利拉鲁肽对晚期糖基化终末产物诱导的人牙周膜细胞的影响
发布时间：2018-04-26 13:23
本文选题：晚期糖基化终末产物 + 利拉鲁肽 ； 参考：《兰州大学》2017年硕士论文
【摘要】：目的:本实验通过检测利拉鲁肽(Liraglutide,Lira)对晚期糖基化终末产物(Advanced glycosylation end products,AGEs)诱导的人牙周膜细胞(periodontal ligament cells,PDLCs)增殖、凋亡、受体表达、成骨分化及炎症因子表达的作用,探讨h PDLCs在AGEs积累时,Lira对h PDLCs的保护及修复作用。方法:采用酶消化法联合组织块培养法分离培养原代h PDLCs,取3-6代细胞用于后续实验,采用细胞化学染色及成骨诱导21天茜素红染色法进行h PDLCs鉴定。采用MTT法筛选Lira对AGEs诱导的h PDLCs的最佳作用浓度及作用时间。采用MTT法检测Lira对AGEs诱导的h PDLCs增殖活性的影响。采用Hoechst33258荧光染色法及Annexin-V-FITC/PI双染检测凋亡水平。采用Realtime PCR及Western Blot检测晚期糖基化终末产物受体RAGE及胰高血糖素样肽受体GLP-1R的表达水平、成骨因子Runx2和ALP的表达水平、炎症因子TNF-α和IL-6的表达水平。采用茜素红染色法检测矿化结节形成水平。结果:1.细胞化学染色:HE染色细胞呈纤维细胞样长梭形外观;抗波形丝蛋白染色阳性,抗角蛋白染色阴性,证明细胞来源为中胚层;茜素红染色见矿化结节形成证明细胞具有成骨分化能力。综上可鉴定细胞为人牙周膜细胞;2.MTT结果:AGEs对h PDLCs细胞增殖抑制作用的最佳作用浓度是100μg/m L,最佳作用时间是48h;Lira对AGEs诱导的h PDLCs的最佳作用浓度是100 nmol/m L,最佳作用时间是24h;与对照组相比,AGEs组的增殖抑制率显著增高(P㩳0.01),Lira组的增殖抑制率显著降低(P㩳0.01),AGEs+Lira组的增殖抑制率显著高于Lira组(P㩳0.01),但低于对照组。3.Western Blot及Real-time PCR结果:(1)受体表达:h PDLCs上RAGE蛋白及m RNA及GLP-1R蛋白及m RNA的表达。相比对照组,AGEs组显著促进RAGE的表达(P㩳0.01),抑制GLP-1R的表达(P㩳0.05);Lira组抑制RAGE的表达(P㩳0.05),促进GLP-1R的表达(P㩳0.05),与AGEs组相比,AGEs+Lira组抑制RAGE的表达(P㩳0.05),促进了GLP-1R的表达(P㩳0.05);(2)成骨因子:相比对照组,AGEs组抑制Runx2和ALP蛋白及m RNA的表达(P㩳0.01,P㩳0.05),Lira组促进Runx2和ALP的表达(P㩳0.05,P㩳0.01),与AGEs组相比,AGEs+Lira组促进Runx2和ALP的表达(P㩳0.05,P㩳0.01);(3)炎症因子:相比对照组,AGEs组促进IL-6及TNF-α蛋白及m RNA的表达(P㩳0.01),Lira组抑制IL-6及TNF-α的表达(P㩳0.05,P㩳0.01),与AGEs组相比,AGEs+Lira组抑制IL-6及TNF-α的表达(P㩳0.01);5.Hoechst33258染色Annexin-V-FITC/PI细胞流式术结果:对照组和Lira组的细胞核均未发生改变;AGEs组细胞核发生皱缩,染色质浓缩或断裂;AGEs+Lira组细胞发生核皱缩,染色质浓缩或断裂的细胞减少。相比对照组,AGEs组的凋亡率显著增高(P㩳0.01);Lira组降低(P㩳0.01);相比AGEs组,AGEs+Lira组的凋亡率显著降低(P㩳0.01),但高于对照组;7.茜素红染色结果:AGEs组的紫红色矿化结节明显少于对照组,AGEs+Lira组的紫红色矿化结节明显多于AGEs组。经Image J软件对染色进行分析,Lira组与对照组,对照组与AGEs组,AGEs+Lira组与AGEs组之间均表现出明显差异(P㩳0.05,P㩳0.01,P㩳0.01)。结论:1.Lira对AGEs诱导的h PDLCs的最佳作用浓度是100 nmol/m L,最佳作用时间是24小时。2.h PDLCs上具有RAGE及GLP-1R受体的表达。Lira可促进正常h PDLCs及AGEs诱导的h PDLCs上GLP-1R的表达,抑制RAGE的表达;3.Lira可促进正常h PDLCs和AGEs诱导的h PDLCs增殖,抑制其凋亡,促进成骨分化,抑制炎症因子的表达。为糖尿病牙周病患者选择Lira保护及修复牙周组织提供实验依据。
[Abstract]:Objective: To investigate the proliferation, apoptosis, receptor expression, osteogenesis and expression of inflammatory factors in human periodontal ligament cells (periodontal ligament cells, PDLCs) induced by advanced glycation end products (Advanced glycosylation end products, AGEs) by Leila Lou (Liraglutide, Lira). Methods: the protection and repair effect of PDLCs. Methods: the original h PDLCs was isolated and cultured by enzyme digestion method combined with tissue mass culture. 3-6 generation of cells were used for follow-up experiments. Cytochemical staining and osteogenesis induced 21 days alizarin red staining for H PDLCs identification. The optimum concentration and effect of Lira on AGEs induced H PDLCs were selected by MTT method. Time. The effect of Lira on the proliferation of H PDLCs induced by AGEs was detected by MTT. Hoechst33258 fluorescence staining and Annexin-V-FITC/PI double staining were used to detect the apoptosis level. The expression level of receptor RAGE and glucagon like peptide in advanced glycosylated end products was detected by Realtime PCR and Western Blot. And expression level of ALP, expression level of inflammatory factor TNF- alpha and IL-6. Using alizarin red staining method to detect the formation level