过表达死亡蛋白相关激酶2对TGF-β_(1)诱导的人肺成纤维细胞自噬和胶原蛋白分泌的影响  

Effect of overexpression of death-associated protein kinase 2 on TGF-β_(1)-induced autophagy and collagen secretion in human lung fibroblasts

作　　者：张江柳 姚君 黄媛媛[1] 何杰[1] Zhang Jiangliu;Yao Jun;Huang Yuanyuan;He Jie(Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital of Chengdu Medical College,Chengdu 610500,China)

机构地区：[1]成都医学院第一附属医院呼吸与危重症医学科,成都610500

出　　处：《国际呼吸杂志》2022年第22期1720-1731,共12页International Journal of Respiration

基　　金：四川省医学科研课题计划基金项目(S21054)。

摘　　要：目的结合生物信息学分析结果研究死亡蛋白相关激酶2(DAPK2)在肺纤维化中的作用及分子机制。方法从GEO数据库下载特发性肺纤维化(IPF)数据集GSE150910进行自噬相关基因的差异表达分析, 利用极端梯度提升法筛选出关键基因。用t分布随机邻域嵌入法在单细胞测序数据集GSE135893中验证关键基因在细胞中的表达, 采用Kaplan-Meier法在GSE28221和GSE93606数据集中绘制DAPK2高、低表达组患者的生存曲线。将12只6~8周龄的C57BL/6小鼠随机分为对照组和实验组, 每组6只, 实验组小鼠气管内滴注博来霉素构建肺纤维化模型, 取小鼠肺组织进行Masson染色、免疫组织化学染色和实时荧光定量聚合酶链式反应(RT-qPCR)检测DAPK2的蛋白和mRNA表达水平。将人肺成纤维细胞分为空载体慢病毒组、转化生长因子β_(1)(TGF-β_(1))组、空载体慢病毒+TGF-β_(1)组、过表达DAPK2慢病毒+TGF-β_(1)组, 应用蛋白质印迹法检测各组自噬相关蛋白以及Ⅰ型胶原蛋白(Collage Ⅰ)、Ⅲ型胶原蛋白(Collage Ⅲ)的表达, 并应用MDC染色检测各组细胞的自噬小体所占比例。结果 GSE150910数据集中筛选出32个差异表达的衰老相关基因, 其中8个上调, 24个下调。极端梯度提升法筛选出关键自噬相关基因DAPK2。单细胞测序分析提示DAPK2主要表达于肺成纤维细胞中, 且在IPF患者肺组织内成纤维细胞的表达量低于正常肺组织(P<0.05);高表达DAPK2的IPF患者总体生存时间较低表达DAPK2的IPF患者更长(P<0.05)。Masson染色提示小鼠肺纤维化模型造模成功, RT-qPCR及免疫组织化学染色结果提示DAPK2在博来霉素诱导的小鼠肺纤维化组织中为低表达(P值均<0.05)。与TGF-β_(1)组和空载体慢病毒+TGF-β_(1)组比较, 过表达DAPK2慢病毒+TGF-β_(1)组细胞中的LC3Ⅱ和Beclin 1蛋白表达水平升高, 而哺乳动物雷帕霉素靶蛋白、Collage Ⅰ、Collage Ⅲ降低(P值均<0.05);自噬相关蛋白及ColObjective To investigate the role and molecular mechanism of death-associated protein kinase 2(DAPK2)in pulmonary fibrosis by bioinformatics analysis.Methods IPF dataset GSE150910 was downloaded from GEO database for differential expression analysis of autophagy-related genes,and the extreme Gradient Boosting algorithm was used to screen out the key genes.T-distributed stochastic neighbor embedding(T-SNE)was used to verify the expression of key genes in cells in the single-cell sequencing dataset GSE135893.Kaplan-Meier method was used to plot the survival curves of patients with high and low DAPK2 expression groups in GSE28221 and GSE93606 datasets.Twelve C57BL/6 mice aged 6-8 weeks were randomly divided into the control group and the experimental group,with 6 mice in each group.The experimental group was injected with Bleomycin(BLM)intratracheally to establish the pulmonary fibrosis model.The expression levels of DAPK2 protein and mRNA were detected by Masson staining,immunohistochemistry,and real-time quantitative polymerase chain reaction(RT-qPCR).Human lung fibroblasts(HLFs)were divided into empty vector lentivirus group,transforming growth factor-β_(1)(TGF-β_(1))group,empty vector lentivirus+TGF-β_(1) group,and overexpressing DAPK2 lentivirus+TGF-β_(1) group.Western blotting was used to detect the expressions of autophagy-related proteins,CollageⅠ,and CollageⅢin each group.MDC staining was used to detect the proportion of autophagosomes in each group.Results A total of 32 differentially expressed aging-related genes were screened from the GSE150910 dataset,among which 8 genes were up-regulated and 24 genes were down-regulated.The key autophagy-related gene DAPK2 was screened by extreme gradient boosting algorithm.Single cell sequencing analysis suggested that DAPK2 was mainly expressed in lung fibroblasts,and the expression of DAPK2 in lung fibroblasts of IPF patients was lower than that in normal lung tissues(P<0.05).The overall survival time of IPF patients with high DAPK2 expression was longer tha

关 键 词：肺纤维化 自噬 死亡蛋白相关激酶2 生物信息学分析 

分 类 号：R563[医药卫生—呼吸系统] Q811.4[医药卫生—内科学]