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目的:探讨肾上腺髓质素(ADM)对血管紧张素Ⅱ(AngⅡ)诱导的血管外膜成纤维细胞(AF)迁移及胶原生成的影响及机制。方法:体外培养大鼠胸主动脉AF,运用Trans well技术测定不同浓度ADM对AF迁移的影响,采用酶联免疫吸附法(ELISA)测定培养上清中Ⅰ、Ⅲ型胶原蛋白含量,用RT-PCR及Western blotting分析AngⅡ(10-6mol/L)及不同浓度ADM干预后大鼠胸主动脉AF内骨桥蛋白(OPN)、转化生长因子β1(TGFβ1)mRNA及蛋白的表达。结果:在AngⅡ(10-6mol/L)趋化作用下,AF迁移数目较对照组显著增多[(30.26±1.08)∶(4.35±1.26),P<0.01]。ADM可抑制AngⅡ刺激的细胞迁移,迁移细胞数目在一定范围内随着ADM浓度增加而减少;AngⅡ(10-6mol/L)诱导OPN呈高表达,ADM可下调这种表达,呈一定剂量依赖性;AngⅡ显著增加培养上清中Ⅰ、Ⅲ型胶原蛋白含量,ADM呈剂量依赖的抑制AngⅡ上述作用,其中10-8mol/LADM组中Ⅰ、Ⅲ型胶原合成分别抑制了30%和31%(P<0.01),10-7mol/LADM组则分别抑制了43%和42%(P<0.01);ADM呈剂量依赖性抑制AngⅡ刺激的TGFβ1mRNA及蛋白表达,其中10-8mol/LADM组中TGFβ1mRNA及蛋白表达分别抑制了55%和45%(P<0.01),10-7mol/LADM组则分别抑制了70%和59%(P<0.01)。结论:ADM对AF的迁移及胶原生成无明显直接影响,但ADM可能通过下调细胞内OPN及TGFβ1表达,抑制AngⅡ诱导的血管AF迁移及胶原生成,从而发挥有效的抗血管重构作用。
Objective: To investigate the effects of adrenomedullin (ADM) on angiotensin Ⅱ-induced migration and collagen production of vascular adventitial fibroblasts (AF) and its mechanism. Methods: Rat thoracic aorta AF was cultured in vitro. Transwell technique was used to determine the effect of different concentrations of ADM on AF migration. The contents of type Ⅰ and type Ⅲ collagen in culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA) The expressions of osteopontin (OPN), transforming growth factor β1 (TGFβ1) mRNA and protein in the thoracic aorta of rats after Ang Ⅱ (10-6 mol / L) and different concentrations of ADM were analyzed by PCR and Western blotting. Results: Under the chemotactic effect of AngⅡ (10-6mol / L), the number of AF migration was significantly increased compared with the control group [(30.26 ± 1.08) vs (4.35 ± 1.26), P <0.01]. ADM could inhibit AngⅡ-stimulated cell migration, the number of migrated cells decreased with the increase of ADM concentration within a certain range; high level of OPN was induced by AngⅡ (10-6mol / L); ADM could down-regulate this expression in a dose-dependent manner ; Ang Ⅱ significantly increased the content of type I and type III collagen in culture supernatant, while ADM inhibited the effect of AngⅡ in a dose-dependent manner. The synthesis of type I and type III collagen in 10-8mol / L ADM group was inhibited by 30% and 31% respectively (P <0.01). The mRNA and protein expression of TGFβ1 stimulated by AngⅡ in a dose-dependent manner were inhibited by ADM, and the expression of TGFβ1 mRNA and protein in 10-8mol / L ADM group was significantly lower than that in 10-7mol / L ADM group The expression was inhibited by 55% and 45%, respectively (P <0.01), while 70% and 59% (P <0.01) by 10-7mol / L ADM group respectively. CONCLUSION: ADM has no direct effect on the migration of AF and collagen production. However, ADM may exert an effective anti-angiogenic effect by down-regulating the expression of OPN and TGFβ1, inhibiting Ang Ⅱ-induced vascular AF migration and collagen production.