IKKα介导的Maspin在人牙周膜细胞中的表达及黄芩苷对其的调控作用

发布时间：2018-04-01 07:39

本文选题：牙周炎　切入点：人牙周膜细胞　出处：《山西医科大学》2017年硕士论文

【摘要】：第一部分实验目的:观察NF-κB非经典通路IKKα介导的Maspin在HPDLCs中的表达。方法:组织块法原代培养HPDLCs,倒置显微镜及SP免疫组化鉴定细胞来源。CCK-8检测LPS对HPDLCs增殖的影响。免疫细胞化学定位Maspin在HPDLCs中的表达位置。实验分空白对照组(NC)、LPS1-4组(LPS终浓度分别为0.1,1,10,50 mg/L),RT-PCR及Western Blot分别检测不同浓度LPS刺激下HPDLCs中Maspin、IKKαmRNA及蛋白的表达,ELISA检测炎性因子TNF-α和IL-1β的分泌。结果:免疫细胞化学显示Maspin在正常与LPS刺激的HPDLCs中均呈胞浆表达。Western Blot结果显示,随着LPS浓度增加Maspin蛋白表达逐渐下调,呈浓度依赖性,LPS1-4组与NC组相比差异具有统计学意义(P0.05);IKKα蛋白表达逐渐上调,呈浓度依赖性,LPS1-4组与NC组相比差异具有统计学意义(P0.05);且二者相关性分析可知,Maspin蛋白与IKKα蛋白表达呈负相关(r=-0.959,P0.05)。mRNA表达与蛋白表达趋势相同。ELISA结果显示,NC组中TNF-α、IL-1β分泌量较低,加入LPS刺激后,TNF-α、IL-1β随着LPS浓度增加分泌增加,与Maspin蛋白表达呈负相关。结论:Maspin在人牙周膜细胞中呈胞浆表达,且LPS刺激下人牙周膜细胞中NF-κB非经典通路IKKα介导的Maspin表达下调,并与TNF-α、IL-1β呈负相关。第二部分实验目的:观察黄芩苷对HPDLCs中IKKα介导的Maspin表达的影响。方法:CCK-8检测黄芩苷对HPDLCs增殖的影响。实验分空白对照组(NC组:黄芩苷终浓度为0 mg/L,LPS终浓度为0 mg/L)、LPS刺激组(黄芩苷终浓度为0 mg/L,LPS终浓度为10 mg/L)、黄芩苷+LPS 1-4组(黄芩苷终浓度依次为0.01,0.1,1,10 mg/L,LPS终浓度为10 mg/L),RT-PCR及Western Blot分别检测药物作用下Maspin、IKKαmRNA及蛋白的表达,ELISA检测炎性因子TNF-α和IL-1β的分泌。结果:LPS组与NC组相比Maspin蛋白表达下调,IKKα蛋白表达上调(P0.01)。黄芩苷+LPS1-4组与LPS组相比,随着黄芩苷浓度的增加,Maspin蛋白表达逐渐上调,呈浓度依赖性,且黄芩苷+LPS1-4组与LPS组相比差异具有统计学意义(P0.05);IKKα蛋白表达逐渐下调,呈浓度依赖性,且黄芩苷+LPS1-4组与LPS组相比差异具有统计学意义(P0.01);同时Maspin蛋白与IKKα蛋白表达呈负相关(r=-0.886,P0.05)。mRNA表达与蛋白表达趋势相同。ELISA结果显示,LPS组与NC组相比TNF-α、IL-1β分泌显著增加,黄芩苷+LPS1-4组与LPS组相比,随着黄芩苷浓度的增加TNF-α、IL-1β分泌逐渐减少,与Maspin蛋白表达呈负相关。结论:黄芩苷介导了人牙周膜细胞中NF-κB非经典通路IKKα调控的Maspin表达上调,同时抑制TNF-α、IL-1β分泌,对LPS刺激引起的细胞炎性损伤起保护作用。
[Abstract]:Part one: to investigate the expression of IKK 伪 -mediated Maspin in HPDLCs via NF- 魏 B nonclassical pathway.Methods: HPDLCs were cultured by tissue mass method. The effect of LPS on HPDLCs proliferation was detected by inverted microscope and SP immunohistochemistry.Immunocytochemistry was used to locate the expression of Maspin in HPDLCs.Results: immunocytochemistry showed that Maspin showed cytoplasmic expression in both normal and LPS stimulated HPDLCs. Western Blot showed that the expression of Maspin protein was down-regulated with the increase of LPS concentration.In a dose-dependent manner, the expression of Ike 伪 in LPS1-4 group was significantly higher than that in NC group.There was a significant difference between LPS1-4 group and NC group (P 0.05), and the correlation analysis showed that there was a negative correlation between Maspin protein and IKK 伪 protein expression. The results of Elisa showed that the secretion of TNF- 伪 IL-1 尾 was lower in NC-treated group, and the expression of Maspin protein was negatively correlated with the expression of IKK 伪 protein.The secretion of TNF- 伪 IL-1 尾 increased with the increase of LPS concentration, and negatively correlated with the expression of Maspin protein.Conclusion the expression of LPS in human periodontal ligament cells is cytoplasmic, and the expression of NF- 魏 B non-classical pathway IKK 伪 -mediated Maspin is down-regulated in human periodontal ligament cells, which is negatively correlated with the expression of TNF- 伪 and IL-1 尾 in human periodontal ligament cells.Part two: to observe the effect of baicalin on Maspin expression mediated by IKK 伪 in HPDLCs.Methods the effect of baicalin on proliferation of HPDLCs was detected by 10% CCK-8.The experiment was divided into blank control group (NC group): baicalin final concentration was 0 mg / L LPs was 0 mg / L LPS-stimulated group (baicalin final concentration was 0 mg / L LPS = 10 mg / L), baicalin LPS 1-4 group (baicalin final concentration was 0.011% 10 mg / L LPS-LPS final concentration was 10 mg 路L ~ (-1) / L ~ (-1)) reverse transcription-polymerase chain reaction (RT-PCR).And Western Blot were used to detect the expression of Maspin Ike 伪 mRNA and protein respectively. The secretion of inflammatory factors TNF- 伪 and IL-1 尾 was detected by Elisa.Results compared with NC group, the expression of Maspin protein was down-regulated and the expression of Ike 伪 protein was up-regulated in the control group compared with that in the control group (P 0.01).Compared with LPS group, baicalin LPS1-4 group increased the expression of baicalin protein in a dose-dependent manner with the increase of baicalin concentration, and the difference between baicalin LPS1-4 group and LPS group was statistically significant.There was a significant difference between baicalin LPS1-4 group and LPS group, and there was a negative correlation between Maspin protein and IKK 伪 protein expression. The results of Elisa showed that the expression of Maspin protein and IKK 伪 protein in Maspin group was significantly higher than that in NC group.Compared with LPS group, the secretion of TNF- 伪 and IL-1 尾 in baicalin LPS1-4 group decreased gradually with the increase of baicalin concentration, which was negatively correlated with the expression of Maspin protein.Conclusion: baicalin mediates the up-regulation of Maspin regulated by IKK 伪 in human periodontal ligament cells, and inhibits the secretion of TNF- 伪 and IL-1 尾, which may protect the cells from inflammatory injury induced by LPS.
【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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