本文選題：地塞米松 + 人牙齦上皮細(xì)胞��； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過檢測(cè)地塞米松(Dex)對(duì)TNF-α誘導(dǎo)的人牙齦上皮細(xì)胞(HGECs)表達(dá)β-防御素-1,2(hBD-1、h BD-2)和炎癥因子IL-1β、IL-6的影響及核轉(zhuǎn)錄因子-κB(nuclear factor-κB,NF-κB)和絲裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)等相關(guān)信號(hào)通路蛋白的活化情況,探討地塞米松對(duì)口腔上皮免疫的影響及作用機(jī)制。方法:1.將臨床取材的正常人牙齦組織采用酶消化培養(yǎng)法獲取原代牙齦上皮細(xì)胞。選用10ng/ml TNF-α誘導(dǎo)第三代牙齦上皮細(xì)胞24h。另一組選用p38MAPK抑制劑或NF-κB抑制劑分別孵育細(xì)胞2h后,再用10ng/ml TNF-α誘導(dǎo)細(xì)胞24h。提取細(xì)胞總RNA檢測(cè)hBD-2 mRNA的表達(dá)量。2.不同濃度Dex(0.1,1,10μM/L)分別培養(yǎng)第三代HGEC 2h后再加入10ng/ml TNF-α誘導(dǎo)細(xì)胞24h。取貼壁細(xì)胞用于提取總RNA進(jìn)行實(shí)時(shí)熒光定量PCR(Real Time-PCR)檢測(cè)hBD-1 mRNA、hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA的表達(dá)。取貼壁細(xì)胞用于提取細(xì)胞總蛋白行Western Blot(WB)檢測(cè)p-p65NF-κB、p65NF-κB和p-p38MAPK、p38MAPK蛋白的活化程度以及p65NF-κB蛋白的核轉(zhuǎn)位情況。所有實(shí)驗(yàn)重復(fù)三次。采用SPSS17.0軟件進(jìn)行分析。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(喁±s)表示,多組間比較采用方差分析,兩組間比較采用獨(dú)立樣本t檢驗(yàn);以P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.10ng/ml TNF-α能誘導(dǎo)HGECs hBD-2 mRNA表達(dá)量明顯升高。而在p38MAPK或NF-κB抑制劑存在的情況下,10ng/ml TNF-α誘導(dǎo)的HGECs h BD-2 mRNA相對(duì)表達(dá)量明顯降低(P0.05)。2.Dex對(duì)TNF-α誘導(dǎo)的HGECs表達(dá)hBD-1 mRNA、hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA的影響。各組間HGECs hBD-1 mRNA相對(duì)表達(dá)量進(jìn)行比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(均P0.05)。TNF-α能誘導(dǎo)HGECs hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA表達(dá)量升高(P0.05);在1、10μM/L Dex存在下,TNF-α誘導(dǎo)的HGECs hBD-2 mRNA相對(duì)表達(dá)量下降(P0.05);0.1、10μM/L Dex也能使TNF-α能誘導(dǎo)的IL-1βmRNA相對(duì)表達(dá)量下降(P0.05);0.1、1、10μM/L Dex均能使TNF-α能誘導(dǎo)的IL-6 mRNA相對(duì)表達(dá)量明顯下降(P0.05)。3.不同濃度Dex對(duì)TNF-α誘導(dǎo)的HGECs hBD-2 mRNA表達(dá)的通路蛋白活化情況。10ng/ml TNF-α能使HGECs p65NF-κB、p38MAPK通路蛋白明顯激活,p-p65NF-κB、p-p38 MAPK蛋白的相對(duì)表達(dá)量明顯升高(P0.05)。在1、10μM/L Dex的存在下,TNF-α誘導(dǎo)的HGECs p-p65NF-κB蛋白相對(duì)表達(dá)量降低(P0.05)。而0.1、1、10μM/L Dex均能使TNF-α能誘導(dǎo)的HGEC p-p38MAPK相對(duì)表達(dá)量明顯下降(P0.05),且隨著Dex濃度的逐漸升高p-p38MAPK蛋白相對(duì)表達(dá)量也逐漸下降。4.TNF-α能誘導(dǎo)p65NF-κB蛋白在HGECs核內(nèi)表達(dá)明顯升高,0.1、1、10μM/L Dex均能使TNF-α誘導(dǎo)的p65NF-κB在HGECs核內(nèi)的表達(dá)明顯降低。結(jié)論:Dex能通過NF-κB、p38MAPK信號(hào)通路下調(diào)TNF-α誘導(dǎo)的HGECs hBD-2 mRNA、IL-1βmRNA、IL-6 mRNA的表達(dá)。TNF-α誘導(dǎo)HGECs hBD-2 mRNA表達(dá)機(jī)制可能與p38MAPK和NF-κB信號(hào)通路有關(guān)。
[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- 偽. 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- 偽 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- 偽 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- 偽 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 鹵standard deviation (MSA 鹵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- 偽 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- 偽 decreased significantly. 2. Dex significantly decreased the expression of hBD-1 mRNA-hBD-2 mRNAIL-1 尾 mRNAIL-6 mRNA in TNF- 偽 -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- 偽 induced IL-1 尾 mRNA relative expression decreased P0.05ML-Dex also decreased the relative expression of IL-1 尾 mRNA induced by TNF- 偽 0.110 渭 ML-Dex in the presence of 1 ~ 10 渭 M / L Dex, TNF- 偽 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- 偽 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- 偽 was decreased in the presence of 10 渭 m / L Dex. The relative expression of p-p38 MAPK in HGEC induced by TNF- 偽 decreased significantly, and the relative expression of p-p38 MAPK protein decreased gradually with the increase of Dex concentration. 4. TNF- 偽 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- 偽 induced HGECs hBD-2 mRNA expression may be related to p38MAPK and NF- 魏 B signaling pathway.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781

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