地塞米松对人牙龈上皮细胞表达β-防御素的影响及其信号通路研究

发布时间：2018-06-16 01:50

本文选题：地塞米松 + 人牙龈上皮细胞　；参考：《广西医科大学》2017年硕士论文

【摘要】：目的:通过检测地塞米松(Dex)对TNF-α诱导的人牙龈上皮细胞(HGECs)表达β-防御素-1,2(hBD-1、h BD-2)和炎症因子IL-1β、IL-6的影响及核转录因子-κB(nuclear factor-κB,NF-κB)和丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)等相关信号通路蛋白的活化情况,探讨地塞米松对口腔上皮免疫的影响及作用机制。方法:1.将临床取材的正常人牙龈组织采用酶消化培养法获取原代牙龈上皮细胞。选用10ng/ml TNF-α诱导第三代牙龈上皮细胞24h。另一组选用p38MAPK抑制剂或NF-κB抑制剂分别孵育细胞2h后,再用10ng/ml TNF-α诱导细胞24h。提取细胞总RNA检测hBD-2 mRNA的表达量。2.不同浓度Dex(0.1,1,10μM/L)分别培养第三代HGEC 2h后再加入10ng/ml TNF-α诱导细胞24h。取贴壁细胞用于提取总RNA进行实时荧光定量PCR(Real Time-PCR)检测hBD-1 mRNA、hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA的表达。取贴壁细胞用于提取细胞总蛋白行Western Blot(WB)检测p-p65NF-κB、p65NF-κB和p-p38MAPK、p38MAPK蛋白的活化程度以及p65NF-κB蛋白的核转位情况。所有实验重复三次。采用SPSS17.0软件进行分析。计量资料以均数±标准差(喁±s)表示,多组间比较采用方差分析,两组间比较采用独立样本t检验;以P0.05为差异有统计学意义。结果:1.10ng/ml TNF-α能诱导HGECs hBD-2 mRNA表达量明显升高。而在p38MAPK或NF-κB抑制剂存在的情况下,10ng/ml TNF-α诱导的HGECs h BD-2 mRNA相对表达量明显降低(P0.05)。2.Dex对TNF-α诱导的HGECs表达hBD-1 mRNA、hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA的影响。各组间HGECs hBD-1 mRNA相对表达量进行比较,差异均无统计学意义(均P0.05)。TNF-α能诱导HGECs hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA表达量升高(P0.05);在1、10μM/L Dex存在下,TNF-α诱导的HGECs hBD-2 mRNA相对表达量下降(P0.05);0.1、10μM/L Dex也能使TNF-α能诱导的IL-1βmRNA相对表达量下降(P0.05);0.1、1、10μM/L Dex均能使TNF-α能诱导的IL-6 mRNA相对表达量明显下降(P0.05)。3.不同浓度Dex对TNF-α诱导的HGECs hBD-2 mRNA表达的通路蛋白活化情况。10ng/ml TNF-α能使HGECs p65NF-κB、p38MAPK通路蛋白明显激活,p-p65NF-κB、p-p38 MAPK蛋白的相对表达量明显升高(P0.05)。在1、10μM/L Dex的存在下,TNF-α诱导的HGECs p-p65NF-κB蛋白相对表达量降低(P0.05)。而0.1、1、10μM/L Dex均能使TNF-α能诱导的HGEC p-p38MAPK相对表达量明显下降(P0.05),且随着Dex浓度的逐渐升高p-p38MAPK蛋白相对表达量也逐渐下降。4.TNF-α能诱导p65NF-κB蛋白在HGECs核内表达明显升高,0.1、1、10μM/L Dex均能使TNF-α诱导的p65NF-κB在HGECs核内的表达明显降低。结论:Dex能通过NF-κB、p38MAPK信号通路下调TNF-α诱导的HGECs hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA的表达。TNF-α诱导HGECs hBD-2 mRNA表达机制可能与p38MAPK和NF-κB信号通路有关。
[Abstract]:Objective: to investigate the effects of dexamethasone Dexon on the expression of 尾 -defensin-1hBD-1hBD-1hBD-2hBD-2hBD-2hBD-2hBD-2) and the related signal pathways such as nuclear transcription factor- 魏 B nuclear factor- 魏 BNF- 魏 B) and mitogenmit-activated protein kinase- MAPK in human gingival epithelial cells induced by TNF- 伪. Activation of proteins, To investigate the effect and mechanism of dexamethasone on oral epithelial immunity. Method 1: 1. Primary gingival epithelial cells were obtained from normal human gingival tissue by enzyme digestion. The third generation gingival epithelial cells were induced by 10ng/ml TNF- 伪 for 24 h. The other group was incubated with p38 MAPK inhibitor or NF- 魏 B inhibitor for 2 h, then induced by 10ng/ml TNF- 伪 for 24 h. Total RNA was extracted to detect the expression of hBD-2 mRNA. The third generation of HGECs were cultured at different concentrations of 0.1 渭 M / L of 10 渭 M / L for 2 h and