Ang1~7对婴幼儿血管瘤内皮细胞增殖及凋亡的影响

Effects of Angiotensin 1-7 on proliferation and apoptosis of hemangioma-derived endothelial cells in vitro

目的研究血管紧张素1~7(Ang1~7)对婴幼儿血管瘤内皮细胞(Hem ECs)的增殖、凋亡及Mas受体mRNA表达的影响。方法收集2016—2017年间在广东医科大学附属医院整形外科经手术切除的临床及病理诊断为增殖期婴幼儿血管瘤组织块3例,采用贴壁法培养并经第Ⅷ因子相关抗原鉴定为Hem ECs,qRT-PCR、Western blot检测Hem ECs中Mas受体mRNA及蛋白质的表达。MTT法检测不同浓度Ang1~7对Hem ECs增殖的影响,qRT-PCR检测Mas受体mRNA表达的影响。采用Annexin V-FITC/PI双染流式细胞仪术检测Ang1~7作用Hem ECs 24 h、48 h后Hem ECs的凋亡率。结果成功培养并经第Ⅷ因子相关抗原鉴定为Hem ECs,qRT-PCR及Western blot检测结果显示:Hem ECs中有Mas受体mRNA及蛋白质的表达,与人脐静脉内皮细胞相比较表达增强,差异有统计学意义(P<0.05);Ang1~7浓度大于1μmol/L抑制Hem ECs的增殖,且呈时间浓度依赖性,可上调Mas受体mRNA表达,差异有统计学意义(P<0.05);Ang1~7作用Hem ECs后24 h细胞总凋亡率为(0.79±0.13)%,48 h细胞总凋亡率为(1.48±0.18)%,Ang1~7作用Hem ECs 48 h细胞凋亡率显著增加,差异有统计学意义(P<0.05)。结论Hem ECs中有Mas受体蛋白及mRNA的表达,Ang1~7可以抑制Hem ECs增殖、促进其凋亡并上调Mas受体mRNA的表达。

Objective To investigate the effects of Angiotensin 1-7(Ang1-7)on proliferation and apoptosis of hemangioma-derived endothelial cells(Hem ECs)and the mRNA expression of Mas receptor in Hem ECs.Methods Three infantile hemangioma tissue blocks were collected from Department of Plastic Surgery,the Affiliated Hospital of Guangdong Medical University from 2016 to 2017,which were resected by surgery and diagnosed as proliferative stage clinically and pathologically.Cells were obtained through the tissue explants adherent method and identified by immunocytochemical staining of anti-factorⅧand morphology as Hem ECs,and then the expression of Mas receptor protein and the mRNA in Hem ECs were examined by Western blot and qRT-PCR,.The effect of different concentrations of Ang1-7 on the proliferation of Hem ECs was determined by MTT,and the expression of MAS receptor mRNA was detected by qRT-PCR.Annexin V-FITC/PI double staining flow cytometry was used to observe the apoptosis of Hem ECs at 24 and 48 hour under the treatment of Ang1-7.Results Hem ECs were successfully cultured and identified by immunocytochemical staining of anti-factorⅧ.qRT-PCR and western blot results showed that there were Mas receptor mRNA and protein expression in Hem ECs,and the expression level was significantly higher than those in human umbilical vein endothelial cells(HUVEC),with statistically significant difference(P<0.05).When the drug concentration was greater than 1μmol/L,Ang1-7 inhibited the proliferation of Hem ECs with dependence of concentration and time and up-regulated Mas receptor mRNA expression(P<0.05).The apoptosis rate of Hem ECs was(0.79±0.13)%at 24 h and(1.48±0.18)%at 48 h under the treatment of Ang1-7.The apoptosis rate of Hem ECs was significantly increased at 48 h by the treatment of Ang1-7(P<0.05).Conclusion There were Mas receptor protein and mRNA expression in Hem ECs.Ang1-7 inhibited cell proliferation of Hem ECs and promoted their apoptosis.Ang1-7 might significantly up-regulate the expression of Mas receptor mRNA in Hem ECs.