rhTNF-α对成骨向分化前后的人脂肪基质细胞分泌血管生成相关生长因子的影响

Effects of rhTNF-α on the secretion of angiogenesis-related growth factors of human adipose tissue-derived stromal cells before and after osteogenic differentiation

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 investigate the proliferation and the secretion of vascular endothelial growth factor（ VEGF）, fibroblast growth factor-2 （FGF-2） and insulin-like growth factor-1 （IGF-1） of human adipose tissue-derived stromal cells （hADSCs）before and after osteogenic differentiation under the stimuli of recombinant human tumor necrosis factor alpha （rhTNF-α）. Methods： hADSCs were obtained from human ]ipoaspirates. All the cells used were at passage four. The proliferation of hADSCs was measured with MTF assays 48, 72, 96 hours after being treated with 0, 1, 5, 10, 50 or 100 μg/L rhTNF-α respectively. The secretion of VEGF, FGF-2 and IGF-1 of the undifferentiated hADSCs under stimuli of rhTNF-α with the above 5 concentration grades was observed and the secretion of these 3 growth factors of hADSCs at different stages of osteogenic differentiation under stimuli of 10 μg/L rhTNF-α was also observed. All the supernatants were harvested for measuring after 24 hours＇ incubation with rhTNF-α. The secretion of VEGF, FGF-2 and IGF-1 was measured with ELISA, and the values were normalized to the cell number of the corresponding wells. Results： The effect of rhTNF-α on the proliferation of hADSCs varied with the concentration and time. Compared with the control（0 μg/L）, 10μg/L rhTNF-α showed no suppression or acceleration on proliferation of hADSCs at hour 48, but significantly promoted the proliferation at hour 96 （0. 903 ±0. 042 vs 0. 810 ±0. 011 , P 〈0. 01）, 100 μg/L rhTNF-α seemed to sup-press the proliferation at hour 48 （0. 317 ±0. 024 vs 0. 458 ±0. 046, P 〈0. 01 ）, but appeared to promote it （0. 956 ±0. 030 vs 0. 810 ±0. 011, P 〈0. 01 ） at hour 96. rhTNF-α（ 1, 5, 10, 50 and 100 μg/ L） significantly increased VEGF, FGF-2 and IGF-1 production of hADSCs versus the control （0μg/L） （P 〈 0. 01 ）. After osteogenie differention, the secretion of the three growth factors of hADSCs （ without rhTNF-μ treated） was elevated with the days increasing. Under the stimulus of 10 μg/L rhTNF-α, the hADSCs after 1 day of osteogenic differentiation significantly increased the secretion of VEGF （ P 〈 0. 01 ） compared with the group without rhTNF-α treated; after 3 and 7 days of osteogenic differentiation, the hADSCs significantly increased the secretion of VEGF （P 〈 0. 01 ）, FGF-2 （ P 〈 0. 05 ） and IGF-1 （ P 〈 0. 05）. However, after 14 days of osteogenic differentiation, 10 μg/L rhTNF-α appeared to suppress the production of VEGF （P 〈0. 01 ）, FGF-2 （P 〈0. 05） and IGF-1 （P 〈0. 05） of the differentiated hADSCs. Conclusion： Within certain concentration range, rhTNF-α can promote the proliferation of hADSCs and the production of VEGF, FGF-2 and IGF-1. The effect of 10 μg/L rhTNF-α on the production of VEGF, FGF- 2 and IGF-Ⅰ of the differentiated hADSCs varied at different stages of osteogenic differentiation.