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目的:研究在重组人肿瘤坏死因子(recombinant human tumor necrosis factor alpha,rhTNF-α)刺激下人脂肪基质细胞(human adipose tissue-derived stromal cells,hADSCs)增殖的改变及成骨向分化前后血管内皮生长因子(vascular endothelial growth factor,VEGF)、成纤维细胞生长因子2(fibroblast growth factor-2,FGF-2)和胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)分泌的变化。方法:采用吸脂术获得的第4代hADSCs,以终浓度为0、1、5、10、50、100μg/L的rhTNF-α刺激hADSCs,分别在48、72、96 h后行MTT法检测不同浓度rhTNF-α对hADSCs增殖的影响;并以该浓度梯度的rhTNF-α刺激hADSCs,24 h后收集上清,以ELISA法检测VEGF、FGF-2和IGF-1的分泌情况;在成骨向诱导的第1、3、7、14天收集细胞上清,ELISA法检测上述3种生长因子分泌的变化,收集细胞上清前24 h更换培养基,并在相应孔中加入rhTNF-α,使其终浓度为10μg/L。所有ELISA数据均以相应培养孔的细胞数进行校正。结果:rhTNF-α能促进hADSCs的增殖,其作用表现为一定的浓度和时间依赖。相比于对照组,48 h时,10μg/LrhTNF-α对增殖的促进作用并不明显,但96 h时则表现为促进作用(P<0.01),而100μg/L rhTNF-α在48 h表现为抑制作用(P<0.01),96 h时则表现为明显的促进作用(P<0.01)。相对于对照组,rhTNF-α可以显著促进hADSCs分泌VEGF、FGF-2和IGF-1(P<0.01);hADSCs经成骨向诱导后,上述3种生长因子的分泌有增加的趋势;不同成骨向分化阶段的hADSCs用10μg/L rhTNF-α刺激后,在诱导第1天,rhTNF-α显著促进VEGF的分泌(P<0.01),但对FGF-2和IGF-1的分泌无显著性影响;在诱导第3天和第7天,rhTNF-α对VEGF(P<0.01)、FGF-2(P<0.05)和IGF-1(P<0.05)的分泌均有促进作用;但在第14天则抑制VEGF(P<0.01)、FGF-2(P<0.05)和IGF(P<0.05)的分泌。结论:一定浓度的rhTNF-α对hADSCs的增殖有促进作用;hADSCs分泌VEGF、FGF-2和IGF-1具有rhTNF-α的浓度依赖;在不同成骨向分化阶段,rhTNF-α对hADSCs分泌VEGF、FGF-2和IGF-1这3种生长因子的作用不同。
OBJECTIVE: To study the changes of proliferation of human adipose tissue-derived stromal cells (hADSCs) stimulated by recombinant human tumor necrosis factor alpha (rhTNF-α) and the effects of osteogenic differentiation on vascular endothelial growth The changes of the secretion of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and insulin-like growth factor-1 (IGF-1) Methods: The hADSCs of the fourth generation were obtained by liposuction. The hADSCs were stimulated with rhTNF-α at the final concentrations of 0, 1, 5, 10, 50 and 100 μg / L, respectively. MTT assay was performed 48, The effect of different concentration of rhTNF-α on the proliferation of hADSCs was observed. The hADSCs were stimulated with rhTNF-α of this concentration gradient. The supernatants were collected 24 h later. The secretions of VEGF, FGF-2 and IGF-1 were detected by ELISA. Cell supernatants were harvested on days 1, 3, 7, and 14, and the secretion of these three growth factors was measured by ELISA. The culture medium was changed 24 h before supernatant was collected and rhTNF-α was added to the corresponding wells. The final concentration of 10μg / L. All ELISA data were corrected for the number of cells in the corresponding culture well. Results: rhTNF-α can promote the proliferation of hADSCs, the role of which showed a certain concentration and time-dependent. Compared with the control group, the effect of 10μg / L rhTNF-α on proliferation was not obvious at 48 h, but it was promoted at 96 h (P <0.01), while that of 100 μg / L rhTNF-α at 48 h (P <0.01), and showed obvious promotion effect at 96 h (P <0.01). Compared with the control group, rhTNF-α can significantly promote the secretion of VEGF, FGF-2 and IGF-1 in hADSCs (P <0.01). After induced by osteogenic induction, hADSCs tended to increase the secretion of these three growth factors; On the 1st day of induction, rhTNF-α significantly promoted the secretion of VEGF (P <0.01), but had no significant difference on the secretion of FGF-2 and IGF-1 after bone differentiation to hADSCs stimulated with 10μg / L rhTNF- (P <0.01), FGF-2 (P <0.05) and IGF-1 (P <0.05) on day 3 and day 7. On the 3rd and 7th day of induction, rhTNF- 14 days inhibited the secretion of VEGF (P <0.01), FGF-2 (P <0.05) and IGF (P <0.05). CONCLUSION: rhTNF-α can promote the proliferation of hADSCs at a certain concentration. The concentration of VEGF, FGF-2 and IGF-1 secreted by hADSCs is dependent on the concentration of rhTNF-α. At different stages of osteogenic differentiation, rhTNF- , FGF-2 and IGF-1 the role of these three kinds of growth factors.