去整合素金属蛋白酶10在糖尿病冠状动脉支架内再狭窄中作用的研究

Effects of a disintegrin and metalloprotease 10 on coronary artery in-stent restenosis in diabetes

目的:探讨去整合素金属蛋白酶10(ADAM10)在糖尿病冠状动脉(冠脉)支架内再狭窄(ISR)中的作用。方法:在17头糖尿病猪和10头正常猪的冠脉内置入雷帕霉素洗脱支架,6个月后行冠脉造影,留取发生和未发生ISR的冠脉组织,Western blot检测ADAM10表达水平。在人主动脉平滑肌细胞(HASMC)中感染ADAM10的过表达和敲减病毒,BrdU检测细胞增殖,划痕实验检测细胞迁移能力。分别用低糖培养基、高糖培养基、晚期糖基化终末产物-牛血清白蛋白(AGE-BSA)、AGE-BSA+AGE受体(RAGE)抗体培养HASMC,实时定量RT-PCR和Western blot检测ADAM10表达水平。结果:在糖尿病组中,未发生ISR和发生ISR的冠脉组织中ADAM10的表达均高于非糖尿病组(3.36±1.27对2.11±2.05,10.48±4.72对6.72±1.36,P均<0.01)。在非糖尿病组和糖尿病组,发生ISR的冠脉组织中ADAM10的表达均高于未发生ISR的冠脉组织(P均<0.01)。BrdU实验显示,在低糖培养基和高糖培养基中,ADAM10过表达的HASMC增殖均明显高于转染空载体的HASMC(2.25±0.07对1.87±0.08,2.47±0.10对2.07±0.10,P均<0.05);而ADAM10敲减的HASMC增殖均明显低于转染空载体的HASMC(1.34±0.10对1.87±0.08,1.46±0.09对2.07±0.10,P均<0.05);ADAM10过表达和ADAM10敲减的HASMC在高糖培养基中的增殖均明显高于低糖培养基(P均<0.05)。细胞划痕实验显示,在低糖培养基和高糖培养基中,ADAM10过表达的HASMC迁移距离均明显大于转染空载体的HASMC[(1.02±0.12)mm对(0.65±0.04)mm,(1.26±0.06)mm对(0.78±0.06)mm,P均<0.05)],而ADAM10敲减的HASMC迁移距离均明显小于转染空载体的HASMC[(0.26±0.06)mm对(90.65±0.04)mm,(0.43±0.14)mm对(0.78±0.06)mm,P均<0.05)];ADAM10过表达和ADAM10敲减的HASMC在高糖培养基中的迁移距离均明显大于低糖培养基(P均<0.05)。与低糖培养基相比,高糖培养基和AGE-BSA中HASMC ADAM10 mRNA和蛋白的相对表达水平均明显升高(P均<0.05);与AGE-BSA相比,AGE-BSA+RAGE抗体中HASMC ADAM10 mRNA和蛋白的相对表达水平均明显降低(P均<0.05)。结论:ADAM10在糖尿病发生ISR的冠脉中表达显著升高,ADAM10高表达促进动脉平滑肌细胞增殖和迁移,高糖环境及AGE均可促进ADAM10的表达,ADAM10可能参与了糖尿病冠脉ISR的发生与发展。

Objective:To investigate the effects of a disintegrin and metalloprotease 10(ADAM10)on coronary artery in-stent restenosis(ISR)in diabetes. Methods:Rapamycin-eluting stents were implanted in the coronary arteries of 17 diabetic and 10 normal minipigs,and angiography was repeated after 6 months.The coronary artery tissues of significant ISR and non-ISR segments in both diabetic and normal minipigs were analyzed by western blot analysis to detect the expression of ADAM10.Overexpression and konckdown of ADAM10 were transfected by retrovirus in human aortic smooth muscle cells(HASMC).The proliferation of HASMC was measured by BrdU assay and migration was detected by scratch test.The ADAM10 expressions of HASMC were analyzed by real time RT-PCR and western blot after treatment with low glucose,high glucose,advanced glycation end products-bovine serum albumin(AGE-BSA),and AGE-BSA+receptor for AGE(RAGE)antibody. Results:The results showed that the expressions of ADAM10 in both non-ISR tissues(3.36±1.27 vs.2.11±2.05,P<0.01)and ISR tissues(10.48±4.72 vs.6.72±1.36,P<0.01)were significantly higher in diabetic minipigs than those in normal minipigs.ADAM10 levels were significantly increased in ISR tissues compared with non-ISR tissues in both normal minipigs and diabetic minipigs(both P<0.01).BrdU assay showed that The proliferation of HASMC with overexpre-ssion of ADAM10 increased in both low glucose culture(2.25±0.07 vs.1.87±0.08,P<0.05)and high glucose culture(2.47±0.10 vs.2.07±0.10,P <0.05)compared with that of HASMC transfected by empty vector,while the proliferation of HASMC with knockdown of ADAM10 significantly inhibited in both low glucose culture(1.34±0.10 vs.1.87±0.08,P<0.05)and high glucose culture(1.46±0.09 vs.2.07±0.10,P<0.05)compared with that of HASMC transfected by empty vector.The cell proliferation in high glucose culture was significantly higher than that in low glucose culture both in HASMC with overexpression and knockdown of ADAM10(both P<0.05).Cell scratch test showed that the cell migration distances were significantly longer in HASMC with overexpression of ADAM10 compared with HASMC transfected by empty vector both in low glucose culture[(1.02±0.12)mm vs.(0.65±0.04)mm,P<0.05)]and high glucose culture[(1.26±0.06)mm vs.(0.78±0.06)mm,P<0.05)],while those were shorter in HASMC with knockdown of ADAM10 compared with HASMC transfected by empty vector both in low glucose culture[(0.26±0.06)mm vs.(0.65±0.04)mm,P<0.05)]and high glucose culture[(0.43±0.14)mm vs.(0.78±0.06)mm,P<0.05)].The cell migration distance in high glucose culture was significantly longer than that in low glucose culture both in HASMC with overexpression and knockdown of ADAM10(both P<0.05).The relative expressions of ADAM10 mRNA and protein were significantly higher in high glucose culture and AGE-BSA than those in low glucose culture(all P<0.05),while significantly lower in AGE-BSA+RAGE antibody than those in AGE-BSA(both P<0.05). Conclusions:ADAM10 expression is significantly increased in coronary artery ISR segments of diabetes.Increased ADAM10 expression promotes the proliferation and migration of arterial smooth muscle cells.High glucose and AGE can both induce the expression of ADAM10.ADAM10 may be involved in the development and progress of diabetic ISR.