TRAF6沉默对LPS刺激牙周膜成纤维细胞成骨分化的影响

发布时间：2018-10-30 20:08

【摘要】：目的:使用小干扰RNA(small interfering RNA,siRNA)沉默人牙周膜成纤维细胞(human periodontal ligament cell,hPDLC)肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor associated factor 6,TRAF6)的基因,观察沉默TRAF6基因对LPS刺激的hPDLC成骨分化的影响。方法:实验分为空白对照组、转染试剂组、TRAF6siRNA组、control siRNA组,采用脂质体法将TRAF6siRNA、control siRNA分别瞬时转染入对应组中,设转染试剂组加入等量的转染试剂,空白对照组不做处理,再使用10 mg/L LPS刺激细胞,RT-PCR、Western blot检测转染后TRAF6的基因及蛋白表达水平,使用LPS+成骨诱导液培养细胞,碱性磷酸酶检测试剂盒检测碱性磷酸酶的活性,RT-PCR检测Runx-2和I型胶原蛋白(Col-I)基因表达情况。结果:TRAF6siRNA组的TRAF6mRNA和蛋白的表达显著降低(P0.001);成骨诱导3d后,LPS刺激下各组ALP的表达量要显著低于对照组(P0.05),TRAF6siRNA组的ALP表达量要高于空白对照、转染试剂组和control siRNA组(P0.05),TRAF6siRNA的Runx-2和Col-I的mRNA表达均明显高于其余3组(P0.05)。结论:沉默TRAF6基因能减轻LPS对hPDLC成骨分化的抑制作用,即沉默TRAF6基因能促进LPS刺激下的hPDLC成骨分化,推测TRAF6可能影响牙周炎的发生发展进程,是牙周炎潜在的治疗靶点。
[Abstract]:Objective: to silencing the gene of tumor necrosis factor receptor related factor 6 (tumor necrosis factor receptor associated factor 6 (TRAF6) in human periodontal ligament fibroblasts (human periodontal ligament cell,hPDLC) by using small interfering RNA (small interfering RNA,siRNA. To observe the effect of silencing TRAF6 gene on the osteogenic differentiation of hPDLC stimulated by LPS. Methods: the experiment was divided into three groups: blank control group, transfection reagent group and, control siRNA group. The TRAF6siRNA,control siRNA was transiently transfected into the corresponding group by liposome method. The transfection reagent group was added with the same amount of transfection reagent, and the blank control group was not treated. Then the cells were stimulated with 10 mg/L LPS. The expression of TRAF6 gene and protein were detected by RT-PCR,Western blot. The cells were cultured with LPS osteoblast and the activity of alkaline phosphatase was detected by alkaline phosphatase assay kit. Runx-2 and type I collagen (Col-I) gene expression were detected by RT-PCR. Results: the expression of TRAF6mRNA and protein in TRAF6siRNA group was significantly decreased (P0. 001). After 3 days of osteogenesis induction, the expression of ALP in LPS group was significantly lower than that in control group (P0.05), ALP expression in TRAF6siRNA group was higher than that in blank control group, transfection reagent group and control siRNA group (P0.05). The expression of Runx-2 and Col-I in TRAF6siRNA was significantly higher than that in the other three groups (P0.05). Conclusion: silencing TRAF6 gene can reduce the inhibitory effect of LPS on hPDLC osteogenic differentiation, that is, silencing TRAF6 gene can promote hPDLC osteogenic differentiation stimulated by LPS. It is speculated that TRAF6 may affect the occurrence and development of periodontitis and may be a potential therapeutic target for periodontitis.
【作者单位】：山西医科大学口腔医学系;
【基金】：山西省科技攻关项目(编号:20150313010-3) 山西医科大学校科技创新基金项目(编号:02101313) 山西医科大学博士启动基金项目(编号:03201321)
【分类号】：R781.42

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