脂多糖诱导牙髓细胞DNA双链断裂的研究

Lipopolysaccharide Stimulation Induces DNA Double-strand Breaks in Dental Pulp Cells

目的:探讨低剂量条件下脂多糖诱导牙髓细胞DNA损伤方法,为研究脂多糖诱导牙髓细胞DNA双链断裂及DNA修复提供实验模型。方法:10μg/L脂多糖连续刺激牙髓细胞1、3、6次后,采取MTT及TUNEL分别检测其对牙髓细胞增殖及凋亡的作用;采用q-PCR及免疫印迹检测γ-H2A.X的mRNA及蛋白水平的表达;使用免疫荧光及免疫组化法观测γ-H2A.X在体内、外牙髓细胞的表达。结果:与对照组相比,10μg/L脂多糖连续刺激对牙髓细胞增殖及凋亡均无显著性差异;脂多糖连续刺激6次后细胞免疫荧光显示在细胞核检测到γ-H2A.X的阳性表达,且蛋白及mRNA水平的表达较对照组有显著性增加(P<0.05);免疫组化结果表明在脂多糖诱导4、6、8d后,γ-H2A.X在大鼠牙髓组织较对照组表达水平显著升高(P<0.05)。结论:低剂量脂多糖连续刺激可诱导牙髓细胞产生DNA双链断裂。

Objective：To investigate whether low dose of lipopolysaccharide（LPS）induces DNA damage in dental pulp cells,and to provide an experimental model for studying the DNA double-strand breaks and DNA repair.Methods：Tenμg/L LPS was added to dental pulp cells constantly once,three times,and six times,respectively.Cell proliferation was detected by MTT assay.Cell apoptosis was detected by TUNEL assay.The mRNA and protein expression levels ofγ-H2 A.X were investigated by q-PCR and Western blot.The expressions ofγ-H2 A.X in vivo or vitro were detected by immunofluorescence and immunohistochemistry.Results：Compared with the control group,the repeated stimulation of 10μg/L LPS had no significantinfluence on cell proliferation or on cell apoptosis.After repeated stimulation of 10μg/L LPS for six times,immunofluorescence revealed the positive expression ofγ-H2 A.X in cell nucleus.Moreover,compared with control groups,the mRNA and protein expression levels ofγ-H2 A.X increased significantly（P0.05）.Immunohistochemistry showedγ-H2 A.X expression level increased remarkably after being induced by LPS for 4,6and 8days,respectively（P0.05）.Conclusion：The repeated stimulation of low dose of LPS may induce the occurrence of DNA double-strand breaks in dental pulp cells.