普萘洛尔对血管瘤中HemSC凋亡和VEGF、bFGF表达的影响及临床意义

Effect of propranolol on apoptosis and VEGF,bFGF expression of hemangioma-derived stem cells

目的:研究普萘洛尔(PRN)治疗血管瘤患儿的药代动力学及其对血管瘤干细胞(HemSC)的作用机制。方法:12例患儿以1 mg/kg.d口服PRN,按每天1次(qd)和每天2次(bid,间隔8 h)随机分为2组,每组6例。用反相高效液相色谱法(RP-HPLC)检测首次服药后2、6、10、24 h的血药浓度。对体外培养的HemSC分别给予0、0.02、0.2、2、20μmol/L浓度的PRN作用后,通过MTT、流式细胞仪分析、反转录PCR、实时荧光定量PCR、酶联免疫吸附试验和免疫印迹分析,检测其对HemSC增殖、凋亡以及对VEGF、bFGF表达的影响。将HemSC经PRN处理后与人脐静脉内皮细胞(HUVEC)联合注入BALB/c-nu裸鼠皮下,以未处理组作为对照,1周后处死取材,行HE染色,测定平均微血管密度(MVD),采用SPSS17.0软件包对数据进行配对t检验。使用免疫组织化学SP法观察PRN对血管瘤动物模型中VEGF、bFGF表达的影响。结果:口服PRN后,qd、bid 2组血药浓度均在2 h达到高峰,qd组的高峰浓度为(207.8±33.9)ng/mL,bid组的高峰浓度为(155.2±40.6)ng/mL,2组消除半衰期(t1/2)超过6 h。qd组在服药10 h时,血药浓度明显下降,而bid组在10 h时血药浓度升高。常规血药浓度的PRN不能抑制HemSC活性和增殖,对凋亡无显著影响,但能显著抑制HemSC表达VEGF、bFGF的mRNA和蛋白质,浓度在0.2～2μmol/L范围内抑制效果最佳。血管瘤动物模型显示,PRN能使MVD显著减少。免疫组化染色显示,VEGF、bFGF在IH组织中高表达;应用PRN后,VEGF表达减弱,而bFGF变化不明显。结论:PRN在常规剂量血药浓度内不能促进HemSC凋亡,但可抑制HemSC表达VEGF、bFGF。

PURPOSE：Propranolol was found by chance very effective in treating hemangioma,yet the therapeutic mechanism remain unclear.This study was aimed to investigate the therapeutic mechanism of propranolol on hemangioma.METHODS：The plasma propranolol concentrations at 2,6,10,24 h were assayed by reversed phase high performance liquid chromatography（RP-HPLC）after oral administration of propranolol（1 mg/kg.d） once a day（qd,n=6） or twice a day（bid,n=6）;The hemangioma-derived stem cells（HemSCs） were isolated from Chinese IH patients.The effect of propranolol on proliferation,apoptosis rate of HemSCs was observed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） method and bi-fluorescence flow cytometry（FCM）.With RT-PCR,real-time PCR,Elisa and Western blot,expression of vascular endothelial growth factor（VEGF） and basic fibroblast growth factor（bFGF） was observed after treatment with different concentrations（0,0.02,0.2,2.20 μM） of PRN at different times.For in vivo study,HemSCs were treated with PRN,and then subcutaneously injected into BALB/c-nu mice with human umbilical vein endothelial cell（HUVEC）.The grafts were harvested after 1 week for HE staining and immunohistological staining.SPSS 17.0 software package was used for statistical analysis.RESULTS：The peak propranolol concentration reached at 2h in both qd group（207.8 ±33.9 ng/mL） and bid group（155.2 ±40.6 ng/mL）,the elimination half-life（t 1/2） was longer than 6h.The mean concentration of 10h was significantly decreased in qd group,while that was elevated in bid group.The proliferation and total apoptosis rate of HemSCs was not obviously affected by PRN in normal concentration;but the expression of VEGF and bFGF in HemSC was suppressed by PRN.In vivo model,the mean microvessel density（MVD） was significantly decreased after HemSCs were treated by PRN.CONCLUSION：PRN can inhibit HemSC to express VEGF and bFGF,but can’t inhibit proliferation or induce apoptosis of HemSCs.