普萘洛尔及异丙肾上腺素对体外培养婴儿血管瘤内皮细胞增殖和血管内皮生长因子、碱性成纤维细胞生长因子表达的影响

In vitro effects of propranolol and isoproterenol on proliferation of cultured infantile hemangioma endothelial cells and expressions of vascular endothelial growth factors and basic fibroblast growth factor

目的：探讨普萘洛尔及异丙肾上腺素在体外对婴儿血管瘤内皮细胞增殖及血管内皮细胞生长因子（VEGF、VEGF-2）、人碱性成纤维细胞生长因子（bFGF）表达的影响。方法将体外培养的2～3代增殖期婴儿血管瘤内皮细胞分为普萘洛尔组和异丙肾上腺素组，其中普萘洛尔组分别换浓度为10、15、20 mg/L 普萘洛尔工作液（10、15、20 mg/L 普萘洛尔组）、空白 EGM-2培养基（空白组）和含0.16% DMSO EGM-2培养基（DMSO组）培养，而异丙肾上腺素组分别换5、10、20 mg/L 异丙肾上腺素工作液（5、10、20 mg/L 异丙肾上腺素组）和空白EGM-2培养基（空白组）培养。采用 MTT 法测定24、48、72、96 h 时各组血管瘤内皮细胞的增殖情况，ELISA 法测定培养24、48 h 时各组细胞培养上清液中 VEGF、VEGF-2、bFGF 的浓度。结果培养24、48 h 时，10、15、20 mg/L 普萘洛尔组血管瘤内皮细胞增殖差异无统计学意义（H 值分别为1.152、2.643，均 P 〉0.05）；72、96 h 时，20 mg/L 普萘洛尔组血管瘤内皮细胞的增殖明显低于空白组（P 〈0.05），而空白组、DMSO 组及10、15 mg/L 普萘洛尔组间差异均无统计学意义。各浓度普萘洛尔、异丙肾上腺素作用24 h 时均对 VEGF、VEGF-2、bFGF 水平有一定影响。48 h 时，15、20 mg/L 普萘洛尔组 VEGF 水平均较空白组显著下降，同时10、15、20 mg/L 普萘洛尔组VEGF-2、bFGF 水平也均较空白组显著下降（P 〈0.05）；而5、10、20 mg/L 异丙肾上腺素组 VEGF 水平均较空白组显著升高（P 〈0.05），20 mg/L 异丙肾上腺素组 VEGF-2、bFGF 水平均显著高于空白组及5、10 mg/L 异丙肾上腺素组（P 〈0.05）。结论一定浓度普萘洛尔作用于增殖期婴儿血管瘤内皮细胞一定时间后能够抑制细胞生长，而异丙肾上腺素反之，两者对细胞因子表达的影响作用也相反，因此推断普萘洛尔治疗婴儿血管瘤的机制可能与β肾上腺素能受体及其下游信号转导影响相关细胞因子表达相关。

Objective To evaluate in vitro effects of propranolol and isoproterenol on proliferation of infantile hemangioma endothelial cells（IHECs）as well as expressions of vascular endothelial growth factors（VEGF and VEGF-2） and human basic fibroblast growth factor （bFGF）. Methods The second - third passage endothelial cells derived from the proliferative phase of infantile hemangioma were divided into propranolol and isoproterenol groups. The propranolol group was further classified into 5 groups to be treated with propranolol solutions at concentrations of 10, 15, 20 mg/L, EGM-2 medium （blank control group 1）, or EGM-2 medium containing 0.16% DMSO （DMSO group）, while the isoproterenol group was classified into 4 groups to be treated with isoproterenol solutions at concentrations of 5, 10, 20 mg/L or EGM-2 medium（blank control group 2）. Methyl thiazolyl tetrazolium（MTT）assay was performed to evaluate cellular proliferative activity in these propranolol groups at 24, 48, 72 and 96 hours separately, enzyme-linked immunosorbent assay （ELISA）to measure VEGF, VEGF-2 and bFGF lev els in culture supernatants of IHECs at 24 and 48 hours separately. Results The proliferative activity of IHECs showed no significant differences between 10-, 15- and 20-mg/L propranolol groups at either 24 or 48 hours （H = 1.152, 2.643, respectively, both P 〉 0.05）, or between the blank control group 1, DMSO group, and 10- and 15-mg/L propranolol groups at either 72 or 96 hours, but significantly decreased in the 20-mg/L propranolol group compared with the blank control group 1 at 72 and 96 hours （both P 〈 0.05）. The 24-hour treatments with propranolol or isoproterenol at the above concentrations all affected the expressions of VEGF, VEGF-2 and bFGF to different degrees. At 48 hours, there was a significant decrease in VEGF levels in 15- and 20-mg/L propranolol groups, as well as in VEGF-2 and bFGF levels in 10-, 15- and 20-mg/L propranolol groups compared with the blank control group 1 （all P 〈 0.05）, but a significant increase in VEGF levels in 5-, 10- and 20-mg/L isoproterenol groups compared with the blank control group 2 （all P 〈 0.05）, as well as in VEGF-2 and bFGF levels in the 20-mg/L isoproterenol group compared with the blank control group 2, 5- and 10-mg/L isoproterenol groups （all P 〈 0.05）. Conclusions The treatment with propranolol at certain concentrations for certain durations can suppress the growth of, as well as expressions of VEGF, VEGF-2 and bFGF in, endothelial cells derived from the proliferative phase of infantile hemangioma, whereas that with isoproterenol has opposite effects. The therapeutic mechanism of propranolol in infantile hemangioma may be associated with expressions of β-adrenergic receptors and their downstream signal transduction-related cytokines.