利拉鲁肽对人牙周膜细胞增殖、迁移、炎症以及成骨分化潜能的影响

发布时间：2018-05-26 08:07

  本文选题：LIRA + LPS　； 参考：《兰州大学》2017年硕士论文

【摘要】：目的本次研究探讨利拉鲁肽(Liraglutide,LIRA)对牙周炎潜在的治疗作用。选用经典革兰阴性菌E.Coli的LPS和人牙周膜细胞(Human periodontal ligament cells,hPDLCs)共培养来模拟牙周炎的病理过程,体外分析LIRA对LPS作用下hPDLCs增殖、迁移、炎症以及成骨分化潜能的影响。方法采用组织块酶消化法原代培养hPDLCs,免疫组织化学染色法鉴定细胞来源;茜素红染色分析原代培养获得的细胞是否拥有骨向分化潜能;q RT-PCR鉴定hPDLCs是否表达胰高血糖素样肽-1受体(Glucagon-like peptide,GLP-1 receptor,GLP-1R);MTT法筛选LIRA和E.Coli LPS最佳作用浓度和时间并分析LIRA对LPS刺激下hPDLCs增殖活性的影响;划痕试验分析LIRA对hPDLCs迁移能力的影响;qRT-PCR和Western Blot分析LIRA对LPS刺激下hPDLCs炎症因子表达(IL-6和TNF-α)以及成骨因子表达(ALP和Runx2)的影响。结果1.组织块酶消化法成功培养获得hPDLCs,经免疫组化染色鉴定:抗波形丝蛋白阳性、抗角形蛋白阴性,证明细胞来源于间充质,茜素红染色证实了hPDLCs的成骨分化能力。2.qRT-PCR结果显示hPDLCs上存在GLP-1R mRNA的表达,并且LIRA可增加GLP-1R mRNA的表达。3.MTT结果:25,50,75,100,125 nM LIRA培养24h后,均可增强hPDLCs的增殖活性(P0.05),并呈剂量依赖性;认为100 nM为LIRA的最佳药物浓度。低浓度LPS(0.1,1,10μg/ml)可促进hPDLCs的增殖活性,而高浓度LPS(100μg/ml)则抑制hPDLCs的增殖活性(P0.05),呈时间依赖性;因此,选择10μg/ml LPS与hPDLCs共培养来模拟牙周炎的发病过程。再者,LIRA可减弱高浓度LPS对hPDLCs增殖活性的抑制作用(P0.05)。4.划痕试验结果:LIRA可促进hPDLCs的迁移能力,呈时间依赖性。5.qRT-PCR结果:炎症因子,与对照组相比,LPS组显著增加IL-6和TNF-αmRNA的表达(P0.01),LIRA组则可抑制IL-6和TNF-αmRNA的表达(P0.05);而与LPS组相比,LPS+LIRA组显著降低IL-6和TNF-αmRNA的表达(P0.01)。成骨因子,与对照组相比,LIRA组和LPS组均可增加ALP和Runx2mRNA的表达(P0.05);而与LPS组相比,LPS+LIRA组则抑制ALP和Runx2mRNA的表达(P0.05)。6.Western Blot结果:炎症因子表达结果与qRT-PCR结果基本一致;成骨因子表达结果则与qRT-PCR结果略有差异:与对照组相比,LIRA明显促进ALP和Runx2蛋白的表达(P0.05),而LPS组则可抑制ALP和Runx2蛋白的表达(P0.05);而与LPS组相比,LPS+LIRA组则明显增加ALP和Runx2蛋白的表达(P0.05)。结论1.hPDLCs上存在GLP-1R的表达,LIRA可促进GLP-1R的表达,并促进hPDLCs的增殖活性和迁移能力;2.LPS可诱导hPDLCs炎症反应,而抑制hPDLCs成骨分化,即LPS与hPDLCs共培养可以模拟牙周炎的病理过程;3.LIRA不仅可以抑制hPDLCs的炎症反应,促进hPDLCs的骨向分化潜能,更能抑制LPS诱导的炎症反应,恢复LPS对hPDLCs骨向分化潜能的损害。这就表明LIRA对牙周炎有潜在的治疗作用,为LIRA作为牙周炎的辅助用药提供了一定的理论依据。
[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- 伪 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- 伪 mRNA was significantly increased in lipopolysaccharide group (P 0.01) and the expression of IL-6 and TNF- 伪 mRNA was inhibited in LIRA group (P 0.05), while the expression of IL-6 and TNF- 伪 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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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