本文選題：腫瘤壞死因子受體相關(guān)因子6　切入點：RNA干擾　出處：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:使用小干擾RNA(siRNA)沉默人牙周膜細胞(HPDLCs)中腫瘤壞死因子受體相關(guān)因子6(TRAF6)的基因;觀察在LPS刺激下,TRAF6基因沉默對HPDLCs增殖能力和成骨分化能力的影響。方法:(1)酶消化聯(lián)合組織塊法體外培養(yǎng)HPDLCs,并進行免疫細胞化學鑒定。(2)實驗分組:TRAF6 siRNA組、Control SiRNA組、轉(zhuǎn)染試劑組、空白對照組,脂質(zhì)體法將TRAF6 siRNA、control siRNA分別轉(zhuǎn)染入對應組HPDLCs中,轉(zhuǎn)染試劑組僅加入等量的轉(zhuǎn)染試劑,空白對照組不做任何處理,再使用10μg/ml LPS刺激HPDLCs,RTPCR、Western blot檢測細胞中TRAF6的mRNA及蛋白的表達情況。(3)CCK-8法檢測TRAF6 siRNA組、Control SiRNA組、轉(zhuǎn)染試劑組在轉(zhuǎn)染后使用含或不含10μg/ml LPS的培養(yǎng)基培養(yǎng)24h、48h時細胞增殖情況。(4)使用10μg/ml LPS+成骨分化誘導培養(yǎng)基培養(yǎng)轉(zhuǎn)染細胞3d,堿性磷酸酶檢測試劑盒檢測ALP的活性,RT-PCR檢測Runx-2和I型膠原蛋白(Col-I)基因表達情況。結(jié)果:(1)免疫細胞化學實驗結(jié)果顯示HPDLCs培養(yǎng)成功;(2)TRAF6 siRNA組的TRAF6 mRNA和蛋白的表達水平與其余三組相比顯著下降,差異有統(tǒng)計學意義(p0.001),表明TRAF6基因沉默的細胞模型構(gòu)建成功;(3)CCK-8法檢測結(jié)果顯示,10μg/ml LPS刺激各組細胞24、48h時,LPS刺激下Control SiRNA組、轉(zhuǎn)染試劑組的A值要高于對照組,差異有統(tǒng)計學意義(p0.001),表明LPS刺激能促進HPDLCs增殖;LPS刺激24h時TRAF6 siRNA組在LPS刺激下A值與未受LPS刺激組無差異,LPS刺激48h時TRAF6siRNA組的A值在LPS刺激組低于未受LPS刺激組,表明TRAF6基因沉默能抑制LPS引起的HPDLCs增殖;(4)10μg/ml LPS+成骨分化誘導培養(yǎng)基培養(yǎng)細胞3d后,TRAF6 siRNA組的ALP表達量要高于空白對照、轉(zhuǎn)染試劑組和control siRNA組,差異有統(tǒng)計學意義(p0.05);TRAF6 siRNA組的Runx-2和Col-I的mRNA表達均明顯高于其余三組,差異有統(tǒng)計學意義(p0.05),表明TRAF6基因沉默能提高LPS刺激下HPDLCs的成骨能力。結(jié)論:TRAF6基因沉默能抑制LPS刺激下HPDLCs的增殖,促進LPS刺激下HPDLCs的成骨分化,推測TRAF6可能是牙周病潛在的治療靶點。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4

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