肿瘤坏死因子α对大鼠骨髓间充质干细胞生物学特性的影响

Effects of TNF-α on Biological Properties in Bone Marrow Mesenchymal Stem Cells

目的:探讨不同浓度肿瘤坏死因子α(TNF-α)对大鼠骨髓间充质干细胞(BMSCs)生物学特性的影响。方法:采用全骨髓贴壁法分离培养大鼠BMSCs,倒置显微镜观察细胞形态学变化,流式细胞仪检测BMSCs表面标记物;分别以10、100、1000 ng/mL的TNF-α完全培养液预处理P3代BMSCs,并设置仅含培养基的空白对照组,24 h后采用ELISA试剂盒检测细胞培养上清液中IL-6、IL-10的表达水平;胰酶消化细胞,用Presto Blue法检测10、100、1000 ng/mL TNF-α完全培养液预处理BMSCs后细胞增殖活性。结果:ELISA检测结果显示,与对照组相比,各浓度TNF-α干预组IL-6、IL-10均有不同程度升高,差异有统计学意义(P<0.05),且1000 ng/mL TNF-α干预组IL-10表达水平最高,其浓度为170.2±11.9 pg/mL,组间比较差异有统计学意义(P<0.05);100 ng/mL TNF-α干预组IL-6表达水平最高,其浓度为144.0±18.6 pg/mL,组间比较差异有统计学意义(P<0.05)。Presto Blue检测结果显示,与对照组相比,10、100、1000 ng/mL TNF-α干预组荧光值均增高,差异有统计学意义(P<0.05),但组间荧光值比较差异无统计学意义(P>0.05)。结论:10~1000 ng/mL TNF-α可促进BMSCs的增殖,且随着TNF-α浓度的升高,BMSCs分泌促炎因子IL-6的能力降低,分泌抑炎因子IL-10的能力升高。

Objective:To investigate the effects of different concentrations of tumor necrosis factor-α(TNF-α)on the release of interleukin-6(IL-6)and interleukin-10(IL-10)from rat bone marrow mesenchymal stem cells(BMSCs).Methods:BMSCs were isolated and cultivated by whole bone marrow adherent culture method.Morphological changes of BMSCs were observed by inverted phase contrast microscope,the surface markers of BMSCswere detected by flow cytometry.The BMSCs were treated with10,100and1000ng/mL TNF-αcomplete culture medium respectively,the control group containing medium only,24hours later,IL-6and IL-10in the cell culture supernatants were detected by ELISA kit.Then the cells were treated with trypsin to detect the cell viabilitywith PrestoBlue method.Results:The levels of IL-6and IL-10in the supernatants of TNF-αtreated group weresignificantly higher than those in the control group(P<0.05).And IL-10(170.2±11.9pg/mL)was the highest in1000ng/mL TNF-αintervention group,IL-6(144.0±18.6pg/mL)was the highest in100ng/mL TNF-αintervention group,the difference was statistically significant among groups(P<0.05).PrestoBlue test showed that the fluorescence values of10,100and1000ng/mL TNF-αgroups were all higher than those of the control group butthere was no significant difference in fluorescence between groups(P>0.05).Conclusion:TNF-αmay influencethe proliferation of BMSCs and the secretion of IL-6and IL-10.With the increase of TNF-αconcentration,theBMSCs can increase the level of proinflammatory cytokine IL-6and decrease anti-inflammatory cytokine IL-10.