本文選題：白及　切入點(diǎn)：肺纖維化　出處：《浙江中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的本實(shí)驗(yàn)采用脂多糖(LPS)刺激小鼠單核巨噬細(xì)胞白血病細(xì)胞(RAW264.7),建立RAW264.7炎癥模型,通過該模型指導(dǎo)白及活性組分分離純化,從分子水平研究白及活性單體成分對LPS刺激RAW264.7炎癥模型的抑制作用,并探討其作用機(jī)制。方法體外培養(yǎng)RAW264.7細(xì)胞,通過LPS刺激RAW264.7細(xì)胞建立巨噬細(xì)胞炎癥模型,采用RT-PCR方法確定LPS誘導(dǎo)RAW264.7細(xì)胞IL-1β mRNA表達(dá)升高的最適濃度和最適作用時(shí)間。運(yùn)用醇提-聚酰胺色譜分離制備白及醇提物,用巨噬細(xì)胞炎癥模型評(píng)價(jià)藥物效應(yīng),指導(dǎo)白及活性組分分離純化,并對單體成分進(jìn)行結(jié)構(gòu)鑒定。采用MTS法檢測細(xì)胞存活率,確定白及單體對LPS刺激RAW264.7細(xì)胞最適作用濃度。光學(xué)顯微鏡下觀察各實(shí)驗(yàn)組RAW264.7形態(tài)學(xué)變化,流式細(xì)胞術(shù)檢測RAW264.7細(xì)胞胞外IL-6、TNF-α和MCP-1 的細(xì)胞因子含量;RT-PCR檢測IL-1β、IL-6、iNOS、COX2、TNF-α、TGF-β的mRNA表達(dá)情況;Western Blot檢測白及單體對LPS刺激RAW264.7細(xì)胞MAPK通路、NF-κB通路、COX2、iNOS、p-IRF3/IRF3的蛋白表達(dá)情況。結(jié)果1.通過RT-PCR法檢測LPS對RAW264.7細(xì)胞IL-1β的mRNA相對表達(dá)量的影響,IL-1β mRNA相對表達(dá)量隨著LPS濃度和作用時(shí)間增加而升高,且呈劑量依賴性,其中200 ng/mL LPS刺激6 h,即可使IL-1β的mRNA相對表達(dá)量提高將近1000倍。2.白及提取物經(jīng)聚酰胺柱色譜分離所得五個(gè)粗分組分經(jīng)HPLC表征,發(fā)現(xiàn)各粗分樣品間交叉峰較少,分離效果較好。通過RAW264.7細(xì)胞炎癥模型,結(jié)合RT-PCR分析表明,除80%乙醇洗脫粗分組分(E80)外,其余各粗分組分均可劑量依賴性降低RAW264.7細(xì)胞IL-1βmRNA相對表達(dá)量,與模型組比較有極顯著性差異(P0.01),其中40%乙醇洗脫組分(E40)抑制效果最為顯著。3.白及40%乙醇洗脫粗分活性組分(E40)再經(jīng)硅膠柱細(xì)分為E40-(1-6)六個(gè)亞組分,并進(jìn)一步采用RAW264.7細(xì)胞炎癥模型和RT-PCR分析IL-Iβ mRNA表達(dá)的抑制活性。結(jié)果表明,不同濃度各亞組分聯(lián)合LPS作用于RAW264.7細(xì)胞6h后,其IL-1βmRNA相對表達(dá)量隨劑量增加而降低;與模型組相比,除E40-6外,其余各亞組分均有極顯著性差異P0.01,其中尤以E40-3、E40-4抑制作用最明顯。4.采用半制備液相色譜法對白及E40-3和E40-4兩個(gè)活性亞組份進(jìn)行分離、純化,核磁共振波譜鑒定RT16.6為貝母蘭寧(2,7-二羥基-4-甲氧基-9,10-二氫菲),RT21.0為 batatasin Ⅲ(3',3"-二羥基-5'-甲氧基聯(lián)芐)。5.MTS檢測不同濃度白及單體及聯(lián)合LPS對RAW264.7細(xì)胞存活率的影響,未加LPS時(shí),與正常對照組相比,單體組分RT16.6和RT21.0在低劑量時(shí)均有一定促進(jìn)增殖作用,但濃度高于15 μg/mL時(shí),對細(xì)胞有一定毒性。不同濃度單體成分聯(lián)合LPS作用于RAW264.7 24 h后,與正常對照組相比,RT16.6在20 μg/mL、RT21.0在25μg/mL時(shí),對細(xì)胞有一定毒性。6.光學(xué)顯微鏡下觀察不同濃度白及單體作用于RAW264.7對其細(xì)胞形態(tài)學(xué)的影響,加入不同濃度白及單體后,與正常細(xì)胞相比,白及單體RT16.6在1-5μg/mL、RT21.0在2.5-10 μg/mL劑量范圍內(nèi)細(xì)胞數(shù)目略有增加,形態(tài)沒有明顯變化。7.采用流式細(xì)胞術(shù)檢測不同濃度白及單體成分對LPS刺激RAW264.7細(xì)胞相關(guān)炎癥因子表達(dá)影響,結(jié)果表明,模型組LPS刺激后可顯著增加RAW264.7細(xì)胞培養(yǎng)上清中MCP-1、TNF-a、IL-6的含量,與正常組比較具有顯著性差異(P0.01)。與模型組相比,白及單體化合物RT16.6可劑量依賴性降低RAW264.7細(xì)胞外上清MCP-1和IL-6的含量,具有顯著性差異(P0.01);低劑量亦可降低TNF-a分泌量,但高劑量反而促進(jìn)TNF-a分泌。白及單體RT21.0也可劑量依賴性降低MCP-1、TNF-a、IL-6的分泌,具有顯著性差異(P0.01)。8.熒光定量PCR檢測不同濃度白及單體對LPS刺激RAW264.7細(xì)胞炎癥相關(guān)基因mRNA表達(dá)的影響,與空白對照組相比,模型組加LPS后可增加IL-1β、IL-6、iNOS、COX2、TNF-α mRNA相對表達(dá)量,具有顯著性差異(P0.01),也增加TGF-βmRNA相對表達(dá)量,具有顯著性差異(P0.05)。白及單體RT16.6可減少各炎癥基因的mRNA相對表達(dá)量,具有劑量依賴性。但在低劑量1 μg/mL時(shí),COX2、TNF-α、TGF-β mRNA相對表達(dá)量比LPS組高,這可能與低劑量促進(jìn)細(xì)胞增殖有關(guān)。白及單體RT21.0也可減少各炎癥基因的mRNA相對表達(dá)量,除了 TNF-α外,均具有劑量依賴性,與模型組比較具有顯著性差異(P0.01)。9.Western Blot檢測白及單體對LPS刺激RAW264.7細(xì)胞MAPK通路、NF-κB通路、COX2、iNOS、p-IRF3/IRF3的蛋白表達(dá)影響。結(jié)果表明,與正常對照組相比,模型組 IKK、IKBα、NF-KB、ERK1/2、JNK、P38、IRF3 磷酸化水平均明顯升高(P0.01),COX2、iNOS蛋白水平均明顯升高(P0.01);而與模型組相比,白及單體RT16.6和RT21.0均能夠顯著減少IKK、IKBα、NF-κB、JNK、IRF3磷酸化水平(P0.01);在一定程度上也可劑量依賴性地抑制iNOS、COX2蛋白表達(dá)(P0.01)。