本文選題：哮喘　切入點(diǎn)：氣道炎癥　出處：《南京醫(yī)科大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：第一部分高良姜素對(duì)小鼠哮喘模型氣道炎癥的影響 目的：研究高良姜素對(duì)小鼠哮喘模型氣道炎癥和氣道高反應(yīng)性的影響及其相關(guān)機(jī)制。 方法：36只BALB/c小鼠按隨機(jī)原則分成對(duì)照組(Control組)、哮喘組(OVA組)、DMSO組(OVA+DMSO組)、高劑量高良姜素組(OVA+GA15組)、低劑量高良姜素組(OVA+GA5組)和地塞米松組(OVA+DXM),每組6只。哮喘組及干預(yù)組小鼠于第0及第14天腹腔內(nèi)注射20μg OVA致敏,第22、23、24天1%OVA連續(xù)霧化三天,每天一次,每次30分鐘。干預(yù)組于OVA激發(fā)前一天開(kāi)始每天腹腔內(nèi)注射DMSO、不同劑量的高良姜素、地塞米松。末次激發(fā)24h后,使用小動(dòng)物肺功能分析系統(tǒng)進(jìn)行氣道反應(yīng)性測(cè)定,取肺組織病理切片行蘇木精-伊紅(HE)染色觀(guān)察肺組織氣管旁及血管旁炎性細(xì)胞浸潤(rùn),PAS染色觀(guān)察杯狀細(xì)胞增生,用酶聯(lián)免疫吸附試驗(yàn)(ELISA)分別檢測(cè)各組小鼠血清OVA特異性IgE和肺泡灌洗液(BALF)中IL-4、IL-4和IL-13的水平。使用免疫組織化學(xué)法(IHC)及蛋白質(zhì)印跡法(Western blot)檢測(cè)肺組織中iNOS及VCAM的蛋白表達(dá)。使用Western blot檢測(cè)肺組織細(xì)胞總蛋白IλBα和磷酸化的p65的蛋白表達(dá),核蛋白和細(xì)胞質(zhì)中p65的蛋白表達(dá)。 結(jié)果：對(duì)小鼠肺阻力(RL)檢測(cè)發(fā)現(xiàn),隨著氯化乙酰膽堿濃度的增加,OVA組小鼠RL明顯增加,Control組小鼠RL輕度增加,各組小鼠的基礎(chǔ)氣道阻力沒(méi)有差異(P0.05),當(dāng)氯化乙酰膽堿濃度在30μg/kg及以上時(shí),OVA組小鼠RL顯著高于Control組小鼠(P0.05),高良姜素組和地塞米松組小鼠與OVA組小鼠相比有統(tǒng)計(jì)學(xué)意義(P0.05); OVA組小鼠BALF中炎性細(xì)胞總數(shù)、EOS細(xì)胞數(shù)和NEUT細(xì)胞數(shù)、LYM細(xì)胞數(shù)高于Control組小鼠(P0.05),高良姜素組和地塞米松組小鼠BALF中炎性細(xì)胞總數(shù)、EOS細(xì)胞數(shù)、NEUT細(xì)胞數(shù)和LYM細(xì)胞明顯降低,與OVA組小鼠相比有統(tǒng)計(jì)學(xué)意義(P0.05); OVA組BALF中IL-4、IL-5和IL-13和血清OVA特異性IgE含量高于Control組(P0.05),高良姜素組和地塞米松組BALF中IL-4、IL-5和IL-13和血清OVA特異性IgE含量低于OVA組小鼠(P0.05)；OVA組小鼠氣道粘液評(píng)分高于Control組小鼠(P0.05),高良姜素組和地塞米松組小鼠氣道粘液評(píng)分低于OVA組小鼠(P0.05); OVA組小鼠肺組織iNOS和VCAM-1的蛋白表達(dá)高于Control組(P0.05),高良姜素和地塞米松組小鼠肺組織iNOS和VCAM-1的蛋白表達(dá)與OVA組相比降低(P0.05); OVA組小鼠肺組織細(xì)胞總蛋白IκBα的表達(dá)低于Control組(P0.05),高良姜素和地塞米松組小鼠肺組織細(xì)胞總蛋白IκBα的蛋白表達(dá)與OVA組相比增高(P0.05); OVA組小鼠肺組織細(xì)胞總蛋白磷酸化的p65表達(dá)高于Control組(P0.05),高良姜素和地塞米松組小鼠肺組織細(xì)胞總蛋白磷酸化的p65表達(dá)與OVA組相比降低(P0.05); OVA組小鼠肺組織核蛋白p65表達(dá)高于Control組(P0.05),高良姜素和地塞米松組小鼠肺組織核蛋白p65表達(dá)與OVA組相比降低(P0.05); OVA組小鼠肺組織胞漿蛋白p65表達(dá)低于Control組(P0.05),高良姜素和地塞米松組小鼠肺組織核蛋白p65表達(dá)與OVA組相比增高(P0.05)。 結(jié)論：高良姜素可在一定程度上抑制哮喘模型小鼠的氣道炎癥,氣道高反應(yīng)性,這種抑制作用可能與抑制NF-κB的活化有關(guān)。 第二部分高良姜素在人氣道平滑肌細(xì)胞中的抗炎作用 目的研究探討高良姜素對(duì)腫瘤壞死因子-α (TNF-α)誘導(dǎo)的人氣道平滑肌細(xì)胞(ASMCs)表達(dá)單核細(xì)胞趨化蛋白-1(MCP-1)、嗜酸性粒細(xì)胞趨化因子(eotaxin)、CXC趨化因子配體10(CXCL10)和血管細(xì)胞黏附分子-1(VCAM-1)的影響及可能的機(jī)制。 方法體外培養(yǎng)原代人氣道平滑肌細(xì)胞,Cell Counting Kit-8(CCK-8)法檢測(cè)不同濃度的高良姜素對(duì)人氣道平滑肌細(xì)胞的活性影響；實(shí)時(shí)熒光定量PCR(Real-time PCR)法檢測(cè)高良姜素對(duì)TNF-α誘導(dǎo)人氣道平滑肌細(xì)胞表達(dá)MCP-1、eotaxin、CXCL10、VCAM-1mRNA的影響；蛋白質(zhì)印跡法(Western blot)檢測(cè)高良姜素對(duì)TNF-α誘導(dǎo)人氣道平滑肌細(xì)胞NF-κB活性的影響。 