复旦学报（医学版）
複旦學報（醫學版）
복단학보（의학판）
FUDAN UNIVERSITY JOURNAL OF MEDICAL SCIENCES
2009年
6期
687-691
,共5页
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
細胞外信號調節MAP激酶類%燐痠二酯酶抑製劑%血管平滑肌%肺動脈%5-羥色胺
세포외신호조절MAP격매류%린산이지매억제제%혈관평활기%폐동맥%5-간색알
extracellular signal regulated MAP kinases%phosphodiesterase inhibitors%vascular smooth muscle%pulmonary artery%serotonin
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、10 μmol/L 5-HT、1 μmol/L西地那非干预;S-HT组提前20 min给予1 μmol/L西地那非干预,再给予10 μmol/L 5-羟色胺处理.细胞干预60 min后采用Western blot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.
目的 探討西地那非增彊5-羥色胺引起的肺動脈平滑肌細胞增殖作用的可能機製.方法 體外原代培養豬肺動脈平滑肌細胞,取第3～5代細胞用于實驗.細胞隨機分為4組:對照組(CON)、5-羥色胺組(HT)、西地那非組(SIL)、西地那非和5-羥色胺聯閤榦預組(S-HT).細胞傳代培養的第3天,給予含0.2%胎牛血清的培養基饑餓72 h,CON組、HT組、SIL組分彆給予PBS、10 μmol/L 5-HT、1 μmol/L西地那非榦預;S-HT組提前20 min給予1 μmol/L西地那非榦預,再給予10 μmol/L 5-羥色胺處理.細胞榦預60 min後採用Western blot法測定各組細胞外信號調節激酶(ERK1/ERK2)的燐痠化水平;加入5-羥色胺24 h後,流式細胞法測定細胞週期併計算S期細胞佔細胞總數的比例;加入5-羥色胺72 h後MTT法檢測細胞增殖程度.結果 與CON組相比,HT組ERK1/ERK2的燐痠化水平、S期細胞比例及細胞增殖率明顯增加(P<0.05),S-HT 組ERK1/ERK2的燐痠化水平、S期細胞比例及細胞增殖率較HT組進一步增加(P<0.05);各組間總ERK1/ERK2水平差異無統計學意義(P>0.05).結論 西地那非增彊5-羥色胺引起的肺動脈平滑肌細胞DNA閤成和增殖,可能與ERK1/ERK2的燐痠化水平上調有關.
목적 탐토서지나비증강5-간색알인기적폐동맥평활기세포증식작용적가능궤제.방법 체외원대배양저폐동맥평활기세포,취제3～5대세포용우실험.세포수궤분위4조:대조조(CON)、5-간색알조(HT)、서지나비조(SIL)、서지나비화5-간색알연합간예조(S-HT).세포전대배양적제3천,급여함0.2%태우혈청적배양기기아72 h,CON조、HT조、SIL조분별급여PBS、10 μmol/L 5-HT、1 μmol/L서지나비간예;S-HT조제전20 min급여1 μmol/L서지나비간예,재급여10 μmol/L 5-간색알처리.세포간예60 min후채용Western blot법측정각조세포외신호조절격매(ERK1/ERK2)적린산화수평;가입5-간색알24 h후,류식세포법측정세포주기병계산S기세포점세포총수적비례;가입5-간색알72 h후MTT법검측세포증식정도.결과 여CON조상비,HT조ERK1/ERK2적린산화수평、S기세포비례급세포증식솔명현증가(P<0.05),S-HT 조ERK1/ERK2적린산화수평、S기세포비례급세포증식솔교HT조진일보증가(P<0.05);각조간총ERK1/ERK2수평차이무통계학의의(P>0.05).결론 서지나비증강5-간색알인기적폐동맥평활기세포DNA합성화증식,가능여ERK1/ERK2적린산화수평상조유관.
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 0.2% 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.
