ADAM10在人动静脉内瘘...平滑肌细胞增殖和迁移的影响_孙利军.pdf

第 49 卷 第 2 期2023年 3 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.49 No.2Mar.2023DOI：10.13481/j.1671587X.20230225ADAM10在人动静脉内瘘狭窄处血管组织中的表达及其对血管平滑肌细胞增殖和迁移的影响孙利军,冯杰,刘小明,任广伟,阮琳（河北医科大学第一医院肾内科，河北 石家庄 050031）摘要 目的目的：探讨去整合素金属蛋白酶 10（ADAM10）在人动静脉内瘘（AVF）狭窄处血管组织中的表达及其对血管平滑 肌 细 胞（VSMCs）增殖和迁移 的 影 响，并 阐 明 其 可 能 的 分子机制。方法方法：收集 42 例因 AVF 狭窄再次接受手术治疗的终末期肾病（ESRD）患者狭窄静脉血管组织（AVF组）及其首次手术的正常静脉血管组织（正常对照组）。采用免疫组织化学法检测 2组患者静脉血管组织中 ADAM10蛋白表达情况，实时荧光定量 PCR（RT-qPCR）法检测 2组患者静脉血管组织中 ADAM10 mRNA 表达水平。将 VSMCs分为对照组、模型组 脂多糖（LPS）组，给予 10 mg L1 LPS、LPS+si-NC组（转染 si-NC质粒+10 mg L1 LPS）、LPS+si-ADAM10组（转染 si-ADAM10质 粒+10 mg L1 LPS）、LPS+Notch 信 号 通 路 抑 制 剂 DAPT 组（10 mg L1 LPS+10 mol L1 Notch 信号通路抑制剂 DAPT）和 LPS+si-ADAM10+DAPT 组（转染 si-ADAM10 质粒+10 mg L1 LPS+10 mol L1 DATP），CCK-8 法检测各组细胞增殖活性，Transwell 法检测各组迁移细胞数，Western blotting 法检测各组细胞中 ADAM10、增殖细胞核抗原（PCNA）、基质金属蛋白酶9（MMP-9）、Notch同源蛋白 1（Notch1）、Notch细胞内片段（NICD）和发状分裂相关增强子 1（Hes1）蛋白表达水平。结果结果：与正常对照组比较，AVF 组患者静脉血管组织中 ADAM10 蛋白表达量明显增加，ADAM10 mRNA 表达水平明显增加（P0.05）。与对照组比较，LPS 组细胞增殖活性和迁移细胞数及细胞中 ADAM10、PCNA、MMP-9、Notch1、NICD 和 Hes1蛋白表达水平均明显升高（P0.05）；与 LPS 组 和 LPS+si-NC 组 比 较，LPS+si-ADAM10 组 细 胞 增 殖 活 性 和 迁 移 细 胞 数 及 细 胞 中ADAM10、PCNA、MMP-9、Notch1、NICD 和 Hes1 蛋白表达水平均明显降低（P0.05）。与 LPS组比较，LPS+si-ADAM10 组和 LPS+DAPT 组细胞增殖活性和细胞迁移数量及细胞中 PCNA、MMP-9、Notch1、NICD 和 Hes1 蛋白表达水平均明显降低（P0.05）；与 LPS+si-ADAM10 组比较，LPS+si-ADAM10+DAPT 组细胞增殖活性和细胞迁移数及细胞中 PCNA、MMP-9、Notch1、NICD 和 Hes1蛋白表达水平均明显降低（P0.05）。结论结论：ADAM10在人 AVF 狭窄处血管组织中高表达，沉默 ADAM10基因表达可抑制 LPS诱导的 VSMCs增殖和迁移，其作用机制可能与阻断 Notch信号通路有关。关键词 去整合素金属蛋白酶 10；动静脉内瘘；血管平滑肌细胞；细胞增殖；细胞迁移中图分类号 R34文献标志码 A文章编号 1671587X(2023)02048210收稿日期 20220330基金项目 河北省科技厅重点研发计划项目（21377747D）作者简介 孙利军（1979），男，河北省石家庄市人，副主任医师，医学硕士，主要从事肾脏病方面的研究。通信作者 阮琳，副主任医师（E-mail：）482孙利军，等.ADAM10在人动静脉内瘘狭窄处血管组织中的表达及其对血管平滑肌细胞增殖Expression of ADAM10 in vascular tissue at stenosis of human arteriovenous fistula and its effect on proliferation and migration of vascular smooth muscle cellsSUN Lijun,FENG Jie,LIU Xiaoming,REN Guangwei,RUAN Lin（Department of Nephrology，First Hospital，Hebei Medical University，Shijiazhuang 050031，China）ABSTRACT Objective：To investigate the expression of a disintegrin and metalloproteinase 10（ADAM10）in vascular tissue at the stenosis of human arteriovenous fistula（AVF）and its effect on the proliferation and migration of the vascular smooth muscle cells（VSMCs），and to clarify its possible molecular mechanism.Methods：The stenotic venous vascular tissue of 42 patients with end-stage renal disease（ESRD）who underwent reoperation due to