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目的:观察肾上腺髓质素N端20肽对血管紧张素Ⅱ剌激血管平滑肌细胞增殖及胶原生成的影响。方法:实验于2003-09/2004-09在南方医科大学珠江医院中心实验室进行。①采用组织贴壁法培养大鼠血管平滑肌细胞。②实验分组:空白对照组;1×10-9,1×10-8,1×10-7,1×10-6mol/L血管紧张素Ⅱ组;1×10-9,1×10-8,1×10-7,1×10-6mol/L肾上腺髓质素N端20肽组;1×10-7mol/L血管紧张素Ⅱ+(1×10-9,1×10-8,1×10-7,1×10-6mol/L)肾上腺髓质素N端20肽组。③以3H脯氨酸掺入试验测定血管平滑肌细胞的胶原合成功能,以四氮唑盐法测定细胞增殖情况。分别观察不同浓度肾上腺髓质素N端20肽、血管紧张素Ⅱ对血管平滑肌细胞增殖及合成胶原的影响。结果:①肾上腺髓质素N端20肽及血管紧张素Ⅱ对血管平滑肌细胞的胶原合成功能的作用:肾上腺髓质素N端20肽组的3H脯氨酸掺入率与对照组相比(P>0.05),差异均无显著性。随着血管紧张素Ⅱ浓度的增高,血管平滑肌细胞的3H脯氨酸掺入率呈递增趋势。血管紧张素Ⅱ+肾上腺髓质素N端20肽组的3H脯氨酸掺入率在血管紧张素Ⅱ浓度不变的情况下,随着肾上腺髓质素N端20肽浓度的增高,血管平滑肌细胞的3H脯氨酸掺入率呈递减趋势,各组之间差异有显著意义(P<0.01)。②肾上腺髓质素N端20肽及血管紧张素Ⅱ对血管平滑肌细胞增殖的影响:随着肾上腺髓质素N端20肽浓度的增高,四氮唑盐比色值差异无显著性(P均>0.05)。随着血管紧张素Ⅱ浓度的增高,四氮唑盐比色值增也明显增高,各组间差异有显著性意义(P均<0.01)。血管紧张素Ⅱ+肾上腺髓质素N端20肽组中,在血管紧张素Ⅱ浓度不变的情况下,随着肾上腺髓质素N端20肽浓度的增高,四氮唑盐比色值均显著降低(P<0.01)。结论:肾上腺髓质素N端20肽对血管紧张素Ⅱ剌激血管平滑肌细胞增生及胶原的生成起明显抑制作用。
Objective: To observe the effects of adrenomedullin N-terminal 20 peptide on angiotensin Ⅱ-induced proliferation and collagen production in vascular smooth muscle cells. Methods: The experiment was performed at Central Laboratory of Zhujiang Hospital, Southern Medical University from September 2003 to September 2004. ① Adopt tissue adherent method to culture rat vascular smooth muscle cells. ② Experimental group: blank control group; 1 × 10-9,1 × 10-8,1 × 10-7,1 × 10-6mol / L angiotensin Ⅱ group; 1 × 10-9,1 × 10-8 , 1 × 10-7, 1 × 10-6mol / L adrenomedullin N-terminal 20 peptide group; 1 × 10-7mol / L angiotensin Ⅱ + (1 × 10-9,1 × 10-8,1 × 10-7,1 × 10-6mol / L) Adrenomedullin N-terminal 20 peptide group. (3) 3H-proline incorporation assay was used to measure the collagen synthesis of vascular smooth muscle cells, and cell proliferation was measured by tetrazolium salt method. Effects of different concentrations of adrenomedullin N-terminal 20 peptide and angiotensin Ⅱ on proliferation of vascular smooth muscle cells and collagen synthesis were observed. Results: ① Adrenomedullin N-terminal 20 peptide and angiotensin Ⅱ on vascular smooth muscle cell collagen synthesis function: adrenomedullin N-terminal 20 peptide group 3H proline incorporation rate compared with the control group ( P> 0.05), the difference was not significant. With the increase of angiotensin Ⅱ concentration, 3H proline incorporation rate of vascular smooth muscle cells showed an increasing trend. The incorporation of 3H proline in the N-terminal 20 peptide group of angiotensin Ⅱ + adrenomedullin increased with the concentration of adrenomedullin N-terminal 20 peptide unchanged at the same concentration of angiotensin Ⅱ, 3H-proline incorporation rate of cells showed a decreasing trend, the difference between the groups was significant (P <0.01). ② Adrenomedullin N-terminal 20 peptide and angiotensin Ⅱ on the proliferation of vascular smooth muscle cells: With the adrenomedullin N-terminal 20 peptide concentration increased, tetrazolium salt color value was no significant difference (P > 0.05). As the concentration of angiotensin Ⅱ increased, the colorimetric value of tetrazolium salt increased significantly, with significant difference between the groups (all P <0.01). Angiotensin Ⅱ + adrenomedullin N-terminal 20 peptide group, in the case of constant concentration of angiotensin Ⅱ, with the adrenomedullin N-terminal 20 peptide concentration increased, tetrazolium salt colorimetric value Significantly lower (P <0.01). Conclusion: Adrenomedullin N-terminal 20 peptide can significantly inhibit angiotensin Ⅱ-induced vascular smooth muscle cell proliferation and collagen production.