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目的:观察肿瘤坏死因子(tumor necrosis factor,TNF)-α预处理骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)后,对其核因子(nuclear factor,NF)-κB通路以及成骨分化能力的影响。方法:实验分为BMMSCs阴性对照组(A组)、TNF-α预刺激1 d组(B组)及TNF-α预刺激7 d组(C组)。预刺激结束后,采用Western blot方法检测各组IκBα和pIκBα蛋白水平的表达;采用RT-PCR方法检测各组BMMSCs中分泌红细胞生成素和肝细胞受体B4(erythroprotein-producing hepatocellular receptor B4,EPHB4)、胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)、骨保持素(osteoprotegerin,OPG)、白细胞介素(interleukin,IL)-7及基质金属蛋白酶(metrix metalloproteinase,MMP)-1 mRNA表达水平的变化;加成骨诱导剂诱导后,茜素红及碱性磷酸酶检测各组成骨分化能力。同时观察加用NF-κB抑制剂吡咯烷二硫氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)对TNF-α作用的影响。结果:与A组相比,B组和C组IκBα蛋白的表达水平降低(P<0.05),p-IκBα蛋白的表达水平增高(P<0.05);与A组相比,B组和C组的EPHB4、IGF-1、OPG mRNA表达水平上调(P<0.05),IL-7及MMP-1mRNA表达水平下调(P<0.05);与A组相比,B组和C组的成骨分化能力增强,而加用NF-κB抑制剂后,B组和C组的成骨分化能力减弱。结论:体外实验表明,BMMSCs经TNF-α预处理后,可以促使BMMSCs成骨分化活性,并且多次刺激较单次刺激效果明显,其促进成骨分化效果至少部分是通过NF-κB通路介导的。
OBJECTIVE: To observe the effects of tumor necrosis factor (TNF) -α preconditioning on bone marrow mesenchymal stem cells (BMMSCs) and its nuclear factor (NF) -κB pathway and osteogenic differentiation The impact of ability. Methods: The experiment was divided into three groups: the negative control group (group A), the pre-stimulation of TNF-α group (group B) and the pre-stimulation of TNF-α for 7 d group (group C). Western blot was used to detect the expression of IκBα and pIκBα protein in each group after the pre-stimulation. RT-PCR was used to detect the expression of erythroprotein-producing hepatocellular receptor B4 (EPHB4) , Insulin-like growth factor 1 (IGF-1), osteoprotegerin (OPG), interleukin (IL) -7 and metrix metalloproteinase mRNA expression level changes; osteogenic differentiation ability of each group was detected by alizarin red and alkaline phosphatase after induced by addition of bone induction agent. Meanwhile, the effect of adding pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on TNF-α was also observed. Results: Compared with group A, the expression of IκBα protein in group B and group C was decreased (P <0.05) and the expression of p-IκBα protein was increased (P <0.05). Compared with group A, group B and group C (P <0.05), while the levels of IL-7 and MMP-1 mRNA were down-regulated (P <0.05). Compared with group A, the osteogenic differentiation ability of group B and group C Enhanced, and with the addition of NF-κB inhibitor, B and C group decreased osteogenic differentiation. CONCLUSIONS: In vitro experiments show that BMMSCs pretreatment with TNF-α can promote the osteogenic differentiation of BMMSCs, and the effect of multiple stimulation is better than that of single stimulation. The effect of osteoblast differentiation is at least partly mediated by NF-κB pathway of.