then induced by 10ng/ml TNF- 伪 for 24 h. The hBD-1 mRNA-hBD-2 mRNA-hB@@ The attachment cells were used to detect the activation of p65NF- 魏 B, p65NF- 魏 B and pp38MAPK p38MAPK protein and the nuclear translocation of p65NF- 魏 B protein. All experiments were repeated three times. SPSS 17.0 software was used to analyze. The measurement data were expressed as mean 卤standard deviation (MSA 卤s), the analysis of variance was used in the comparison between the two groups, and the independent sample t test was used in the comparison between the two groups. The difference was statistically significant with P0.05 as the difference. Results: 1. 10 ng / ml TNF- 伪 induced a significant increase in HGECs hBD-2 mRNA expression. In the presence of p38 MAPK or NF- 魏 B inhibitor, the relative expression of HGECs hBD-2 mRNA induced by 10 ng / ml TNF- 伪 decreased significantly. 2. Dex significantly decreased the expression of hBD-1 mRNA-hBD-2 mRNAIL-1 尾 mRNAIL-6 mRNA in TNF- 伪 -induced HGECs. The relative expression of HGECs hBD-1 mRNA was compared among groups. There was no significant difference in HGECs hBD-2 mRNA-IL-1 尾 mRNA-IL-6 mRNA expression induced by HGECs hBD-2 mRNA-IL-1 尾 mRNA-IL-6 mRNA expression in HGECs, and in the presence of 1 ~ 10 渭 M / L Dex, HGECs hBD-2 mRNA relative expression decreased P0.05ML-Dex and TNF- 伪 induced IL-1 尾 mRNA relative expression decreased P0.05ML-Dex also decreased the relative expression of IL-1 尾 mRNA induced by TNF- 伪 0.110 渭 ML-Dex in the presence of 1 ~ 10 渭 M / L Dex, TNF- 伪 decreased the relative expression of HGECs hBD-2 mRNA in the presence of 1 ~ 10 渭 M / L Dex. The relative expression of IL-6 mRNA decreased significantly (P 0.05). 3. The activation of HGECs hBD-2 mRNA induced by different concentrations of Dex. 10 ng / ml TNF- 伪 could significantly activate the p65NF- 魏 B p38MAPK pathway protein of HGECs and increase the relative expression of p-p65NF- 魏 Bmp-p38 MAPK protein. The relative expression of HGECs p-p65NF- 魏 B protein induced by TNF- 伪 was decreased in the presence of 10 渭 m / L Dex. The relative expression of p-p38 MAPK in HGEC induced by TNF- 伪 decreased significantly, and the relative expression of p-p38 MAPK protein decreased gradually with the increase of Dex concentration. 4. TNF- 伪 could induce the expression of p65NF- 魏 B protein in HGECs nucleus and increase the expression of p65NF- 魏 B protein in HGECs nucleus. The expression of p65NF- 魏 B in HGECs nucleus was significantly decreased. Conclusion the down-regulation of HGECs hBD-2 mRNA-IL-1 尾 mRNA-IL-6 mRNA expression induced by TGECs hBD-2 mRNA.TNF- 伪 induced HGECs hBD-2 mRNA expression may be related to p38MAPK and NF- 魏 B signaling pathway.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781

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