CAJ | 학술논문

目的观察LY294002、雷帕霉素、曲安奈德对体外培养血管瘤内皮细胞中p110、p-Akt、Akt调控的影响,探讨调控PI3K/Akt信号通路在小儿血管瘤的发生、发展及消退过程中的作用.方法用组织块结合酶消化法培养血管瘤内皮细胞,建立血管瘤血管内皮细胞系,并用第Ⅷ凝血因子相关抗原进行鉴定.待细胞处于对数生长期,换无血清培养液孵育48 h使细胞同步化,然后加入25μmol/L LY294002、10 nmol/L雷帕霉素、1.0 g/L的曲安奈德继续孵育24h,检测血管瘤内皮细胞中p110、p-Akt及Akt的蛋白表达水平,同时检测血管瘤内皮细胞周期分布及细胞凋亡率,分析p110、p-Akt及Akt的表达水平与体外培养血管瘤内皮细胞周期分布及凋亡的相关性.结果干预24 h后,LY294002组p110和p-Akt的表达水平分别为0.188±0.012和0.091±0.021,雷帕霉素组p110和p-Akt的表达水平分别为0.145±0.007和0.093±0.004,与对照组0.371±0.036和0.224±0.013比较,差异均有统计学意义(P＜0.01);曲安奈德组干预24 h后p110蛋白表达水平为0.100±0.004,与对照组比较,差异有统计学意义(P＜0.01),而p-Akt蛋白则未见表达.细胞周期阻滞于G0/G1期的比例增高:LY294002组为(93.813±2.656)％、雷帕霉素组为(88.307±3.667)％、曲安奈德组为(92.937±0.595)％,与对照组(60.883±6.231)％比较,差异均有统计学意义(P＜0.05).凋亡细胞比例增加:LY294002组(11.243±2.347)％,雷帕霉素组(14.407±1.771)％,曲安奈德组(3.983±0.666)％,与对照组(0.507±0.035)％比较,差异有统计学意义(P＜0.01).而总Akt蛋白表达水平无变化.结论 PI3K/Akt信号通路可能参与体外培养血管瘤内皮细胞凋亡的调控.LY294002、雷帕霉素、曲安奈德可能通过干预PI3K/Akt信号通路中相应的靶点,抑制p110激活,阻碍Akt磷酸化,使体外培养血管瘤内皮细胞停滞于G0/G1期并诱导其凋亡.
목적 관찰LY294002、뢰파매소、곡안내덕대체외배양혈관류내피세포중p110、p-Akt、Akt조공적영향,탐토조공PI3K/Akt신호통로재소인혈관류적발생、발전급소퇴과정중적작용.방법 용조직괴결합매소화법배양혈관류내피세포,건립혈관류혈관내피세포계,병용제Ⅷ응혈인자상관항원진행감정.대세포처우대수생장기,환무혈청배양액부육48 h사세포동보화,연후가입25μmol/L LY294002、10 nmol/L뢰파매소、1.0 g/L적곡안내덕계속부육24h,검측혈관류내피세포중p110、p-Akt급Akt적단백표체수평,동시검측혈관류내피세포주기분포급세포조망솔,분석p110、p-Akt급Akt적표체수평여체외배양혈관류내피세포주기분포급조망적상관성.결과 간예24 h후,LY294002조p110화p-Akt적표체수평분별위0.188±0.012화0.091±0.021,뢰파매소조p110화p-Akt적표체수평분별위0.145±0.007화0.093±0.004,여대조조0.371±0.036화0.224±0.013비교,차이균유통계학의의(P＜0.01);곡안내덕조간예24 h후p110단백표체수평위0.100±0.004,여대조조비교,차이유통계학의의(P＜0.01),이p-Akt단백칙미견표체.세포주기조체우G0/G1기적비례증고:LY294002조위(93.813±2.656)％、뢰파매소조위(88.307±3.667)％、곡안내덕조위(92.937±0.595)％,여대조조(60.883±6.231)％비교,차이균유통계학의의(P＜0.05).조망세포비례증가:LY294002조(11.243±2.347)％,뢰파매소조(14.407±1.771)％,곡안내덕조(3.983±0.666)％,여대조조(0.507±0.035)％비교,차이유통계학의의(P＜0.01).이총Akt단백표체수평무변화.결론 PI3K/Akt신호통로가능삼여체외배양혈관류내피세포조망적조공.LY294002、뢰파매소、곡안내덕가능통과간예PI3K/Akt신호통로중상응적파점,억제p110격활,조애Akt린산화,사체외배양혈관류내피세포정체우G0/G1기병유도기조망.
Objective To observe the expressions of PI3Kp110 catalytic subunit,p-Akt and Akt in cultured vascular endothelial cells and to explore the effect of PI3K/Akt signal pathway in different phases of hemangioma in children.Methods The hemangioma vascular endothelial cells derived from the sterile hemangioma were cultured to establish hemangioma endothelial cell lines which was identified by immunohistochemistry of Factor Ⅷ antigen.Cell lines were treated by LY294002 (25 μmol/L),rapamycin (10 nmol/L) and triamcinolone acetonide (1.0 g/L),respectively.PI3Kp110,pAkt and Akt were detected by Western blotting.Cell cycle and cell apoptosis were examined by flow cytometry technique.Results The levels of p110 and p-Akt were lower in cell lines treated by LY294002 (p110 0.188 ± 0.012,p-Akt 0.091 ± 0.021) and rapamycin (p110 0.145 ± 0.007,p-AKT 0.093 ± 0.004) than those in control group (p110 0.371 ± 0.036,p-Akt 0.224 ± 0.013) (P＜0.01).The p110 protein expression level in the triamcinolone acetonide group (0.100 ± 0.004) was lower than that in control group (0.371 ± 0.036),while the p-Akt protein did not express.More Cells were arrested in G0/G1 phase in intervention group [control group (60.883± 6.231)％,LY294002(93.813 ±2.656)％,rapamycin (88.307 ± 3.667)％,triamcinolone acetonide (92.937 ± 0.595)％,P＜ 0.05].The rates of apoptosis cells were increased in intervention group[control group (0.507 ± 0.035) ％,LY294002 (11.243 ± 2.347) ％,rapamycin (14.407 ± 1.771) ％,triamcinolone acetonide (3.983± 0.666)％,P＜0.01].Total Akt protein expression levels showed no significant change.Conclusions PI3K/Akt signaling pathway may be involved in the regulation of apoptosis in cultured hemangioma vascular endothelial cells.LY294002,Rapamycin and triamcinolone acetonide might interfere with PI3K/Akt signaling pathway and down-regulate p-Akt phosphorylation,and arrest cells in G0/G1 phase,thus promoting cell apoptosis.