摘要目的 研究普萘洛尔对体外培养的血管瘤内皮细胞(HemEC)增殖及凋亡的影响,探讨其分子机制.方法 收集7例增生期血管瘤患儿的手术切除血管瘤组织用于体外培养HemEC,同时培养人脐静脉内皮细胞(HUVEC)作为对照.采用0、25、50、75、100、125、150 μmol/L普萘洛尔分别处理两种细胞24、48、72 h.MTT法检测各组细胞的存活率,流式细胞仪检测凋亡率.采用含或不含100 μmol/L普萘洛尔的培养基分别培养HemEC(分别为普萘洛尔干预组及空白对照组)18h,提取两组细胞总RNA,采用表达谱基因芯片技术检测两组HemEC中差异表达的基因,并通过实时定量PCR验证.结果 25 μmol/L普萘洛尔作用24、48 h可引起HemEC轻微增殖(P＜0.05),而≥100 μmol/L普萘洛尔作用≥24 h可引起HemEC存活率下降,作用≥48 h可引起HUVEC存活率下降.100 ～150 μmol/L普萘洛尔作用24～72 h,HemEC细胞存活率比HUVEC低(P＜0.05).100 ～ 150 μmol/L普萘洛尔作用下,HemEC凋亡率随普萘洛尔作用时间及浓度逐渐升高(均P＜0.05).普萘洛尔干预HemEC后,表达谱基因芯片技术筛选出186个表达差异倍数≥1.5的基因,其中表达明显上调的基因128个,明显下调的58个.实时定量PCR显示,普萘洛尔干预组前蛋白转化酶枯草溶菌素9 mRNA表达水平为空白对照组的(9.88士2.19)倍,脂肪酸结合蛋白3 mRNA为(21.90±8.18)倍,差异均有统计学意义(t=7.028、4.427,P＜0.05).结论 高浓度普萘洛尔对HemEC及HUVEC的增殖均有抑制作用,且对HemEC的抑制作用更强.普萘洛尔抑制HemEC的增长可能与抑制HemEC增殖及促进其凋亡有关.

abstractsObjective To evaluate effects of propranolol on the proliferation and apoptosis of in vitro cultured hemangioma endothelial cells (HemEC),and to explore their molecular mechanisms.Methods Hemangioma tissues were resected from 7 children with proliferative hemangioma,and used for in vitro culture of HemEC.Meanwhile,cultured human umbilical vein endothelial cells (HUVEC) served as controls.The 2 kinds of cells were treated with propranolol at different concentrations of 0,25,50,75,100,125 and 150 μmol/L for 24,48 and 72 hours separately.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,and flow cytometry to determine the apoptosis rate.Some cultured HemEC were divided into 2 groups to be treated with 100 μmol/L propranolol-containing culture medium (propranolol group) and culture medium alone (blank control group),respectively,for 18 hours.Total RNA in the 2 groups was extracted separately.Differentially expressed genes in HemEC between the above 2 groups were identified by DNA microarray technology,and verified by real-time quantitative PCR.Results The treatment with 25 μmol/L propranolol for 24 and 48 hours caused a slight proliferation of HemEC (P ＜ 0.05).The survival rate of HemEC was decreased after the treatment with propranolol at the concentration of ≥ 100 μmol/L for more than 24 hours,while the proliferation of HUVEC was inhibited by the treatment with propranolol at the concentration of ≥ 100 μ mol/L for more than 48 hours.During 24-72 hours of treatment with 100-150 μmol/L propranolol,the survival rates of HemEC were significantly lower than those of HUVEC (P ＜ 0.05).After the treatment with 100-150 μmol/L propranolol,the apoptosis rate of HemEC gradually increased with the increase in treatment duration and concentrations of propranolol (all P ＜ 0.05).Compared with the blank control group,186 differentially expressed genes (＞ 1.5-fold changes) were screened out by DNA microarray technology,including 128 upregulated genes and 58 down-regulated genes.Real-time quantitative PCR showed that the mRNA expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) and fatty acid binding protein 3 (FABP3) in the propranolol group were (9.88 ± 2.19) and (21.90 ± 8.18) times that in the blank control group respectively (t =7.028,4.427 respectively,P ＜ 0.05).Conclusions Propranolol at high concentrations can inhibit the proliferation of HemEC and HUVEC,and its inhibitory effect on HemEC is stronger than that on HUVEC.The inhibitory effect of propranolol on HemEC may be related to the inhibition of HemEC proliferation and promotion of HemEC apoptosis.