TRAF6基因沉默对LPS刺激下人牙周膜细胞增殖和成骨分化的影响

发布时间：2018-04-01 21:20

  本文选题：肿瘤坏死因子受体相关因子6　切入点：RNA干扰　出处：《山西医科大学》2017年硕士论文

【摘要】：目的:使用小干扰RNA(siRNA)沉默人牙周膜细胞(HPDLCs)中肿瘤坏死因子受体相关因子6(TRAF6)的基因;观察在LPS刺激下,TRAF6基因沉默对HPDLCs增殖能力和成骨分化能力的影响。方法:(1)酶消化联合组织块法体外培养HPDLCs,并进行免疫细胞化学鉴定。(2)实验分组:TRAF6 siRNA组、Control SiRNA组、转染试剂组、空白对照组,脂质体法将TRAF6 siRNA、control siRNA分别转染入对应组HPDLCs中,转染试剂组仅加入等量的转染试剂,空白对照组不做任何处理,再使用10μg/ml LPS刺激HPDLCs,RTPCR、Western blot检测细胞中TRAF6的mRNA及蛋白的表达情况。(3)CCK-8法检测TRAF6 siRNA组、Control SiRNA组、转染试剂组在转染后使用含或不含10μg/ml LPS的培养基培养24h、48h时细胞增殖情况。(4)使用10μg/ml LPS+成骨分化诱导培养基培养转染细胞3d,碱性磷酸酶检测试剂盒检测ALP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:(1)免疫细胞化学实验结果显示HPDLCs培养成功;(2)TRAF6 siRNA组的TRAF6 mRNA和蛋白的表达水平与其余三组相比显著下降,差异有统计学意义(p0.001),表明TRAF6基因沉默的细胞模型构建成功;(3)CCK-8法检测结果显示,10μg/ml LPS刺激各组细胞24、48h时,LPS刺激下Control SiRNA组、转染试剂组的A值要高于对照组,差异有统计学意义(p0.001),表明LPS刺激能促进HPDLCs增殖;LPS刺激24h时TRAF6 siRNA组在LPS刺激下A值与未受LPS刺激组无差异,LPS刺激48h时TRAF6siRNA组的A值在LPS刺激组低于未受LPS刺激组,表明TRAF6基因沉默能抑制LPS引起的HPDLCs增殖;(4)10μg/ml LPS+成骨分化诱导培养基培养细胞3d后,TRAF6 siRNA组的ALP表达量要高于空白对照、转染试剂组和control siRNA组,差异有统计学意义(p0.05);TRAF6 siRNA组的Runx-2和Col-I的mRNA表达均明显高于其余三组,差异有统计学意义(p0.05),表明TRAF6基因沉默能提高LPS刺激下HPDLCs的成骨能力。结论:TRAF6基因沉默能抑制LPS刺激下HPDLCs的增殖,促进LPS刺激下HPDLCs的成骨分化,推测TRAF6可能是牙周病潜在的治疗靶点。
[Abstract]:Objective: to silencing the gene of tumor necrosis factor receptor related factor 6 (TRAF6) in human periodontal ligament cells (HPDLCs) by small interference RNA-siRNAs (siRNAs). To observe the effect of LPS stimulated silencing on the proliferation and osteogenic differentiation of HPDLCs. Methods HPDLCs were cultured in vitro by the enzyme digestion and tissue block method, and were identified by immunocytochemistry. The cells were divided into two groups: control SiRNA group and transfection reagent group. In the blank control group, the TRAF6 siRNA-control siRNA was transfected into the HPDLCs of the corresponding group by liposome method. The transfection reagent group only added the same amount of transfection reagent, while the blank control group did not do any treatment. Then 10 渭 g/ml LPS was used to stimulate the expression of mRNA and protein of TRAF6 in TRAF6 siRNA group. The expression of TRAF6 mRNA and protein in TRAF6 siRNA group was detected by CCK-8 method. In the transfection reagent group, the cells were cultured in the culture medium containing or without 10 渭 g/ml LPS for 24 h or 48 h after transfection. The cells were transfected with 10 渭 g/ml LPS osteogenic induction medium for 3 days. The activity of ALP was detected by alkaline phosphatase assay kit and RT-PCR. The expression of Runx-2 and Collagen I Col-I gene was measured. Results: the results of immunocytochemistry showed that the expression of TRAF6 mRNA and protein in the HPDLCs culture group was significantly lower than that in the other three groups, and the expression of TRAF6 mRNA and protein in the siRNA group was significantly lower than that in the other three groups. The difference was statistically significant (P 0.001), which indicated that the cell model of TRAF6 gene silencing was successfully constructed by CCK-8 method. The results of CCK-8 method showed that the A value of the transfected reagent group was higher than that of the control group after stimulation of 10 渭 g/ml LPS for 24 h for 48 h. The difference was statistically significant (p 0.001), which indicated that LPS stimulation could promote the proliferation of TRAF6 siRNA for 24 h. There was no difference in A value of TRAF6 siRNA group under LPS stimulation and that in TRAF6siRNA group at 48 h after stimulation by LPS. The A value of TRAF6siRNA group was lower than that of non-#en8# stimulated group at 48 h after LPS stimulation. The results showed that TRAF6 gene silencing could inhibit the HPDLCs proliferation induced by LPS (10 渭 g/ml LPS). After 3 days, the expression of ALP in the TRAF6 siRNA group was higher than that in the blank control group. The expression of ALP in the transfection reagent group and control siRNA group was higher than that in the control group. The expression of Runx-2 and Col-I in TRAF6 siRNA group was significantly higher than that in the other three groups. The difference was statistically significant (p 0.05), which indicated that TRAF6 gene silencing could improve the osteogenic ability of HPDLCs stimulated by LPS. Conclusion the silencing of TRAF6 gene can inhibit the proliferation of HPDLCs stimulated by LPS and promote the osteogenic differentiation of HPDLCs stimulated by LPS. It is speculated that TRAF6 may be a potential therapeutic target for periodontal disease.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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