摘要：

目的：探究TARDNA结合蛋白-43（TARDNA binding protein-43，TDP-43）对脂多糖（lipopolysaccharides，LPS）诱导的炎症微环境中小鼠牙龈成纤维细胞(mouse gingival fibroblasts cells，MGFs)的影响。方法：分离培养野生型C57BL/6J小鼠的牙龈成纤维细胞。设置不同浓度及处理时间的LPS刺激，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）技术检测相关炎症因子的mRNA表达量，筛选出最佳处理条件。对细胞进行免疫荧光染色，观察炎症微环境下MGFs中TDP-43的变化。使用小干扰RNA（small interfering RNA，siRNA）转染技术敲低MGFs中的TDP-43，并测定转染效率。设置3个实验组：阴性对照组（NC组）、LPS诱导对照组（NL组）和TDP-43敲低+LPS诱导组（SL组）。利用RT-qPCR技术检测部分炎症因子、基质金属蛋白酶（matrix metalloproteinases，MMPs）、黏附相关因子mRNA的表达情况。应用蛋白质印迹法（western blotting）检测部分炎症因子、黏附相关因子蛋白表达情况。使用天狼星红染色法探究细胞内胶原沉积情况。结果：最佳LPS刺激的浓度为100 ng/mL，时间为6 h；在无LPS刺激的状态下，MGFs中的TDP-43基本在细胞核内，LPS刺激后，TDP-43出现在细胞核和细胞质中；成功使用siRNA转染MGFs构建敲低TDP-43的细胞模型，转染效率接近50%；在LPS刺激下，TDP-43敲低的实验组（SL组）与对照实验组（NL组）相比，炎症因子、基质金属蛋白酶、黏附相关因子mRNA的表达量出现下调（P<0.05），且部分黏附相关蛋白及炎症因子蛋白的表达量也显著下调；在胶原沉积方面，SL组和NL组相较于NC组都有减少（P<0.05），但NL组和SL组差异不大，无统计学意义（P>0.05）。结论：当LPS诱导MGFs处于炎症微环境时，TDP-43出现由核内向核外移动的趋势；且TDP-43对LPS诱导的MGFs炎症微环境下炎症因子、黏附相关因子及基质金属蛋白酶表达起正向调控作用，对胶原沉积的影响不明显。

关键词: TDP-43, 脂多糖, 牙周炎, 小鼠牙龈成纤维细胞

Abstract:

Objective: To explore the effects of TARDNA binding protein-43 (TDP-43) on mouse gingival fibroblasts (MGFs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment. Methods: The gingival fibroblasts of wild-type C57BL/6J mice were cultured. LPS of different concentrations and treatment time were set, mRNA expression levels of related inflammatory factors were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and the optimal treatment conditions were selected. The cells were stained with immunofluorescence to observe the changes of TDP-43 in MGFs under inflammatory microenvironment. TDP-43 in MGFs was knocked down by small interfering RNA (siRNA) transfection technique, and transfection efficiency was measured. Three experimental groups were set up: Negative control group (NC group), LPS induced control group (NL group) and TDP-43 knockdown +LPS induced group (SL group). RT-qPCR was used to detect the mRNA expression of some inflammatory factors, matrix metalloproteinases (MMPs) and adhesion related factors. Western blotting was used to detect the expression of some inflammatory factors and adhesion related factors. Picro sirius red staining was used to investigate intracellular collagen deposition. Results: The optimal concentration of LPS stimulation was 100 ng/mL for 6 h. In the absence of LPS stimulation, TDP-43 in MGFs was basically in the nucleus, and it appears in the nucleus and the cytoplasm after LPS stimulation. The cell model with TDP-43 knockdown was successfully constructed by siRNA transfection with MGFs, and the transfection efficiency was nearly 50%. Compared with the control group (NL group), the mRNA expressions of inflammatory factors, MMPs and adhesion-related factors were significantly down-regulated in the TDP-43 down-knocked experimental group (SL group) under LPS stimulation (P<0.05). The protein expressions of some adhesion related factors and inflammatory factors were also significantly down-regulated. Collagen deposition in SL group and NL group was decreased compared with NC group (P<0.05), but there was no striking difference between the NL group and SL group (P>0.05). Conclusion: When MGFs were in the inflammatory microenvironment induced by LPS, TDP-43 showed a tendency to move from inside the nucleus to outside. TDP-43 positively regulates the expression of inflammatory factors, adhesion related factors and MMPs in the LPS-induced inflammatory microenvironment of MGFs, but has no significant effect on collagen deposition.

Key words: TDP-43, lipopolysaccharides, periodontitis, mouse gingival fibroblasts cells

中图分类号: 

• R782