辛伐他汀对大鼠心肌细胞肥大及PTEN表达的影响

Effects of simvastatin on the hypertrophy and PTEN expression of rat cardiac myocytes

目的观察抑癌基因-第十号染色体缺失的张力蛋白同源区（PTEN）在辛伐他汀调控心肌细胞肥大中的信号转导作用。方法以培养的新生Sprague-Dawley（SD）大鼠心肌细胞为实验模型，应用图像分析系统测定心肌细胞表面积，[^3H]-亮氨酸掺入法测定心肌细胞蛋白合成速率，逆转录聚合酶链式反应（RT-PCR）检测心钠素（ANP）mRNA表达，RT-PCR和蛋白免疫印迹法（Western blot）检测PTEN的mRNA和蛋白表达水平。结果（1）15％血清组干预24h后心肌细胞表面积[（1611．16±160．75）[Am。]显著高于无血清对照组[（538．04±118．60）μm^2]（P〈0．01）；给予辛伐他汀和血清共同干预后，随着辛伐他汀浓度的增加，心肌细胞表面积呈递减趋势，其中10^-5和10^-6mol／L组分别为（799．84±167、70）μm^2和（1076．88±199．28）μm^2，均显著低于血清组（P〈0．01）。（2）在15％血清刺激后，心肌细胞[^3H]-亮氨酸掺入率为（2360±106）μm／well，明显高于无血清对照组[（1305±92）cpm／well]（P〈0．01）；10^-5和10^-6moL／L辛伐他汀组[^3H]-亮氨酸掺入率分别为（1707±101）cpm／well和（1962±125）cpm／well，均较血清组明显降低（P〈0．01）。（3）随着辛伐他汀浓度的增加，心肌细胞ANP的mRNA表达水平逐渐降低，其中10^-5和10^-6moL／L辛伐他汀组分别为0．29±0．03和0．40±0.03，与单纯血清干预组0．60±0．03比较明显降低（P〈0．01）。（4）心肌细胞PTEN的mRNA和蛋白表达水平均随辛伐他汀浓度的增加而呈递增趋势，其中10^-7、10^-6、10^-5mol／L辛伐他汀组PTENmRNA表达水平为0．38±0．03、0．83±0．04和0．85±0．05，明显高于血清干预组（0．29±0．04）（P〈0．05，P〈0．01）。上述3组PTEN蛋白表达水平分剐为39．25±3．41、46．35±1．78和47．22±2．39，也较血清组（32．21±4．06）显著升高（P〈0．05，P〈0．01）。结论辛伐他汀能够抑制血清诱导的心肌细胞肥大，升高PTEN表达水平可能是其分子生物学机制之一。

Objective To study the role of phosphatase and tensin homolog deleted on chromosome ten （PTEN） in the effect of simvastatin on the hypertrophy of cultured rat cardiac myocytes induced by serum. Methods Cultured neonatal Sprague-Dawley （SD） rat cardiac myocytes were used as the experimental model. Image analysis system was used to measure cell surface area. Protein synthesis of myocytes was measured via [ ^3H ]-leucine incorporation. The expression level of atrial natriaretic peptide（ANP） mRNA in myocytes was determined with reverse transcription polymerase chain reaction（RT-PCR）. To study the mRNA and protein expression level of PTEN in cardiac myocytes, RT-PCR and Western blot were applied respectively. Results （1）Cardiac myocytes surface areas in serum-free group and 15% serum group were [ （538.04 ± 118.60） μm^2 and （ 1611.16 ± 160.75） μm^2]. It was much higher in serum group （ P 〈 0.01 ）. With the increase of serum concentrations, simvastatin decreased ceil surface areas in a dose dependent manner when co-intervention with serum. Cell surface areas in 10^-5 and 10^-6 mol/L simvastatin groups were （799. 84 ± 167. 70）μm^2 and （ 1076. 88 ± 199. 28） μm^2, which were both significantly lower than that in serum group（both P 〈0.01 ）. （2）Incorporation rate of [^3H] -leucine in serum-free group was 1305 ±92cpm/well. It was much higher in 15% serum group（2360 ± 106）cpm/well than that in serum-free group（ P 〈0. 01 ）. Incorporation rate of [^3H] -leucine in 10^-3 and 10^-6 mol/L simvastatin groups were （1707 ± 101 ）cpm/well and （1962 ± 125）cpm/well, respectively, which were both lower than that in serum group（ P 〈 0. 01 ）. （3）With the increase of simvatatain＇s concentrations, the mRNA expression level of ANP was decreased gradually compared with that of serum group（0. 60 ±0. 03）. The levels of mRNA in 10 ^-5 and 10^ -6 mol/L simvastatin groups were 0. 29 ± 0.03 and 0.40 ±0.03, which were both lower that that in serum group（ P 〈0. 01 ）. （4）Simvastatin increased the expression of mRNA and protein of PTEN in cardiac myocytes inhibited by serum in a dose dependent manner. The mRNA expression level of PTEN in serum group was 0. 29 ±0.04. The mRNA level in 10 ^-7 , 10^ -6and 10 ^-5mol/L simvastatin groups were 0. 38 ±0.03, 0. 83 ±0. 04 and 0. 85 ±0. 05, respectively, which were both higher than that of serum group （ P 〈 0. 05, P 〈 0.01 ）. Similarly, compared with serum group （ 32. 21 ± 4. 06 ）, the protein level of PTEN in 10^ - 7 , 10^ -6 and 10^ -5 mol/L simvastatin groups （39. 25 ± 3.41,46. 35 ± 1.78 and 47.22 ± 2. 39） were also both significantly increased（ P 〈0. 05, P 〈0.01 ）. Conclusion Simvastatin can inhibit the hypertrophy of cultured rat cardiac myo- cytes induced by serum, the mechanism of which might be related with the increase of PTEN expression.