結(jié)果研究發(fā)現(xiàn)高良姜素濃度為1μM～20～M時(shí),對(duì)人氣道平滑肌細(xì)胞的活性無(wú)明顯影響,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),而高良姜素濃度為50μM、100μM時(shí),人氣道平滑肌細(xì)胞活性明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);10ng/mL TNF-α可以誘導(dǎo)人氣道平滑肌細(xì)胞MCP-1、Eotaxin、CXCL10和VCAM-1mRNA的表達(dá)(P0.05);1μM、10μM高良姜素及10μN(yùn)F-κB特異性抑制劑TPCA-1均能有效抑制MCP-1、Eotaxin、CXCL10和VCAM-1mRNA的表達(dá)(P0.05);1μM、10μM高良姜素均能抑制TNF-a誘導(dǎo)的人平滑肌細(xì)胞NF-κB p65亞單位的核易位(P0.05)。 結(jié)論高良姜素能通過(guò)抑制NF-κB活性,下調(diào)TNF-α誘導(dǎo)的人氣道平滑肌細(xì)胞MCP-1、Eotaxin、CXCL10和VCAM-1mRNA的表達(dá),發(fā)揮其抗炎作用。
[Abstract]:Part 1 Effect of galangin on airway inflammation in asthmatic mice model
Objective: To study the effect of Takara Jiangso on airway inflammation and airway hyperresponsiveness in mice model of asthma and its mechanism.
Methods: 36 BALB/c mice were randomly divided into control group (Control group), asthma group (OVA group), DMSO group (OVA+DMSO group), high dose of galangin group (OVA+GA15 group), low dose of galangin group (OVA+GA5 group) and dexamethasone group (OVA+DXM), and the intervention group with 6 rats in each group. The asthma group mice at zeroth and 14 days by intraperitoneal injection of 20 g OVA sensitized 22,23,24 days 1%OVA continuous spray for three days, once a day, 30 minutes each time. The intervention group was treated with intraperitoneal injection of OVA activated DMSO the day before, different doses of galangin, dexamethasone. 24h after the last challenge. Determination of airway reactivity using small animal lung function analysis system, the lung pathological section with hematoxylin and eosin (HE) staining of tracheal and lung tissue adjacent to vascular inflammatory cell infiltration, goblet cell hyperplasia were observed with PAS staining, adsorption test by enzyme-linked immunosorbent assay (ELISA) were detected in mice Serum OVA specific IgE and bronchoalveolar lavage fluid (BALF) in IL-4, IL-4 and IL-13. Using immunohistochemistry method (IHC) and Western blotting (Western blot) expression of iNOS and VCAM in lung tissues was detected. The protein expression in lung tissue of total cell protein using Western blot detection I lambda B alpha and phosphate the expression of p65 protein, nuclear protein and p65 protein in the cytoplasm.
