过表达低氧诱导因子1α对乳牙牙髓干细胞向血管内皮细胞分化影响的实验研究  

Effect of hypoxia inducible factor 1α overexpression on differentiation of stem cells derived from human exfoliated deciduous teeth into vascular endothelial cells

作　　者：李媛媛 陈丹 吴蓓 LI Yuanyuan;CHEN Dan;WU Bei(Department of Stomatology,Chengdu Women's and Children's Central Hospital,School of Medicine,University of Electronic Science and Technology of China,Chengdu Sichuan,610036,P.R.China;Department of Stomatology,Qingyang District Hospital of Traditional Chinese Medicine,Chengdu Sichuan,610036,P.R.China)

机构地区：[1]电子科技大学医学院附属妇女儿童医院·成都市妇女儿童中心医院口腔科,成都610036 [2]成都市青羊区中医医院口腔科,成都610036

出　　处：《中国修复重建外科杂志》2021年第6期761-768,共8页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的探讨过表达低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)对乳牙牙髓干细胞(stem cells derived from human exfoliated deciduous teeth,SHED)向血管内皮细胞分化的影响。方法取自愿捐赠的健康儿童滞留乳牙,采用胶原酶消化法分离培养SHED并传代。取第3代细胞经流式细胞术以及成骨诱导分化并茜素红和ALP染色鉴定后,分为空白对照组(未经任何处理)、空载组(感染空载慢病毒)、HIF-1α过表达组(感染HIF-1α过表达慢病毒)、Wnt抑制剂组[经Wnt/β-连环蛋白(β-catenin)信号通路抑制剂IWR-1干预]和联合组(感染HIF-1α过表达慢病毒并经IWR-1干预),并对应处理。其中,取空白对照组、空载组及HIF-1α过表达组细胞采用实时荧光定量PCR和Western blot法检测HIF-1αmRNA和蛋白表达水平,观察转染效率。将5组细胞向血管内皮细胞定向诱导分化14 d后,采用流式细胞术检测细胞表面标记分子血管性血友病因子(von Willebrand factor,vWF)和CD31表达水平;实时荧光定量PCR检测细胞中血管细胞黏附分子1(vascular cell adhesion protein 1,VACM-1)、VEGF受体-2 KDR(Kinase-inserted domain containing receptor)和血管内皮钙黏蛋白(VEcadherin,VE)mRNA表达水平;Western blot法检测细胞中Wnt/β-catenin信号通路相关蛋白磷酸化糖原合酶激酶3β(phosphate-glycogen synthasc kinase 3β,p-GSK3β)及β-catenin表达水平;Matrigel基质胶成管实验检测细胞体外小管形成情况;DiI标记的乙酰化低密度脂蛋白(DiI-labeled acetylated low density lipoprotein,DiI-Ac-LDL)吞噬实验检测细胞吞噬脂质能力。结果经鉴定分离得到的细胞为SHED。慢病毒转染后,与空白对照组和空载组比较,HIF-1α过表达组HIF-1αmRNA和蛋白表达水平均明显增加(P<0.05)。向血管内皮细胞定向诱导分化14 d后,与空白对照组和空载组比较,HIF-1α过表达组细胞中VCAM-1、KDR和VE mRNA相对表达量、vWF和CD31阳性细胞百分比、β-catenin蛋白相对表达Objective To investigate the effects of hypoxia inducible factor 1α(HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth(SHED) into vascular endothelial cells.Methods SHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase(ALP)staining after osteogenic differentiation culture. The SHED were divided into blank control group(SHED without any treatment), empty group(SHED infected with empty lentivirus), HIF-1α overexpression group(SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group(SHED interfered by IWR-1), and combination group(HIF-1αoverexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor(vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1(VCAM-1), KDR(Kinase-inserted domain containing receptor), and VE-cadherin(VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β(p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein(DiI-Ac-LDL) phagocytosis. Results After identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly(P<0.05)

关 键 词：乳牙牙髓干细胞 低氧诱导因子1Α WNT/Β-CATENIN信号通路 细胞分化 

分 类 号：R781.3[医药卫生—口腔医学]