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目的在血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)存在情况下,探讨血管紧张素1-7(angiotensin1-7,Ang1-7)对SD大鼠心室成纤维细胞的影响。方法采用新生1~3 d的SD乳鼠,用胰蛋白酶和Ⅱ型胶原酶消化心室,采用差速贴壁法获取成纤维细胞,将细胞完全随机化分组为对照组(不予处理)、AngⅡ组、Ang1-7组、AngⅡ+Ang1-7组(先用Ang1-7预处理30 min,再加入AngⅡ处理)。采用免疫荧光鉴定细胞,CCK-8检测细胞增殖,Western blot检测细胞Rac1、Rad、gp91phox(Nox2)、P65、结缔组织生长因子(connective tissue growth factor,CTGF)蛋白的表达,实时荧光定量PCR测定Ⅰ型、Ⅲ型胶原蛋白(ColⅠ、ColⅢ)和纤维连接蛋白(fibronectin)mRNA的表达。结果 AngⅡ能够促进SD大鼠心室成纤维细胞增殖,Ang1-7能减弱AngⅡ的促增殖作用(P<0.05);与对照组比较,AngⅡ组Rac1、Nox2、P65、CTGF表达增加,Rad表达下降,ColⅠ、ColⅢ、fibronectin转录增加(P<0.05);与AngⅡ组比较,AngⅡ+Ang1-7组Rac1、Nox2、P65、CTGF表达减少,Rad表达升高,ColⅠ、ColⅢ、fibronectin转录下降(P<0.05);Ang1-7组上述各项指标与对照组比较,差异无统计学意义(P>0.05)。结论 AngⅡ存在时,Ang1-7对心室成纤维细胞具有保护作用。Ang1-7通过调节Rac1、Rad及下游蛋白的表达,能够抑制成纤维细胞合成细胞外基质。
Objective To investigate the effects of angiotensin 1-7 (Ang1-7) on ventricular fibroblasts in the presence of angiotensin Ⅱ (AngⅡ). Methods Newborn SD rats aged 1 to 3 days were used to digest the ventricle with trypsin and type Ⅱ collagenase. Fibroblasts were obtained by differential adherence method. Cells were completely randomized into control group (untreated), Ang Ⅱ Group, Ang1-7 group, AngⅡ + Ang1-7 group (pretreated with Ang1-7 for 30 min and then treated with AngⅡ). The cells were identified by immunofluorescence and the cell proliferation was detected by CCK-8. The expression of Rac1, Rad, gp91phox (Nox2), P65 and connective tissue growth factor (CTGF) Type, type Ⅲ collagen (Col Ⅰ, Col Ⅲ) and fibronectin mRNA expression. Results AngⅡ could promote the proliferation of SD rat ventricular fibroblasts, while Ang1-7 could decrease the promoting effect of AngⅡ (P <0.05). Compared with the control group, the expression of Rac1, Nox2, P65 and CTGF increased and the expression of Rad decreased, (P <0.05). Compared with AngⅡgroup, the expression of Rac1, Nox2, P65 and CTGF in AngⅡ + Ang1-7 group decreased and the expression of Rad increased, Col Ⅰ, Col Ⅲ and fibronectin transcription decreased (P <0.05) ); Ang1-7 group of the above indicators compared with the control group, the difference was not statistically significant (P> 0.05). Conclusions Ang1-7 has a protective effect on ventricular fibroblasts in the presence of AngⅡ. Ang1-7 can inhibit the synthesis of extracellular matrix by fibroblasts by regulating the expression of Rac1, Rad and downstream proteins.