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和迁移的影响

Expression of ADAM10 in vascular tissue at stenosis of human arteriovenous fistula and its effect on proliferation and migration of vascular smooth muscle cells

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: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·L^(-1)LPS]、LPS+si-NC组(转染si-NC质粒+10 mg·L^(-1) LPS)、LPS+si-ADAM10组(转染si-ADAM10质粒+10mg·L^(-1)LPS)、 LPS+Notch信号通路抑制剂DAPT组(10mg·L^(-1)LPS+10μmol·L^(-1)Notch信号通路抑制剂DAPT)和LPS+si-ADAM10+DAPT组(转染si-ADAM10质粒+10 mg·L^(-1)LPS+10μmol·L^(-1) 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: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(P<0.05)。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(P<0.05);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(P<0.05)。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(P<0.05);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(P<0.05)。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。

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·L^(-1) LPS],LPS+si-NC group(transfected with si-NC plasmid+10 mg·L^(-1)LPS),LPS+si-ADAM10 group(transfected with si-ADAM10 plasmid+10 mg·L^(-1) LPS),LPS+Notch signal pathway inhibitor DAPT group(10 mg·L^(-1) LPS+10μmol·L^(-1) Notch signal pathway inhibitor DAPT),and LPS+si-ADAM10+DAPT group(transfected with si-ADAM10 plasmid+10 mg·L^(-1) LPS+10μmol·L^(-1) 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(P<0.05).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(P<0.05);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(P<0.05).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(P<0.05);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(P<0.05).Conclusion:ADAM10 is highly expressed in the vascular tissue at the stenosis of AVF,and silencing the expression of ADAM10 gene can inhibit the proliferation and migration of the VSMCs induced by LPS;its mechanism may be related to blocking the Notch signal pathway.