西地那非通过上调ERK1/ERK2磷酸化增强5-羟色胺的促肺动脉平滑肌细胞增殖作用  

The potential role of sildenafil in serotonin-induced pulmonary arterial SMCs proliferation is mediated via upregualtion of ERK1/ERK2 phosphorylation

作　　者：李冰冰[1] 姜桢[2] 沈建颖[3] 

机构地区：[1]南京大学医学院附属鼓楼医院麻醉科,南京210008 [2]复旦大学附属中山医院麻醉科,上海200032 [3]同济大学附属上海市第十人民医院心内科,上海200072

出　　处：《复旦学报（医学版）》2009年第6期687-691,共5页Fudan University Journal of Medical Sciences

摘　　要：目的探讨西地那非增强5-羟色胺引起的肺动脉平滑肌细胞增殖作用的可能机制。方法体外原代培养猪肺动脉平滑肌细胞,取第3～5代细胞用于实验。细胞随机分为4组：对照组（CON）、5-羟色胺组（HT）、西地那非组（SIL）、西地那非和5-羟色胺联合干预组（S-HT）。细胞传代培养的第3天,给予含0.2%胎牛血清的培养基饥饿72 h,CON组、HT组、SIL组分别给予PBS、10μmol/L 5-HT、1μmol/L西地那非干预;S-HT组提前20 min给予1μmol/L西地那非干预,再给予10μmol/L 5-羟色胺处理。细胞干预60 min后采用Westernblot法测定各组细胞外信号调节激酶（ERK1/ERK2）的磷酸化水平;加入5-羟色胺24 h后,流式细胞法测定细胞周期并计算S期细胞占细胞总数的比例;加入5-羟色胺72 h后MTT法检测细胞增殖程度。结果与CON组相比,HT组ERK1/ERK2的磷酸化水平、S期细胞比例及细胞增殖率明显增加（P〈0.05）,S-HT组ERK1/ERK2的磷酸化水平、S期细胞比例及细胞增殖率较HT组进一步增加（P〈0.05）;各组间总ERK1/ERK2水平差异无统计学意义（P〉0.05）。结论西地那非增强5-羟色胺引起的肺动脉平滑肌细胞DNA合成和增殖,可能与ERK1/ERK2的磷酸化水平上调有关。Objective To explore the underlying mechanism of potential effect of sildenafil on porcine pulmonary artery smooth muscle cells （SMCs） proliferation induced by serotonin. Methods Porcine pulmonary artery SMCs at 3 - 5 passages were randomly divided into 4 groups： control group （CON）, 5-HT group （HT）, sildenafil group （SIL） and sildenfil-5HT combined group （S-HT）. Pulmonary artery SMCs at exponential growth phase were serum starved with 0.2G FBS for 72 h, followed by sterile PBS, serotonin （10 μmol/L） and sildenafil （1 μmol/L） incubation in CON group, HT group and SIL group, respectively. In combined group （S-HT） ： pulmonary artery SMCs were serum starved and then exposed to sildenafil for 20 min, followed by serotonin incubation for indicated time. The phosphorylation of extracellular signal regulated kinase （ERK1/ERK2） was measured by Western blot at indicated time points. Flow active cell sorting （FACS） was used to evaluate the ratio of S phase cells in the cell cycle after 24 h of treatment. MTT color metric method was used to analyze SMCs proliferative index after 72 h of treatment. Results Compared with CON group, the phosphorylation of ERK1/ERK2, the percentage of cells in S phase, and the cell proliferation index increased remarkably after incubation with 5-HT （P〈0.05）. Preincubation with sildenafil followed by serotonin enhanced the phosphorylation of ERK1/ERK2 （p-ERK1/ERK2） and further elevated the percentage of cells in S phase and the cell proliferation index compared with that of HT group. While the total ERK1/ERK2 （t-ERK1/ERK2） was not statistically different among these groups. Conclusions Sildenafil potentiates the proliferative effect of serotonin on pulmonary arterial SMCs via upregulating phosphorylation of ERK1/ERK2.

关 键 词：细胞外信号调节MAP激酶类 磷酸二酯酶抑制剂 血管平滑肌 肺动脉 5-羟色胺 

分 类 号：R614.1[医药卫生—麻醉学]