【摘要】 目的:探讨血管紧张素Ⅱ受体1反义寡核苷酸(AT1-AS-ODNs)对血管紧张素Ⅱ诱导心肌细胞心钠素合成的生物学效应。方法:实验于2004-07/2005-07在武汉大学人民医院心血管国家重点实验室进行。体外培养乳鼠心肌细胞,将其分为4组:①对照组:无血清Opti-MEM培养48h。②血管紧张素Ⅱ组:无血清Opti-MEM培养24h+10-6mol/L血管紧张素Ⅱ刺激24h。③AT1-AS-ODNs组:200nmol/LAT1-AS-ODNs孵育24h+10-6mol/L血管紧张素Ⅱ刺激24h。④顺义序列组:200nmol/LAT1-S-ODNs孵育24h+10-6mol/L血管紧张素Ⅱ刺激24h。显微荧光技术检测脂质体包裹的AT1-AS-ODNs在心肌细胞内分布;免疫沉淀法检测血管紧张素Ⅱ受体1,2蛋白表达水平;放射性免疫法检测心钠素(ANF)表达。结果:①在脂质体的包裹下,AT1-AS-ODNs成功转染心肌细胞,120min转染效率为60%。②血管紧张素Ⅱ组血管紧张素Ⅱ受体1,2蛋白表达水平较对照组低25%,21%(P<0.05),AT1R-AS-ODNs组血管紧张素Ⅱ受体1蛋白表达水平下调60.7%(P<0.01),血管紧张素Ⅱ受体2蛋白表达水平无改变。③血管紧张素Ⅱ组心钠素表达明显高于对照组[(780±38),(430±23)μg/L,P<0.01]。AT1-AS-ODNs组心钠素表达为(589±19)μg/L,明显低于血管紧张素Ⅱ组(P<0.01),顺义序列组与对照组相比差异不显著(P>0.05)。结论:AT1-AS-ODNs可成功转染心肌细胞,通过特异性抑制血管紧张素Ⅱ受体1蛋白表达,拮抗血管紧张素Ⅱ的生物学效应。更多还原

【Abstract】 AIM: To investigate the biological effect of angiotensin Ⅱ (Ang Ⅱ) receptor 1 antisen oligodexynucleotides (AT1-AS-ODNs) on expression of atrial natriuretic factor (ANF) induced by angiotensin Ⅱ in cardiomyocytes. METHODS: The experiment was conducted in the State Key Laboratory of Cardiovascular Disease in Wuhan University Renmin Hospital. Neonatal cardiomyocytes of rats were cultured in vitro, which were assigned randomly to four groups. ①Control group: Cultured with Opti-MEM for 48 hours; ②Ang Ⅱ group: Treated with Opti-MEM for 24 hours plus 10-6 mol/L AngⅡ for 24 hours; ③AT1-AS-ODNs group: Incubated with 200 nmol/L AT1-AS-ODNs for 24 hours plus 10-6 mol/L Ang Ⅱ for 24 hours; ④Sense control group: Incubated with 200 nmol/L AT1-S-ODNs for 24 hours, continued with 10-6 mol/L Ang Ⅱ for 24 hours. The distribution of AT1-AS-ODNs in the cardiomyocytes was detected by microfluorescent technology; the protein expressions of AT1, AT2 were detected by Westernblot; Radioimmunoassay was used to evaluate the protein expression of ANF. RESULTS: ①Liposome-encapsulated AT1-AS-ODNs could be delivered into ventricular cardiocytes and the transfection efficiency was 60% in 120 minutes. ②Compared with the control group, the AT1R and AT2R protein expression was decreased by 25% and 21% in the Ang Ⅱ group, respectively (P < 0.05), the AT1R protein expression in the AT1R-AS-ODNs group was decreased by 60.7% (P < 0.01), but no change of AT2R was detected. ③The ANF protein in the Ang Ⅱ group was markedly higher than that in the control and AT1-AS-ODNs groups [(780±38), (430±23), (589±19) μg/L, P < 0.01]; there was no significant difference between the sense control group and the control group (P > 0.05). CONCLUSION: AT1-AS-ODNs can be successfully delivered into the cardiomyocytes, and inhibit the biological effect of angiotensin Ⅱ by decreasing expression of AT1 receptor.更多还原