AVF stenosis（AVF group）and their normal venous vascular tissue during the first operation（normal control group）were collected.Immunohistochemistry assay was used to detect the expressions of ADAM10 protein in venous vascular tissue of the patients in two groups；real-time fluorescence quantitative PCR（RT-qPCR）assay was used to detect the expression levels of ADAM10 mRNA in venous vascular tissue of the patients in two groups；the VSMCs were divided into control group and model group lipopoly saccharide（LPS）group，given 10 mg L1 LPS，LPS+si-NC group（transfected with si-NC plasmid+10 mg L1LPS），LPS+si-ADAM10 group（transfected with si-ADAM10 plasmid+10 mg L1 LPS），LPS+Notch signal pathway inhibitor DAPT group（10 mg L1 LPS+10 mol L1 Notch signal pathway inhibitor DAPT），and LPS+si-ADAM10+DAPT group（transfected with si-ADAM10 plasmid+10 mg L1 LPS+10 mol L1 DATP）；CCK-8 method was used to detect the proliferation activities of the cells in various groups；Transwell assay was used to detect the numbers of the migration cells in various groups；Western blotting method was used to detect the expression levels of ADAM10，proliferating cell nuclear antigen（PCNA），matrix metalloproteinase-9（MMP-9），Notch homolog protein 1（Notch1），Notch intracellular domain（NICD），and hairy and enhancer of split 1（Hes1）proteins in the cells in various groups.Results：Compared with normal control group，the expression levels of ADAM10 protein and ADAM10 mRNA in venous vascular tissue of the patients in AVF group were significantly increased（P0.05）.Compared with control group，the proliferation activity，number of the migration cells and expression levels of ADAM10，PCNA，MMP-9，Notch1，NICD，and Hes1 proteins in the cells in LPS group were significantly increased（P0.05）；compared with LPS and LPS+si-NC group，the proliferation activity and number of the migration cells and expression levels of ADAM10，PCNA，MMP-9，Notch1，NICD，and Hes1 proteins in the cells in LPS+si-ADAM10 group were significantly decreased（P0.05）.Compared with LPS group，the proliferative activities，numbers of the migration cells and expression levels of PCNA，MMP-9，Notch1，NICD，and Hes1 proteins in the cells in LPS+si-ADAM10 group and LPS+DAPI group were significantly decreased（P0.05）；compared with LPS+si-ADAM10 group，the proliferation activity，number of the migration cells and expression levels of PCNA，MMP-9，Notch1，NICD，and Hes1 proteins in the cells in LPS+si-ADAM10+DAPT group were significantly decreased（P0.05）.Conclusion：ADAM10 is highly expressed in the vascula