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目的 :探讨低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)基因对体外诱导牙髓干细胞(dental pulp stem cells,DPSCs)成血管分化方法并进行鉴定。方法 :取拔除的健康、完整的前磨牙标本20例,提取DPSCs细胞,采用Strol-1、CD146分子进行鉴定。根据DPSCs是否转染HIF-1α基因分为实验组和对照组,RT-PCR法测定HIF-1αm RNA表达,Western印迹检测培养1、4、7、14 d不同时间段HIF-1α及成血管相关因子VEGF、SDF-1、Ang-2及PDGF的表达情况。采用SPSS16.0软件包对数据进行统计学分析。结果 :倒置相差显微镜观察,DPSCs多数细胞呈圆形、椭圆形类,免疫荧光观察到Strol-1、CD146均呈绿色荧光。实验组HIF-1α蛋白、m RNA随着时间的延长,表达水平越来越高,差异显著(P<0.05)。实验组转染后1、4、7、14 d后与对照组相比,HIF-1α蛋白、m RNA显著增高(P<0.05)。对照组血管相关因子VEGF、SDF-1、Ang-2和PDGF随时间的延长,其表达水平无显著差异(P>0.05)。实验组VEGF、SDF-1、Ang-2和PDGF随培养时间延长,其表达水平逐渐升高,不同时间点间差异显著(P<0.05)。与对照组相比,转染后1、4、7、14 d差异显著(P<0.05)。结论:HIF-1α基因修饰DPSCs能够成功体外诱导其血管分化作用,为进一步血管形成研究奠定了基础。
AIM: To investigate and identify the vascular differentiation of dental pulp stem cells (DPSCs) induced by hypoxia inducible factor 1α (HIF-1α) gene in vitro. Methods: Twenty healthy and intact premolar specimens were removed and DPSCs were isolated and identified by Strol-1 and CD146. The expression of HIF-1αmRNA was determined by RT-PCR and Western blotting was used to detect the expression of HIF-1α and angiogenesis at different time points after 1, 4, 7 and 14 days of culture in accordance with whether DPSCs transfected HIF-1α gene into experimental group and control group Factors VEGF, SDF-1, Ang-2 and PDGF expression. SPSS16.0 software package for statistical analysis of the data. Results: By inverted phase contrast microscope, most of the cells in DPSCs were round and oval, and Strol-1 and CD146 were observed by immunofluorescence. The expression of HIF-1αprotein and mRNA in the experimental group was higher and higher with the time increasing, the difference was significant (P <0.05). Compared with the control group, HIF-1αprotein and m RNA were significantly increased (P <0.05) at 1, 4, 7 and 14 days after transfection in experimental group. The expression levels of VEGF, SDF-1, Ang-2 and PDGF in the control group were not significantly different with time prolonging (P> 0.05). The expression of VEGF, SDF-1, Ang-2 and PDGF in the experimental group increased gradually with the prolongation of culture time, and the expression levels of VEGF, SDF-1, Ang-2 and PDGF were significantly increased at different time points (P <0.05). Compared with the control group, there was significant difference at 1, 4, 7 and 14 days after transfection (P <0.05). CONCLUSION: HIF-1α gene-modified DPSCs can successfully induce vascular differentiation in vitro, which lays the foundation for further study of angiogenesis.