此外,白及單體RT16.6顯著增加ERK、P38磷酸化(P0.01);白及單體RT21.0能夠顯著減少P38磷酸化(P0.01),增加ERK磷酸化;結(jié)論1.LPS濃度200 ng/mL刺激6 h即可顯著增加RAW264.7細(xì)胞相關(guān)炎癥基因表達(dá)水平,可用于相關(guān)藥物抗炎活性篩選及相關(guān)分子機(jī)理研究。2.用上述條件建立的RAW264.7巨噬細(xì)胞炎癥模型評(píng)價(jià)藥物抗炎效應(yīng),結(jié)合柱色譜技術(shù)方法,指導(dǎo)白及活性組分分離純化,獲得兩個(gè)單體成分,經(jīng)核磁共振波譜鑒定RT16.6為貝母蘭寧(2,7-二羥基-4-甲氧基-9,10-二氫菲),RT21.0為batatasin Ⅲ(3',3"-二羥基-5'-甲氧基聯(lián)芐)。3.白及藥效單體成分治療肺纖維化疾病的可能分子機(jī)制為通過作用于NF-κB、p-IRF3/IRF3、MAPK、iNOS 和 COX2 等多個(gè)信號(hào)通路及靶點(diǎn),降低 IL-1β、IL-6、iNOS、COX2、TNF-α和TGF-β等炎癥基因表達(dá)和抑制MCP-1、TNF-α、IL-6等炎癥因子分泌而實(shí)現(xiàn)。
[Abstract]:The purpose of this experiment using lipopolysaccharide (LPS) stimulation of leukemic cells in mouse macrophage (RAW264.7), a RAW264.7 model of inflammation, purified by the Rhizoma Bletillae model to guide the active component from the inhibition of the molecular level Rhizoma Bletillae active monomer composition on LPS stimulated RAW264.7 inflammation model, and explore its mechanism. Methods RAW264.7 cells were cultured in vitro, establishment of macrophage inflammatory model by LPS stimulation of RAW264.7 cells, using RT-PCR method to determine the expression of the optimal concentration and the best time of RAW264.7 cells induced by LPS. IL-1 beta mRNA with ethanol and separated polyamide chromatography prepared by Rhizoma Bletillae alcohol extract, evaluation of drug effect of macrophage inflammatory model, guide the Rhizoma Bletillae active component separation purification and structural identification of the monomer composition. Cell viability was determined by MTS assay, determine the Rhizoma Bletillae monomer on the LPS optimal stimulation of RAW264.7 cells The concentration of each experimental group. Changes of RAW264.7 morphology observed under light microscope, flow cytometry was used to detect RAW264.7 cell extracellular IL-6, cytokine levels of TNF- alpha and MCP-1; RT-PCR IL-6, detection of IL-1 beta, iNOS, COX2, TNF- alpha, TGF- beta mRNA expression; Western Blot test Rhizoma Bletillae monomer on LPS stimulation of RAW264.7 cells MAPK NF- pathway, B pathway, COX2, iNOS, expression of p-IRF3/IRF3 protein. Results 1. detected by LPS RT-PCR method on RAW264.7 cells of IL-1 beta mRNA expression effects of IL-1 beta mRNA expression increased with LPS concentration and action time increased in a dose-dependent manner, which 200 ng/mL LPS 6 h stimulation, can make the IL-1 beta mRNA expression increased nearly 1000 times the Rhizoma Bletillae.2. extract by polyamide column chromatography separation of the five crude grouping was characterized by HPLC, found that the crude sample cross peak is less, the separation effect Good. By RAW264.7 inflammation model, combined with RT-PCR analysis showed that in 80% ethanol elution coarse grouping (E80), the rest of the points can be roughly divided into groups dose dependently reduced the relative expression of RAW264.7 cells IL-1 beta mRNA, there was a significant difference compared with the model group (P0.01), 40% ethanol fraction (E40) the most significant inhibitory effect of Rhizoma Bletillae.3. 