膀胱平滑肌细胞在牵拉作用下变化及机制  

Scenario of the response in human bladder smooth muscle cells under stretch

作　　者：何敏坷 张帆 李耀辉 林文耀[4] 项卓仪 王杭 He Minke;Zhang Fan;Li Yaohui;Lin Wenyao;Xiang Zhuoyi;Wang Hang(Department of Urology,Minghang Hospital,Fudan University,Shanghai 201199,China;Department of Urology,Affiliated Cixi Hospital,Wenzhou Medical University,Cixi 315300,China;Department of Urology,Zhongshan Hospital,Fudan University,Shanghai 200032,China;Department of Urology,Zhongshan Hospital Xuhui Branch,Shanghai 200020,China)

机构地区：[1]上海市闵行区中心医院泌尿外科,201199 [2]温州医科大学附属慈溪医院泌尿外科,315300 [3]复旦大学附属中山医院泌尿外科,上海200032 [4]上海市徐汇区中心医院泌尿外科,200020

出　　处：《中华实验外科杂志》2021年第6期1037-1041,共5页Chinese Journal of Experimental Surgery

摘　　要：目的:探索膀胱平滑肌细胞在外力牵拉作用下功能改变过程中关键基因和通路的作用。方法:运用生物信息学的方法,包括不同表达分析(DEA)、基因本体(GO)/京都基因与基因组百科全书(KEGG)、激酶和转录因子富集分析、基因富集分析(GSEA)、蛋白互作网络(PPI)等,对编号为GSE1595的基因芯片进行分析筛选出相应的关键基因,随后通过构建小鼠膀胱出口梗阻(BOO)及膀胱出口梗阻解除模型和膀胱平滑肌细胞牵拉实验,通过抽签法将小鼠分为对照组,牵拉组和牵拉+释放组,每组8只C57小鼠,提取相应RNA后进行反转录,随后进一步进行运用独立样本t检验及曼-惠特尼U检验进行分析验证。结果:通过DEA分析共筛选出321个目的基因,其中有180个上调基因和141个下调基因,GO分析显示上调基因主要富集在蛋白去甲基化,调控其他基因表达和代谢过程,而下调基因主要富集在多细胞生物发育、细胞分化和Notch信号通路等。并最终筛选出乳腺癌易感基因1(BRCA1),周期蛋白依赖激酶抑制因子1B(CDKN1B),拟南芥驱动蛋白2B基因(KAT2B),前列腺素内过氧化物酶2(PTGS2),S期激酶相关蛋白1(SKP1)这五个关键基因。并且发现在细胞实验中发现与对照组比较,牵拉膀胱细胞组BRCA1,KAT2B,PTGS2以及SKP1表达高于对照组(BRCA1为1.336比1.000,t=4.221,P<0.05;PTGS2为1.338比1.000,t=4.222,P<0.05);KAT2B为1.581比1.000,t=2.863,P<0.05;SKP1为1.466比1.000,t=4.300,P<0.05),差异均有统计学意义。在小鼠动物模型中,通过进行外科手术操作,成功造模膀胱梗阻模型,使小鼠膀胱持续充盈牵拉,并随后解除梗阻。进一步检验发现,牵拉组BRCA1,KAT2B以及PTGS2表达高于对照组(BRCA1为1.332比1.000,t=9.421,P<0.05;KAT2B为1.363比1.000,t=2.478,P<0.05;PTGS2为2.211比1.000,t=3.403,P<0.05),差异均有统计学意义,牵拉组CDKN1B和SKP1表达低于对照组(CDKN1B为0.856比1.000,t=2.451,P<0.05;SKP1为0.669比1.000,t=6.814,P<0.05),差异Objective To identify key genes and pathways in human bladder smooth muscle cells(HBSMCs)under stretch.Methods Data in GSE1595 were used in our study.Differentially expressed genes(DEGs)were identified by limma package under the R platform.Gene ontology/kyoto encyclopedia of genes and genomes(GO/KEGG)pathway enrichment,kinases and transcription factor analysis,protein to protein interaction(PPI)analysis and gene sets enrichment analysis were performed.We also identified the results by experiments both in vivo and vitro.Then,the Student′s t test and U test were used to analyze the data.Results A total of 321 DEGs were identified,including 180 up-regulated genes and 141 down-regulated genes.GO analysis showed that up-regulated DEGs were enriched in protein demethylation,positive regulation of gene expression,and metabolic process,while downregulated DEGs were enriched in multicellular organism development,cell differentiation,and Notch signaling pathway.KEGG analysis revealed that upregulated DEGs were enriched in biosynthesis of amino acids,carbon metabolism,metabolic regulation,and pluripotency of stem cells regulation,while the down-regulated DEGs were enriched in Wnt signaling pathway,pathways in cancer,Ras signaling pathway,and Notch signaling pathway.We found that breast cancer 1(BRCA1),prostaglandin-endoperoxide synthase 2(PTGS2),KAT2B and S-phase kinase-associated protein 1(SKP1)expression was up regulated(BRCA11.336 vs.1.000,t=4.221,P<0.05;PTGS21.338 vs.1.000,t=4.222,P<0.05;KAT2B 1.581 vs.1.000,t=2.863,P<0.05;SKP11.466 vs.1.000,t=4.300,P<0.05).Besides,BRCA1 and PTGS2 were identified and verified both in vitro and vivo experiments.Conclusion Stretch induced corresponding changes of gene expression and pathway activity in HBSMCs.We provided a set of useful targets for future investigation of progress of bladder outlet obstructiom.In future,further studies are required to validate our assumption.

关 键 词：生物信息学 膀胱 平滑肌细胞 

分 类 号：Q811.4[生物学—生物工程] R694[医药卫生—泌尿科学]