本文選題：牙周炎　切入點：人牙周膜細胞　出處：《山西醫(yī)科大學》2017年碩士論文

【摘要】：第一部分實驗目的:觀察NF-κB非經典通路IKKα介導的Maspin在HPDLCs中的表達。方法:組織塊法原代培養(yǎng)HPDLCs,倒置顯微鏡及SP免疫組化鑒定細胞來源。CCK-8檢測LPS對HPDLCs增殖的影響。免疫細胞化學定位Maspin在HPDLCs中的表達位置。實驗分空白對照組(NC)、LPS1-4組(LPS終濃度分別為0.1,1,10,50 mg/L),RT-PCR及Western Blot分別檢測不同濃度LPS刺激下HPDLCs中Maspin、IKKαmRNA及蛋白的表達,ELISA檢測炎性因子TNF-α和IL-1β的分泌。結果:免疫細胞化學顯示Maspin在正常與LPS刺激的HPDLCs中均呈胞漿表達。Western Blot結果顯示,隨著LPS濃度增加Maspin蛋白表達逐漸下調,呈濃度依賴性,LPS1-4組與NC組相比差異具有統(tǒng)計學意義(P0.05);IKKα蛋白表達逐漸上調,呈濃度依賴性,LPS1-4組與NC組相比差異具有統(tǒng)計學意義(P0.05);且二者相關性分析可知,Maspin蛋白與IKKα蛋白表達呈負相關(r=-0.959,P0.05)。mRNA表達與蛋白表達趨勢相同。ELISA結果顯示,NC組中TNF-α、IL-1β分泌量較低,加入LPS刺激后,TNF-α、IL-1β隨著LPS濃度增加分泌增加,與Maspin蛋白表達呈負相關。結論:Maspin在人牙周膜細胞中呈胞漿表達,且LPS刺激下人牙周膜細胞中NF-κB非經典通路IKKα介導的Maspin表達下調,并與TNF-α、IL-1β呈負相關。第二部分實驗目的:觀察黃芩苷對HPDLCs中IKKα介導的Maspin表達的影響。方法:CCK-8檢測黃芩苷對HPDLCs增殖的影響。實驗分空白對照組(NC組:黃芩苷終濃度為0 mg/L,LPS終濃度為0 mg/L)、LPS刺激組(黃芩苷終濃度為0 mg/L,LPS終濃度為10 mg/L)、黃芩苷+LPS 1-4組(黃芩苷終濃度依次為0.01,0.1,1,10 mg/L,LPS終濃度為10 mg/L),RT-PCR及Western Blot分別檢測藥物作用下Maspin、IKKαmRNA及蛋白的表達,ELISA檢測炎性因子TNF-α和IL-1β的分泌。結果:LPS組與NC組相比Maspin蛋白表達下調,IKKα蛋白表達上調(P0.01)。黃芩苷+LPS1-4組與LPS組相比,隨著黃芩苷濃度的增加,Maspin蛋白表達逐漸上調,呈濃度依賴性,且黃芩苷+LPS1-4組與LPS組相比差異具有統(tǒng)計學意義(P0.05);IKKα蛋白表達逐漸下調,呈濃度依賴性,且黃芩苷+LPS1-4組與LPS組相比差異具有統(tǒng)計學意義(P0.01);同時Maspin蛋白與IKKα蛋白表達呈負相關(r=-0.886,P0.05)。mRNA表達與蛋白表達趨勢相同。ELISA結果顯示,LPS組與NC組相比TNF-α、IL-1β分泌顯著增加,黃芩苷+LPS1-4組與LPS組相比,隨著黃芩苷濃度的增加TNF-α、IL-1β分泌逐漸減少,與Maspin蛋白表達呈負相關。結論:黃芩苷介導了人牙周膜細胞中NF-κB非經典通路IKKα調控的Maspin表達上調,同時抑制TNF-α、IL-1β分泌,對LPS刺激引起的細胞炎性損傷起保護作用。
[Abstract]:Part one: to investigate the expression of IKK 偽 -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 偽 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 偽 protein expression. The results of Elisa showed that the secretion of TNF- 偽 IL-1 尾 was lower in NC-treated group, and the expression of Maspin protein was negatively correlated with the expression of IKK 偽 protein.The secretion of TNF- 偽 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 偽 -mediated Maspin is down-regulated in human periodontal ligament cells, which is negatively correlated with the expression of TNF- 偽 and IL-1 尾 in human periodontal ligament cells.Part two: to observe the effect of baicalin on Maspin expression mediated by IKK 偽 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 偽 mRNA and protein respectively. The secretion of inflammatory factors TNF- 偽 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 偽 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 偽 protein expression. The results of Elisa showed that the expression of Maspin protein and IKK 偽 protein in Maspin group was significantly higher than that in NC group.Compared with LPS group, the secretion of TNF- 偽 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 偽 in human periodontal ligament cells, and inhibits the secretion of TNF- 偽 and IL-1 尾, which may protect the cells from inflammatory injury induced by LPS.
【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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