40% ethanol crude active component (E40) by silica gel column E40- (1-6) divided into six subgroups, and further the inhibitory activity of RAW264.7 inflammation model and RT-PCR analysis of IL-I beta mRNA expression. The results showed that different concentrations of each sub group of joint the effect of LPS on RAW264.7 6h cells after the IL-1 beta mRNA relative expression decreased with the dose increased; compared with the model group, except E40-6, the other subfractions showed very significant difference between P0.01, especially E40-3, E40-4 inhibited by the most obvious.4. by semi system Purification by liquid chromatography on E40-3 and E40-4 and two active subfractions were isolated, nuclear magnetic resonance spectroscopy of RT16.6 was identified as Lan Ning Fritillary (2,7- two hydroxy -4- methoxy -9,10- two RT21.0 batatasin hydrogen Philippines), III (3', 3 "- two hydroxy -5'- methoxy bibenzyl).5.MTS detection of different concentrations of monomer and Rhizoma Bletillae combined with LPS on the survival rate of RAW264.7 cells, without LPS, compared with the normal control group, single component RT16.6 and RT21.0 were at a low dose could promote the proliferation, but the concentration is higher than 15 g/mL, have a certain toxicity to the cells. Different concentrations of monomer composition combined with the effect of LPS on RAW264.7 24 h, compared with the normal control group, RT16.6 20 g/mL, RT21.0 25 g/mL,.6. have a certain toxicity under optical microscope observation of different concentrations of Rhizoma Bletillae monomer in RAW264.7 affects the cell morphology of cells with different concentrations of Rhizoma Bletillae The monomer, compared with normal cells, Rhizoma Bletillae monomer RT16.6 1-5 g/mL, RT21.0 cells in the 2.5-10 range of g/mL number increased slightly, morphology did not change significantly.7. influence on the expression of LPS in RAW264.7 cells stimulated by different concentrations of inflammatory cytokines in Rhizoma Bletillae flow cytometry monomer composition. The results showed that the model group after LPS stimulation can significantly increase the RAW264.7 cell culture supernatant of MCP-1, TNF-a, IL-6 content, compared with the normal group with significant difference (P0.01). Compared with the model group, Rhizoma Bletillae monomer compound RT16.6 can dose dependently reduced RAW264.7 content of extracellular supernatant of MCP-1 and IL-6, with significant difference (P0.01) low dose; reduce TNF-a secretion, but high dose but to promote the secretion of TNF-a. Rhizoma Bletillae RT21.0 monomer also dose dependently decreased MCP-1, TNF-a, IL-6 secretion, with significant difference (P0.01).8. fluorescence quantitative PC Effect of R to detect different concentrations of Rhizoma Bletillae monomers on LPS stimulated mRNA gene RAW264.7 expression of inflammatory cells, compared with the control group, the model group after