Results: the pulmonary resistance in mice (RL) showed that with the increase of acetylcholine chloride concentration, OVA mice RL mice in group Control increased significantly, RL increased slightly, no differences in baseline airway resistance in mice (P0.05), when the concentration of acetylcholine chloride at 30 g/kg and above, OVA group were significantly higher than that of Control RL groups of mice (P0.05), galangin group and dexamethasone group compared with OVA group were statistically significant (P0.05); the number of inflammatory cells in OVA mice BALF, EOS cell count and NEUT cell number, LYM cell number is higher than that of mice in group Control (P0.05), the number of inflammatory cells of galangin group and dexamethasone group in BALF mice, EOS cells, NEUT cells and LYM cells were significantly decreased, which was statistically significant compared with OVA group (P0.05); IL-4 OVA group BALF, IL-5 and IL-13 and serum OVA specific IgE were higher than group Control (P0.05), Gaoliangjiang IL-4 group and dexamethasone group BALF, IL-5 and IL-13 and serum OVA specific IgE was lower than that in OVA group (P0.05); OVA group of mice airway mucus was higher than that of mice in group Control (P0.05), the Takara Jiangso group and dexamethasone group mice airway mucus score was lower than that of mice in OVA group (P0.05); the expression of OVA in lung tissues of mice iNOS and VCAM-1 protein is higher than that of Control group (P0.05), galangin and dexamethasone in lung tissue of mice iNOS and VCAM-1 protein expression decreased compared with OVA group (P0.05); group the expression of OVA in lung tissues of mice with total cell protein I kappa B alpha was lower than that of Control group (P0.05), the expression of Takara Jiangso and dexamethasone in lung tissue of mice total cell protein I kappa B alpha protein compared with OVA group (P0.05); OVA group of mouse lung cell total protein phosphorylation of p65 was higher than that in group Control (P0.05), Takara Jiangso and dexamethasone in mice lung tissue cells The expression of protein phosphorylation of p65 decreased compared with OVA group (P0.05); nuclear protein p65 in lung tissue of mice in group OVA was higher than that in Control group (P0.05), the expression of nuclear protein p65 Takara Jiangso and dexamethasone group of mice lung tissue compared with the OVA group decreased (P0.05); OVA group of mice lung tissue cytosolic protein expression of p65 was lower than that of Control group (P0.05), nuclear protein p65 Takara Jiangso and dexamethasone group of mice lung tissue expression compared with OVA group (P0.05).
Conclusion: alpinin can inhibit airway inflammation and airway hyperresponsiveness to a certain extent, which may be related to inhibiting the activation of NF- kappa B.
The second part of the anti-inflammatory effect of galangin in human airway smooth muscle cells.
Objective to investigate the galangin on tumor necrosis factor alpha (TNF- alpha) induced human airway smooth muscle cells (ASMCs) expression of monocyte chemoattractant protein -1 (MCP-1), eosinophil chemotactic factor (eotaxin), CXC chemokine ligand 10 (CXCL10) and vascular cell adhesion molecule (-1 VCAM-1) the effects and possible mechanism.
Primary cultured human airway smooth muscle cells in vitro, Cell Counting Kit-8 (CCK-8) method was used to detect the effects of different concentrations of Takara Jiangso activity on human airway smooth muscle cells; real time fluorescence quantitative PCR (Real-time PCR) method for the detection of galangin on TNF- induced MCP-1 expression in human airway smooth muscle cells eotaxin, CXCL10, VCAM-1mRNA; protein Western blotting (Western blot) detection effect of galangin induced kappa B activity in human airway smooth muscle cells of NF- TNF- alpha.
Results galangin concentration is 1 ~ M to 20 ~ M, had no significant effect on human airway smooth muscle cells, there was no statistically significant difference (P0.05), and galangin concentration was 50 M, 100 M, the activity of human airway smooth muscle cells decreased significantly, the difference was statistically significant (P0.05 10ng/mL TNF-); alpha can induce airway smooth muscle cells MCP-1, Eotaxin, expression of CXCL10 and VCAM-1mRNA (P0.05); 1 M, 10 M and 10 NF- of galangin kappa B inhibitor TPCA-1 could effectively inhibit the MCP-1, Eotaxin, CXCL10 and VCAM-1mRNA expression (P0.05); 1 M the nuclear translocation of vascular smooth muscle cells, NF- kappa B subunit p65 10 M galangin can inhibit TNF-a induced by (P0.05).
Conclusion galangin can inhibit NF- kappa B activity, reduced human airway smooth muscle cells induced by TNF- MCP-1, Eotaxin, expression of CXCL10 and VCAM-1mRNA, exert its anti-inflammatory effect.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R562.25

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 蔣雷服;;呼出氣一氧化氮測(cè)定在支氣管哮喘診療中的應(yīng)用[J];中國(guó)實(shí)用內(nèi)科雜志;2014年S1期

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