螺内脂抑制醛固酮诱导大鼠心肌细胞中钙调神经磷酸酶的表达

Expression of aldosterone-induced calcineurin in myocardial cells of rats inhibited by spirone

目的:在引起心肌细胞肥大的刺激因素醛固酮作用下,应用醛固酮受体拮抗剂螺内脂进行共同培养,观察培养的原代大鼠心肌细胞钙调神经磷酸酶蛋白及mRNA表达水平。方法:实验于2004-07/12在基础医学研究所组织工程研究中心实验室进行。取出生1～3d的Wistar乳鼠,进行心肌细胞培养。将培养的乳鼠心肌细胞分为4组:①空白对照组:不干预。②醛固酮组:予以醛固酮至浓度为1nmol/L。③环孢素A组:予以醛固酮至1nmol/L,同时给予环孢酶素A至5mmol/L。④螺内脂组:予以醛固酮至1nmol/L,同时给予螺内脂至3μmol/L。通过免疫组织化学染色观察钙调神经磷酸酶Aβ蛋白表达,反转录-聚合酶链反应方法检测钙调神经磷酸酶蛋白AβmRNA的表达。结果:①钙调神经磷酸酶Aβ蛋白表达:免疫组织化学染色结果发现醛固酮组心肌细胞胞浆中阳性颗粒(棕色)多,表达增高,用环孢素A及螺内酯干预的大鼠心肌胞浆中阳性颗粒显著少于醛固酮组,与空白对照组接近。②钙调神经磷酸酶AβmRNA表达:醛固酮组心肌细胞表达显著高于空白对照组(P<0.05),环孢素A及螺内酯组显著低于醛固酮组,与空白对照组无明显差异。结论:在醛固酮诱导下钙调神经磷酸酶蛋白及mRNA表达水平均升高,螺内酯通过拮抗醛固酮与受体结合可减少钙调神经磷酸酶蛋白及mRNA表达,其效果与钙调神经磷酸酶特异性抑制剂环孢素A相似。

AIM： To observe the level of calcineurin protein and mRNA expressions in primarily cultured rat myocardial cells under tbe action of aldosterone （the stimulative factor of myocardial hypertraphy） and the co-culture with spirone （the aldosterone receptor antagonist）. METHODS： From July and December 2004, the experiment was performed in the Tissue Engineering Research Center, Institute of Basic Medical Sciences. Wistar rats of 1-3 days old were selected for the culture of myocardial cells, and the cultured cells were divided into four groups： ① blank control group： no intervention; ② aldosterone group： treatment of aldosterone （1 nmol/L）; ③ cyelosporin A group： aldosterone （1 nmol/L）＋ cyclosporin A （5 mmol/L）; ④spirone group： aldosterone （1 nmol/L）＋ spirone （3 μmol/L）. The expression of calcineurin Aβ protein was detected with immunohistochemical method and the expression of calcineurin Aβ mRNA was deteced with reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： ① The expression of calcineurin Aβ protein： The results of immunohistochemical staining showed that there were more （brown） positive granules in the myocardial plasm and increased expression in the aldosterone group, and the those were significantly fewer after the interventions of cyclosporin A and spirone, and close to that in the blank control group. ② The expression of caleineurin Aβ mRNA： It was significantly higher in the aldosterone group than in the blank control group （P 〈 0.05）, but those in the cyclosporin A group and spirone group were significantly lower than that in the aldosterone group, but had no obvious difference from that in the blank control group. CONCLUSION： The levels of aldosterone-induced calcineurin protein and mRNA expressions are increased, which can be reduced by spirone through the combination of antagonized aldosterone and receptor, and the effects are similar to cyclosporin A, which is the calcineurin specific inhibitor.