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

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蛋白相对表达量均增加,p-GSK3β蛋白相对表达量降低,细胞小管形成数量及吞噬脂质能力升高,差异均有统计学意义(P<0.05);而Wnt抑制剂组各指标与HIF-1α过表达组均相反(P<0.05)。与HIF-1α过表达组比较,联合组细胞中VCAM-1、KDR和VE mRNA表达水平、vWF和CD31阳性细胞百分比、β-catenin蛋白相对表达量均降低,p-GSK3β蛋白相对表达量升高,细胞小管形成数量及吞噬脂质能力降低,差异均有统计学意义(P<0.05)。结论过表达HIF-1α可通过激活Wnt/β-catenin信号通路,促进SHED向血管内皮细胞定向诱导分化。

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). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher(P<0.05), the relative expression of p-GSK3β protein was significantly lower(P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased(P<0.05) in the HIF-1α overexpression group;while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group(P<0.05). Compared with the HIF-1αoverexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower(P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups(P<0.05). Conclusion Overexpression of HIF-1αcan promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.