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目的研究肿瘤坏死因子-α(TNF-α)预处理对脐带间充质干细胞(UC-MSCs)成骨分化能力的影响。方法细胞生长至80%融合时,随机分为3组,每组3瓶,以DMEM/F12培养基培养的UC-MSCs为空白组;以促成骨分化培养基(OS)培养的UC-MSCs为对照组;以经TNF-α预处理的促成骨分化培养基培养的UC-MSCs为实验组。培养第7天,对各组细胞行碱性磷酸酶染色;培养第21天,进行茜素红染色检测成骨分化情况。取培养7,21 d的各组细胞,用实时定量-PCR和免疫印迹法检测各组转录因子Runx2和Osterix的mRNA及蛋白水平的表达变化。结果 TNF-α抑制碱性磷酸酶活性并抑制钙化骨节的形成。Runx2 mRNA的表达,空白组为1.0,对照组的3次表达分别是1.94,2.15,2.34,实验组的3次表达分别是1.27,1.39,1.45。空白组的Runx2蛋白表达为1.0,在7 d,对照组的3次表达分别是1.83,1.95,1.60,实验组的3次表达分别是1.29,1.44,1.61;在21 d,对照组的3次表达分别是1.78,2.25,2.01,实验组的3次表达分别是1.21,1.30,1.16。空白组的Osterix mRNA表达为1.0,对照组的3次表达分别是2.68,2.25,2.01,实验组的3次表达分别是1.21,1.30,1.16;空白组的Osterix的蛋白表达为1.0,在7 d,对照组的3次表达分别是1.83,1.93,2.05,实验组的3次表达分别是1.20,1.32,1.27;在21 d,对照组的3次表达分别是1.91,1.74,1.84,实验组的3次表达分别是1.23,1.50,1.31;与空白组相比较,对照组和实验组的Runx2和Osterix的mRNA及蛋白的表达均上调,差异有统计学意义(P<0.05);但与对照组相比,实验组的Runx2和Osterix的mRNA及蛋白的表达下调,差异有统计学意义(P<0.05)。结论 TNF-α可通过对转录因子Runx2和Osterix在分子水平的抑制作用,从而抑制UC-MSCs的成骨分化。
Objective To investigate the effect of tumor necrosis factor-α (TNF-α) pretreatment on osteogenic differentiation of umbilical cord mesenchymal stem cells (UC-MSCs). Methods When the cells were 80% confluent, they were randomly divided into three groups with 3 bottles in each group. UC-MSCs cultured in DMEM / F12 medium were used as blank group. UC-MSCs cultured in osteogenic differentiation medium Control group; UC-MSCs cultured in osteogenic differentiation medium pretreated with TNF-α were used as the experimental group. On the 7th day, alkaline phosphatase staining was performed on each group of cells. On the 21st day of culture, alizarin red staining was performed to detect osteogenic differentiation. The cells in each group were cultured for 7 and 21 days. The mRNA and protein levels of Runx2 and Osterix in each group were detected by real-time quantitative PCR and Western blotting. Results TNF-α inhibited alkaline phosphatase activity and inhibited the formation of calcified nodules. The expression of Runx2 mRNA in the blank group was 1.0. The expression of Runx2 in the control group was 1.94, 2.15 and 2.34 respectively. The expression of Runx2 in the control group was 1.27, 1.39 and 1.45 respectively. The expression of Runx2 in the blank group was 1.0. On the seventh day, the expression of Runx2 protein in the control group was 1.83, 1.95 and 1.60, respectively. The three expressions in the experimental group were 1.29, 1.44 and 1.61 respectively. On the 21st day, The expressions were 1.78, 2.25 and 2.01, respectively. The three times of expression in the experimental group were 1.21, 1.30 and 1.16 respectively. Osterix mRNA expression was 1.0 in control group, 2.68,2.25,2.01 in control group, and 1.21,1.30 and 1.16 in experimental group respectively. The protein expression of Osterix in control group was 1.0. After 7 days , Respectively. The expression of three times in the control group was 1.83,1.93,2.05 respectively, and the three times in the experimental group were 1.20,1.32,1.27 respectively. On the 21st day, the three times in the control group were 1.91,1.74 and 1.84 respectively. The expression of Runx2 and Osterix mRNA and protein in the control group and experimental group were up-regulated, the difference was statistically significant (P <0.05), but compared with the control group Compared with the experimental group, the mRNA and protein expressions of Runx2 and Osterix in the experimental group were significantly decreased (P <0.05). Conclusion TNF-α can inhibit the osteogenic differentiation of UC-MSCs by inhibiting the transcription factors Runx2 and Osterix at the molecular level.