the addition of LPS can increase the IL-1 beta, IL-6, iNOS, COX2, the relative expression of TNF- alpha mRNA, with significant difference (P0.01), also increased the relative expression of TGF- beta mRNA, with a significant difference (P0.05). Rhizoma Bletillae RT16.6 monomer can reduce the inflammatory gene relative expression of mRNA, in a dose dependent manner. But in the low dose 1 g/mL, COX2, TNF- alpha, TGF- beta mRNA relative expression quantity is higher than the LPS group, which may promote cell proliferation associated with low dose Rhizoma Bletillae RT21.0 monomer can also reduce the inflammatory gene relative expression of mRNA, TNF- in addition to alpha, both in a dose dependent manner. Compared with the model group with significant difference (P0.01).9.Western Blot detection of Rhizoma Bletillae monomer on LPS RAW264.7 cells stimulated MAPK pathway, NF- B pathway, COX2, iNOS, The expression of p-IRF3/IRF3 protein. Results show that, compared with the normal control group, model group, IKK, NF-KB, ERK1/2, IKB alpha, JNK, P38, IRF3 phosphorylation levels were significantly increased (P0.01), COX2, iNOS protein levels were significantly increased (P0.01); compared with the model group, Rhizoma Bletillae and RT16.6 monomer RT21.0 could significantly reduce IKK, IKB alpha, NF- kappa B, JNK, phosphorylation of IRF3 (P0.01); to a certain extent also dose dependently inhibited the expression of COX2 (iNOS, P0.01). In addition, Rhizoma Bletillae monomer RT16.6 significantly increased ERK, phosphorylation of P38 (P0.01); Rhizoma Bletillae monomer RT21.0 can significantly reduce the phosphorylation of P38 (P0.01), increased the phosphorylation of ERK; conclusion 1.LPS concentration of 200 ng/mL 6 h stimulation significantly increased RAW264.7 gene expression level of inflammatory cells, can be used to study the anti-inflammatory activity of related drug screening and related molecular mechanism of.2. with RAW264.7 macrophage inflammatory conditions above established The evaluation model of drug anti-inflammatory effect, combined with column chromatography method, guiding Rhizoma Bletillae active component separation and purification, two monomer composition, the NMR spectrum of RT16.6 was identified as Lan Ning Fritillary (2,7- two hydroxy -4- methoxy -9,10- two RT21.0 batatasin hydrogen Philippines), III (3', 3 "- two hydroxy -5'- methyl oxy bibenzyl) possible molecular mechanism of.3. Rhizoma Bletillae efficacy monomer composition for treatment of diseases of pulmonary fibrosis by acting on NF- kappa B, p-IRF3/IRF3, MAPK, iNOS and COX2 multiple signaling pathways and targets, reduce IL-1 beta, IL-6, iNOS, COX2, expression of TNF- alpha and TGF- beta gene and inhibiting inflammation MCP-1, TNF- alpha, IL-6 and other inflammatory cytokines secretion.

【學(xué)位授予單位】：浙江中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R563

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4 李

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