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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 role of integrin adhesion protein 10 (ADAM10) in the coronary stent restenosis (ISR) in diabetic patients. METHODS: Rapamycin-eluting stent was placed in the coronary arteries of 17 diabetic pigs and 10 normal pigs. Coronary angiography was performed 6 months after coronary angiography. Coronary arteries with and without ISR were collected. Western blot was used to detect ADAM10 The expression level. In human aortic smooth muscle cells (HASMC) were infected with ADAM10 overexpression and knockdown of virus, BrdU detection of cell proliferation, scratch test to detect cell migration. HASMC were cultured in low glucose medium, high glucose medium, advanced glycation end products (AGE-BSA) and AGE-BSA + AGE receptor (RAGE) blot detection of ADAM10 expression levels. Results: In diabetic group, the expression of ADAM10 in coronary artery tissue without ISR and ISR was higher than that in non-diabetic group (3.36 ± 1.27 vs 2.11 ± 2.05, 10.48 ± 4.72 vs. 6.72 ± 1.36, P <0.01 respectively). In non-diabetic and diabetic groups, the expression of ADAM10 in ISR-producing coronary arteries was higher than that in non-diabetic and diabetic coronary arteries (all P <0.01). BrdU assay showed that the proliferation of HASMC overexpressing ADAM10 was significantly higher than that of HASMC transfected with empty vector (2.25 ± 0.07 versus 1.87 ± 0.08, 2.47 ± 0.10 vs. 2.07 ± 0.10, P <0.05) in both low glucose and high glucose media <0.05). However, the proliferation of HASMC knocked out by ADAM10 was significantly lower than that of HASMC transfected with empty vector (1.34 ± 0.10 vs. 1.87 ± 0.08, 1.46 ± 0.09 vs. 2.07 ± 0.10, P <0.05), ADAM10 overexpression and ADAM10 knockdown The proliferation of HASMC in high glucose medium was significantly higher than that in low glucose medium (all P <0.05). Cell scratch assay showed that the migration distance of ADAM10 overexpressing HASMC was significantly higher than that of HASMC [(1.02 ± 0.12) mm vs (1.26 ± 0.12) mm vs (1.26 ± 0.12) mm) in low glucose and high glucose medium (0.26 ± 0.06) mm vs (0.78 ± 0.06) mm, P <0.05). However, the migration distance of HASMC knocked out by ADAM10 was significantly lower than that of HASMC [(0.26 ± 0.06) mm vs (90.65 ± 0.04) mm , (0.43 ± 0.14) mm vs (0.78 ± 0.06) mm, P <0.05). The migration distance of HASMC in ADAM10 overexpression and ADAM10 knockdown was significantly higher than that in low glucose medium (P < 0.05). Compared with low glucose medium, the relative expression levels of ADAM10 mRNA and protein in HASMC in high glucose medium and AGE-BSA were significantly increased (all P <0.05). Compared with AGE-BSA, AGE-BSA + RAGE antibody HASMC ADAM10 mRNA and protein relative expression levels were significantly lower (P all <0.05). Conclusion: The expression of ADAM10 in the coronary artery of diabetes mellitus with ISR increased significantly. High expression of ADAM10 promoted the proliferation and migration of arterial smooth muscle cells. High glucose and AGE promoted the expression of ADAM10. ADAM10 may be involved in the occurrence of ISR in coronary artery development of.