發(fā)布時(shí)間：2018-10-30 20:08

【摘要】：目的:使用小干擾RNA(small interfering RNA,siRNA)沉默人牙周膜成纖維細(xì)胞(human periodontal ligament cell,hPDLC)腫瘤壞死因子受體相關(guān)因子6(tumor necrosis factor receptor associated factor 6,TRAF6)的基因,觀察沉默TRAF6基因?qū)PS刺激的hPDLC成骨分化的影響。方法:實(shí)驗(yàn)分為空白對(duì)照組、轉(zhuǎn)染試劑組、TRAF6siRNA組、control siRNA組,采用脂質(zhì)體法將TRAF6siRNA、control siRNA分別瞬時(shí)轉(zhuǎn)染入對(duì)應(yīng)組中,設(shè)轉(zhuǎn)染試劑組加入等量的轉(zhuǎn)染試劑,空白對(duì)照組不做處理,再使用10 mg/L LPS刺激細(xì)胞,RT-PCR、Western blot檢測(cè)轉(zhuǎn)染后TRAF6的基因及蛋白表達(dá)水平,使用LPS+成骨誘導(dǎo)液培養(yǎng)細(xì)胞,堿性磷酸酶檢測(cè)試劑盒檢測(cè)堿性磷酸酶的活性,RT-PCR檢測(cè)Runx-2和I型膠原蛋白(Col-I)基因表達(dá)情況。結(jié)果:TRAF6siRNA組的TRAF6mRNA和蛋白的表達(dá)顯著降低(P0.001);成骨誘導(dǎo)3d后,LPS刺激下各組ALP的表達(dá)量要顯著低于對(duì)照組(P0.05),TRAF6siRNA組的ALP表達(dá)量要高于空白對(duì)照、轉(zhuǎn)染試劑組和control siRNA組(P0.05),TRAF6siRNA的Runx-2和Col-I的mRNA表達(dá)均明顯高于其余3組(P0.05)。結(jié)論:沉默TRAF6基因能減輕LPS對(duì)hPDLC成骨分化的抑制作用,即沉默TRAF6基因能促進(jìn)LPS刺激下的hPDLC成骨分化,推測(cè)TRAF6可能影響牙周炎的發(fā)生發(fā)展進(jìn)程,是牙周炎潛在的治療靶點(diǎn)。
[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.
【作者單位】： 山西醫(yī)科大學(xué)口腔醫(yī)學(xué)系;
【基金】：山西省科技攻關(guān)項(xiàng)目(編號(hào):20150313010-3) 山西醫(yī)科大學(xué)�？萍紕�(chuàng)新基金項(xiàng)目(編號(hào):02101313) 山西醫(yī)科大學(xué)博士啟動(dòng)基金項(xiàng)目(編號(hào):03201321)
【分類號(hào)】：R781.42

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