of mineralized nodules. Results: 1. cell chemical staining: HE stained cells showed fibrous cell like long spindle shape; anti wave silk protein staining positive, anti keratin staining negative, proved cell source was mesoderm; alizarin red staining found The formation of mineralized nodules proved that the cells had osteogenic differentiation ability. In addition, the identification cells were human periodontal cells; 2.MTT results: the optimum concentration of AGEs to h PDLCs cell proliferation inhibition was 100 mu g/m L, the best time was 48h, and the optimum concentration of Lira for AGEs induced H PDLCs was 100 nmol/m. Compared with the control group, the proliferation inhibition rate of the AGEs group was significantly increased (P 0.01), the proliferation inhibition rate in the Lira group was significantly lower (P? 0.01), and the proliferation inhibition rate in AGEs+Lira group was significantly higher than that in the Lira group (P? 0.01), but it was lower than the control group.3.Western Blot and Real-time PCR results: (1) receptor expression. Compared with the control group, AGEs group significantly promoted the expression of RAGE (P? 0.01), inhibited the expression of GLP-1R (P? 0.05), the Lira group inhibited the expression of RAGE (P? 0.05), and promoted the expression of GLP-1R (P? 0.05). The expression of ALP and m RNA (P? 0.01, P? 0.05), Lira group promoting the expression of Runx2 and ALP (P? 0.05, P? 0.01). 01), compared with the AGEs group, the AGEs+Lira group inhibited the expression of IL-6 and TNF- alpha (P? 0.01); 5.Hoechst33258 stained Annexin-V-FITC/PI cell flow cytometry results: the nuclei of the control group and the Lira group were not changed; the nucleus of the AGEs group crinkled, chromatin concentration or fracture; the AGEs+Lira group had nuclear crinkle, chromatin concentration or fracture Compared with the control group, the apoptosis rate in the AGEs group was significantly higher (P? 0.01) and the group Lira decreased (P? 0.01); compared with the AGEs group, the apoptosis rate of the AGEs+Lira group was significantly lower (P? 0.01), but higher than the control group; 7. alizarin red staining results: the purple red mineralized nodules in the AGEs group were significantly less than those in the control group, and the purple red mineralized nodules in the AGEs+Lira group were significantly more than those in the AGEs group. The staining was analyzed by Image J software, the Lira group and the control group, the control group and the AGEs group, the AGEs+Lira group and the AGEs group showed significant difference (P? 0.05, P? 0.01, P? 0.01). Conclusion: the best concentration of 1.Lira to AGEs induced H is 100. The best time is 24 hours. .Lira can promote the expression of GLP-1R on normal h PDLCs and AGEs induced H PDLCs and inhibit the expression of RAGE. 3.Lira can promote normal h PDLCs and AGEs induced proliferation, inhibit its apoptosis, promote osteogenic differentiation and inhibit the expression of inflammatory factors. It provides experimental basis for the selection of periodontitis patients to protect and repair periodontal tissues of diabetic 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】：R587.2;R781.4
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