DMOG对人牙周膜干细胞成骨分化和成血管能力影响的体外研究

Effect of dimethyloxalylglycine on osteogenic differentiation and angiogenesis of human periodontal ligament stem cells in vitro

目的探讨二甲氧已二酰甘氨酸(DMOG)对人牙周膜干细胞(h PDLSCs)体外成骨分化和成血管能力的影响。方法采用组织块法分离培养h PDLSCs,应用流式细胞仪进行鉴定。给予0、0.1、1、10、100μmol/L的DMOG处理,MTT法分析DMOG对h PDLSCs增殖能力和细胞活力影响,RTPCR检测核心结合因子2(RUNX2)、碱性磷酸酶(ALP)、骨钙素(OCN)、血管内皮生长因子(VEGF)基因mRNA表达,Western blot法检测低氧诱导因子-1α(HIF-1α)、VEGF蛋白表达。成骨诱导14 d后行ALP染色及茜素红染色。结果从人牙周膜组织中分离培养的细胞经鉴定为h PDLSCs。MTT结果显示,DMOG可呈剂量依赖性地抑制h PDLSCs的增殖(P<0.05),DMOG能提高缺血清条件下h PDLSCs的细胞活力(P<0.05)。RT-PCR和Western blot结果显示,10μmol/L的DMOG可显著上调RUNX2、ALP、OCN基因mRNA表达(P<0.05),而100μmol/L的DMOG可显著上调VEGF基因mRNA和HIF-1α、VEGF蛋白水平的表达(P<0.05)。ALP和茜素红染色显示,10μmol/L的DMOG可显著促进h PDLSCs成骨分化中ALP的表达和钙结节的形成。结论DMOG通过上调HIF-1α的表达,促进h PDLSCs的成骨分化和成血管能力。

Objective To investigate the effect of DMOG on osteogenic differentiation and angiogenesis of h PDLSCs in vitro. Methods h PDLSCs was isolated and cultured by tissue block method and identified by flow cytometry.h PDLSCs was treated with DMOG of 0,0. 1,1,10 and 100 μmol/L,effect of DMOG on proliferation and cell viability of h PDLSCs by MTT assay. The expression level of RUNX2,ALP,OCN,VEGF gene were detected by Real time-PCR and Western blot were used to detect protein levels of HIF-1α and VEGF. After 14 days of osteogenic induction,alkaline phosphatase（ ALP） staining and alizarin red staining were performed. Results The cells isolated from human periodontal ligament tissues were identified as h PDLSCs. MTT results showed that DMOG inhibited proliferation of h PDLSCs in a dose-dependent manner and increased cell viability in serum deficient state（ P〈0. 05）. RT-PCR and Western blot showed that the expression of 10 μmol/L DMOG group significantly increased RUNX2,ALP,OCN mRNA expression（ P〈0. 05）;100 μmol/L DMOG group significantly up-regulated the expression of VEGF mRNA and HIF-1α,VEGF protein（ P〈0. 05）. ALP and alizarin red staining showed that 10 μmol/L DMOG group could significantly promoted the expression of ALP and the formation of calcium nodules in osteogenic differentiation of h PDLSCs. Conclusion DMOG promotes the osteogenic differentiation and angiogenesis of h PDLSCs by up-regulating the expression of HIF-1α.