一步根据差异代谢物信息于京都基因与基因组百科全书数据库(KEGG)中寻找其可能参与的代谢通路。[结果]研究对象的一般情况如体重、身高、年龄、工龄等因素在尘肺壹期、贰期、叁期和对照组4组间,差异均无统计学意义(P>0.05)。煤工尘肺壹期患者与对照组之间共筛选出48种差异代谢物,其中,上调差异代谢物14种,下调差异代谢物34种;煤工尘肺贰期患者与对照组之间共筛选出66种差异代谢物,上调差异代谢物14种,下调差异代谢物有52种;煤工尘肺叁期患者与对照组之间共筛选出63种差异代谢物,上调差异代谢物11种,下调差异代谢物有52种。经汇总分析,在上述比较组中均表达差异的代谢物,即可能与煤工尘肺疾病发生有关的差异代谢物有36种,其中,上调差异代谢物11种,下调差异代谢物有25种。通过查询KEGG数据库共找到4条有意义的差异代谢通路,为亚油酸代谢通路、丙氨酸代谢通路、鞘脂代谢通路及甘油磷脂代谢通路。[结论]肺灌洗回收液代谢组学研究结果显示,煤工尘肺疾病发生发展过程中可能有36种差异代谢物,主要涉及亚油酸代谢、丙氨酸代谢、鞘脂代谢、甘油磷脂代谢通路。

[Background]It is a research hotspot to study the changes of metabolites and metabolic pathways in the process of coal worker's pneumoconiosis(CWP)by metabonomics and to explore its pathogenesis.[Objective]To study the change of metabolites in bronchoalveolar lavage fluid(BALF)of patients with CWP and explore the metabolic regulation mechanism of the disease.[Methods]Patients with CWP who met the national diagnostic criteria according to Diagnosis of occupational pneumoconiosis(GBZ 70-2015)and underwent massive whole lung lavage were selected as the case group,and patients with tracheostenosis who underwent bronchoscopy were selected as the control group.BALF samples were collected from the cases and the controls.After filtering out large particles and mucus,the supernatant was stored in a−80℃refrigerator.The samples were detected and analyzed by liquid chromatography-mass spectrometry after adding extraction solution,cold bath ultrasonication,and high-speed centrifugation,and the metabolic profiles and related data of CWP patients were obtained.The differential metabolites related to the occurrence and development of CWP were screened by multiple statistical analysis;furthermore,we searched the Kyoto Encyclopedia of Genes and Genomes(KEGG)database for potential metabolic pathways involved in the progression.[Results]There was no significant difference in the general conditions of the subjects,such as weight,height,age,and length of service among the stage I group,the stage II group,the stage III group,and the control group(P˃0.05).When comparing the CWP stage I group with the control group,48 differential metabolites were screened out,among which 14 were up-regulated and 34 were down-regulated.A total of 66 differential metabolites were screened out between the patients with CWP stage II and the controls,14 up-regulated and 52 down-regulated differential metabolites.Compared with the control group,63 differential metabolites were screened out in the patients with CWP stage III,including 11 up-regulated and 52 down-regulated differential metabolites.There were 36 differential metabolites that may be related to the occurrence of CWP,among which 11 differential metabolites were up-regulated,and 25 were down-regulated.Four significant differential metabolic pathways were identified through KEGG database query:linoleic acid metabolic pathway,alanine metabolic pathway,sphingolipid metabolic pathway,and glycerophospholipid metabolic pathway.[Conclusion]The metabolomic study of BALF show that there are 36 different metabolites in the occurrence and development of CWP,mainly associating with linoleic acid metabolism,alanine metabolism,sphingolipid metabolism,and glycerophospholipid metabolism pathways.