摘要
背景:课题组前期研究发现气虚血瘀证是脊髓型颈椎病各种中医证型中的主要证型。然而,用于气虚血瘀证发育性颈椎管狭窄向脊髓型颈椎病转化的早期诊断蛋白组学标志物未见相关报道。目的:探讨发育性颈椎管狭窄与脊髓型颈椎病的血清蛋白组学差异质,寻找并鉴定两者之间的潜在血清生物学标志物。方法:分别采集气虚血瘀证脊髓型颈椎病患者(实验组)血清9例、气虚血瘀证发育性颈椎管狭窄(对照组)血清9例,选用同位素相对标记与绝对定量技术(TMT)联合液相色谱-串联质谱(LC-MS/MS)技术进行蛋白组学分析,以此寻找并鉴定差异表达的蛋白质。结果与结论:①TMT技术共筛选出有意义差异蛋白1027种,最终鉴定出显著性差异蛋白89种(P<0.05),其中与对照组比较,实验组中α-肌动蛋白4、α-肌动蛋白1、细胞分裂控制蛋白42同系物、整合素连接蛋白激酶、Β-肌动蛋白等45种蛋白表达上调;纤维连接蛋白、纤维蛋白原γ链、纤维蛋白原α链、纤维蛋白原β链等44种蛋白表达下调;②基于GO富集分析,这些差异蛋白参与了信号受体结合、激酶结合、蛋白激酶活性、整合素结合、肌动蛋白丝结合等分子功能;③KEGG通路分析,筛选20条主要共同差异信号/代谢通路,分别为粘着斑、紧密连接、Rap1信号通路、血小板活化、肌动蛋白细胞骨架的调节等信号通路;④PPI分析表明,气虚血瘀证发育性颈椎管狭窄与脊髓型颈椎病之间的共同差异蛋白中ILK,FGA,FGB,FGG,FN1,CDC42,ACTN1,ACTN4,ACTB等位于蛋白质互作网络的节点,且与骨生成与破坏系统、神经系统、凝血系统、细胞炎症等系统关系密切;⑤结论:采用TMT联合LC-MS/MS技术成功筛选出了气虚血瘀证发育性颈椎管狭窄与脊髓型颈椎病之间的血清差异表达蛋白质,明确了ILK、FN1、CDC42、ACTN4是气虚血瘀证发育性颈椎管狭窄向脊髓型颈椎病转化的特异性标志物,为进一步阐明其转化机制提供了依据。
BACKGROUND:Previous studies have found that qi deficiency and blood stasis syndrome is the main syndrome among various TCM syndromes of cervical spondylotic myelopathy.However,there is no report on proteomic markers as early diagnosis indicators for the transformation of developmental cervical spinal stenosis with qi deficiency and blood stasis syndrome to cervical spondylotic myelopathy.OBJECTIVE:To explore serum proteomics difference between developmental cervical spinal stenosis and cervical spondylotic myelopathy and to find and identify the potential serum biomarkers between them.METHODS:Serum samples of nine patients with cervical spondylotic myelopathy of qi deficiency and blood stasis syndrome(experimental group)and nine patients with developmental cervical spinal stenosis of qi deficiency and blood stasis syndrome(control group)were collected.The proteomic analysis was carried out by Tandem Mass Tag combined with liquid chromatography tandem mass spectrometry,so as to find and identify differentially expressed proteins.RESULTS AND CONCLUSION:A total of 1027 significantly differential proteins were initially screened by TMT technology and 89 significantly differential proteins were finally identified(P<0.05).Compared with the control group,there were 45 up-regulated proteins in the experimental group,such asα-actinin-4,α-actinin-1,cell division control protein 42 homolog,integrin-linked protein kinase and B-actin.Conversely,there were 44 down-regulated proteins in the experimental group compared with the control group,such as fibronectin,fibrinogenγchain,fibrinogenαchain,fibrinogenβchain.Gene ontology enrichment analysis indicated that these differential proteins were involved in signal receptor binding,kinase binding,protein kinase activity,integrin binding,actin filament binding and other molecular functions.Based on the Kyoto Encyclopedia of Genes and Genomes pathway analysis,20 common differential signal/metabolic pathways were identified,including Rap1 signaling pathway,adherens junction,tight junction,platelet activation,and regulation of actin cytoskeleton.Proteinprotein interaction analysis showed that ILK,FGA,FGB,FGG,FN1,Cdc42,ACTN1,ACTN4 and ACTB were located at the nodes of protein-protein interaction network and were closely related to bone formation and destruction system,nervous system,coagulation system,cellular inflammation and other systems.To conclude,the serum differentially expressed proteins between developmental cervical spinal stenosis and cervical spondylotic myelopathy can be successfully screened by Tandem Mass Tag combined with liquid chromatography tandem mass spectrometry.ILK,FN1,CDC42 and ACTN 4 are identified as specific markers for the transformation of developmental cervical spinal stenosis with qi deficiency and blood stasis syndrome into cervical spondylotic myelopathy.These findings provide a basis for further clarifying the transformation mechanism.
作者
卜献忠
卜保献
许伟
李智斐
杨汉立
王微微
周劲衍
钟远鸣
Bu Xianzhong;Bu Baoxian;Xu Wei;Li Zhifei;Yang Hanli;Wang Weiwei;Zhou Jinyan;Zhong Yuanming(Graduate School of Guangxi University of Chinese Medicine,Nanning 530001,Guangxi Zhuang Autonomous Region,China;Luoyang Zhenggu Hospital of Henan Province/Orthopedic Hospital of Henan Province,Luoyang 471002,Henan Province,China;First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,Guangxi Zhuang Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第11期1704-1711,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金项目(81760874),项目负责人:钟远鸣
广西重点研发计划项目(桂科AB20159018),项目负责人:钟远鸣
中医学广西一流学科项目(桂教科研[2018]12号),项目参与者:钟远鸣
河南省中医药科学研究专项课题(20-21ZY2083),项目负责人:卜保献
广西研究生教育创新计划资助项目(YCBXJ2021009),项目负责人:卜献忠
广西研究生教育创新计划资助项目(YCBSZ2020001),项目负责人:许伟。
关键词
发育性颈椎管狭窄
脊髓型颈椎病
蛋白组学
TMT技术
差异蛋白
血清特异性标志物
developmental cervical spinal stenosis
cervical spondylotic myelopathy
proteomics
TMT technology
differential protein
serum specific marker
