本文選題：LIRA + LPS　； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：目的本次研究探討利拉魯肽(Liraglutide,LIRA)對牙周炎潛在的治療作用。選用經(jīng)典革蘭陰性菌E.Coli的LPS和人牙周膜細(xì)胞(Human periodontal ligament cells,hPDLCs)共培養(yǎng)來模擬牙周炎的病理過程,體外分析LIRA對LPS作用下hPDLCs增殖、遷移、炎癥以及成骨分化潛能的影響。方法采用組織塊酶消化法原代培養(yǎng)hPDLCs,免疫組織化學(xué)染色法鑒定細(xì)胞來源;茜素紅染色分析原代培養(yǎng)獲得的細(xì)胞是否擁有骨向分化潛能;q RT-PCR鑒定hPDLCs是否表達(dá)胰高血糖素樣肽-1受體(Glucagon-like peptide,GLP-1 receptor,GLP-1R);MTT法篩選LIRA和E.Coli LPS最佳作用濃度和時間并分析LIRA對LPS刺激下hPDLCs增殖活性的影響;劃痕試驗分析LIRA對hPDLCs遷移能力的影響;qRT-PCR和Western Blot分析LIRA對LPS刺激下hPDLCs炎癥因子表達(dá)(IL-6和TNF-α)以及成骨因子表達(dá)(ALP和Runx2)的影響。結(jié)果1.組織塊酶消化法成功培養(yǎng)獲得hPDLCs,經(jīng)免疫組化染色鑒定:抗波形絲蛋白陽性、抗角形蛋白陰性,證明細(xì)胞來源于間充質(zhì),茜素紅染色證實了hPDLCs的成骨分化能力。2.qRT-PCR結(jié)果顯示hPDLCs上存在GLP-1R mRNA的表達(dá),并且LIRA可增加GLP-1R mRNA的表達(dá)。3.MTT結(jié)果:25,50,75,100,125 nM LIRA培養(yǎng)24h后,均可增強(qiáng)hPDLCs的增殖活性(P0.05),并呈劑量依賴性;認(rèn)為100 nM為LIRA的最佳藥物濃度。低濃度LPS(0.1,1,10μg/ml)可促進(jìn)hPDLCs的增殖活性,而高濃度LPS(100μg/ml)則抑制hPDLCs的增殖活性(P0.05),呈時間依賴性;因此,選擇10μg/ml LPS與hPDLCs共培養(yǎng)來模擬牙周炎的發(fā)病過程。再者,LIRA可減弱高濃度LPS對hPDLCs增殖活性的抑制作用(P0.05)。4.劃痕試驗結(jié)果:LIRA可促進(jìn)hPDLCs的遷移能力,呈時間依賴性。5.qRT-PCR結(jié)果:炎癥因子,與對照組相比,LPS組顯著增加IL-6和TNF-αmRNA的表達(dá)(P0.01),LIRA組則可抑制IL-6和TNF-αmRNA的表達(dá)(P0.05);而與LPS組相比,LPS+LIRA組顯著降低IL-6和TNF-αmRNA的表達(dá)(P0.01)。成骨因子,與對照組相比,LIRA組和LPS組均可增加ALP和Runx2mRNA的表達(dá)(P0.05);而與LPS組相比,LPS+LIRA組則抑制ALP和Runx2mRNA的表達(dá)(P0.05)。6.Western Blot結(jié)果:炎癥因子表達(dá)結(jié)果與qRT-PCR結(jié)果基本一致;成骨因子表達(dá)結(jié)果則與qRT-PCR結(jié)果略有差異:與對照組相比,LIRA明顯促進(jìn)ALP和Runx2蛋白的表達(dá)(P0.05),而LPS組則可抑制ALP和Runx2蛋白的表達(dá)(P0.05);而與LPS組相比,LPS+LIRA組則明顯增加ALP和Runx2蛋白的表達(dá)(P0.05)。結(jié)論1.hPDLCs上存在GLP-1R的表達(dá),LIRA可促進(jìn)GLP-1R的表達(dá),并促進(jìn)hPDLCs的增殖活性和遷移能力;2.LPS可誘導(dǎo)hPDLCs炎癥反應(yīng),而抑制hPDLCs成骨分化,即LPS與hPDLCs共培養(yǎng)可以模擬牙周炎的病理過程;3.LIRA不僅可以抑制hPDLCs的炎癥反應(yīng),促進(jìn)hPDLCs的骨向分化潛能,更能抑制LPS誘導(dǎo)的炎癥反應(yīng),恢復(fù)LPS對hPDLCs骨向分化潛能的損害。這就表明LIRA對牙周炎有潛在的治療作用,為LIRA作為牙周炎的輔助用藥提供了一定的理論依據(jù)。
[Abstract]:Objective to investigate the potential therapeutic effect of liraglutide lira on periodontitis. The LPS of classical gram-negative bacteria E.Coli and human periodontal ligament cells were co-cultured to simulate the pathological process of periodontitis. The effects of LIRA on hPDLCs proliferation, migration, inflammation and osteogenic differentiation potential of LPS were analyzed in vitro. Methods the primary culture of hPDLCswas carried out by tissue mass enzyme digestion, and the origin of the cells was identified by immunohistochemical staining. Alizarin red staining analysis of primary cultured cells with bone differentiation potential Q RT-PCR identification of hPDLCs expression of glucagon-like peptide-1 receptor Glucagon-like GLP-1 receptor GLP-1R MTT assay screening LIRA and E.Coli LPS the best concentration and time of action and analysis of LIRA on LPS Effects of stimulation on proliferation of hPDLCs; The effects of LIRA on the migration of hPDLCs were analyzed by scratch test and Western Blot. The effects of LIRA on the expression of IL-6 and TNF- 偽 in hPDLCs induced by LPS and the expression of osteogenic factors in hPDLCs were analyzed by QRT-PCR and Western Blot. Result 1. HPDLCswere successfully cultured by tissue mass enzyme digestion. The results of immunohistochemical staining showed that the cells were positive for vimentin and negative for anti-keratin, which proved that the cells originated from mesenchymal cells. Alizarin red staining confirmed the osteogenic differentiation ability of hPDLCs. 2. QRT-PCR results showed that there was GLP-1R mRNA expression on hPDLCs, and LIRA could increase the expression of GLP-1R mRNA. 3. The results showed that LIRA could enhance the proliferation activity of hPDLCs in a dose-dependent manner after 24 hours of incubation with 1: 2550NM 75100125nM LIRA. It is considered that 100nM is the best concentration of LIRA. The low concentration of LPSG 0.1 渭 g / ml promoted the proliferation of hPDLCs, while the high concentration of LPS(100 渭 g / ml inhibited the proliferation of hPDLCs in a time-dependent manner. Therefore, 10 渭 g/ml LPS co-cultured with hPDLCs was selected to simulate the pathogenesis of periodontitis. Furthermore, LIRA could attenuate the inhibitory effect of high concentration of LPS on the proliferation of hPDLCs. The results of scratch test showed that: LIRA could promote the migration ability of hPDLCs in a time dependent manner. Compared with the control group, the expression of IL-6 and TNF- 偽 mRNA was significantly increased in lipopolysaccharide group (P 0.01) and the expression of IL-6 and TNF- 偽 mRNA was inhibited in LIRA group (P 0.05), while the expression of IL-6 and TNF- 偽 mRNA was significantly decreased in lipopolysaccharide group (P 0.01) compared with LPS group. Compared with the control group, the expression of ALP and Runx2mRNA was increased in LIRA group and LPS group, while the expression of ALP and Runx2mRNA was inhibited in LPS group. 6. Western Blot results showed that the expression of inflammatory factor was consistent with that of qRT-PCR. The expression of osteogenic factor was slightly different from that of qRT-PCR: compared with the control group, LIRA could significantly promote the expression of ALP and Runx2 protein, while the LPS group could inhibit the expression of ALP and Runx2 protein, while the expression of ALP and Runx2 protein in LPS group was significantly higher than that in LPS group, and the expression of ALP and Runx2 protein was significantly increased in LIRA group compared with the control group. Conclusion the expression of GLP-1R on 1.hPDLCs can promote the expression of GLP-1R, promote the proliferation and migration of hPDLCs. 2. LPS-induced inflammation of hPDLCs and inhibit the osteogenic differentiation of hPDLCs. That is to say, co-culture of LPS and hPDLCs can mimic the pathological process of periodontitis. Lira can not only inhibit the inflammatory response of hPDLCs, promote the bone differentiation potential of hPDLCs, but also inhibit the inflammatory response induced by LPS, and recover the damage of LPS to hPDLCs bone differentiation potential. This suggests that LIRA has a potential therapeutic effect on periodontitis and provides a theoretical basis for LIRA as an adjuvant drug for periodontitis.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.4

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本文編號：1936449