發(fā)布時(shí)間：2018-03-01 17:25

  本文關(guān)鍵詞： PM2.5 哮喘 炎癥反應(yīng) ICAM-1 NF-κB　出處：《天津醫(yī)科大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:研究細(xì)顆粒物PM2.5對(duì)哮喘模型大鼠氣道炎癥的影響,觀察PM2.5對(duì)NF-κB和ICAM-1表達(dá)的調(diào)控,進(jìn)而探討PM2.5促進(jìn)炎癥的可能機(jī)制。方法:1.使用PM采樣器收集空氣中PM2.5樣本,樣本洗脫后過(guò)濾,真空冷凍干燥后,用磷酸鹽緩沖溶液配制PM2.5懸液。2.50只Wistar大鼠隨機(jī)分組,共計(jì)5組:PM2.5高劑量組(0.24mg/次)、PM2.5中劑量組(0.16mg/次)、PM2.5低劑量組(0.08mg/次)、生理鹽水對(duì)照組和正常對(duì)照組。模型大鼠予卵蛋白皮下和腹腔注射兩次致敏,14天后再行卵蛋白霧化吸入連續(xù)14天,建立大鼠過(guò)敏性哮喘模型。從霧化開(kāi)始第1天,氣管注入PM2.5懸液,每三天一次,共計(jì)5次,觀察各組大鼠哮喘發(fā)作表現(xiàn)。霧化結(jié)束2天后取材,進(jìn)行外周血白細(xì)胞計(jì)數(shù)和BALF嗜酸性粒細(xì)胞(EOS)計(jì)數(shù),HE染色觀察氣道病理改變,ELISA試劑盒檢測(cè)血清總Ig E。3.應(yīng)用MTS法觀察PM2.5對(duì)RAW264.7細(xì)胞增殖的影響。RAW264.7細(xì)胞接種于培養(yǎng)板,8個(gè)復(fù)孔。分別加入PM2.5懸液終濃度為3.2、1.6、0.8、0.4、0.2mg/ml,培養(yǎng)24h后,4個(gè)復(fù)孔MTS法檢測(cè)OD值觀察不同濃度PM2.5對(duì)RAW264.7細(xì)胞增殖的影響。同時(shí),提取另外4個(gè)復(fù)孔培養(yǎng)液中的上清液,用ELISA方法檢測(cè)IL-1β和TNF-α濃度。ELISA方法檢測(cè)PM2.5對(duì)哮喘大鼠血清和BALF中IL-1β和TNF-α濃度的影響。4.應(yīng)用RT-PCR(實(shí)時(shí)熒光定量PCR)和Western Blot方法,觀察PM2.5體外濃度為3.2、1.6、0.8mg/ml時(shí)對(duì)RAW264.7細(xì)胞ICAM-1和NF-κB的m RNA和蛋白表達(dá)的影響。應(yīng)用實(shí)時(shí)熒光定量PCR和Western Blot方法檢測(cè)哮喘模型大鼠肺組織和脾組織,觀察PM2.5的體內(nèi)作用對(duì)ICAM-1和NF-κB的m RNA和蛋白表達(dá)的影響。結(jié)果:1.PM2.5收集共計(jì)2.5g,磷酸鹽緩沖溶液配置懸液100mg/ml,-20℃冷凍保存?zhèn)溆谩?.成功建立大鼠哮喘模型。3.PM2.5高、中、低劑量(0.24、0.16、0.08mg/次)能夠加重哮喘大鼠的哮喘發(fā)作癥狀,PM2.5高劑量和中劑量能夠明顯增加外周血白細(xì)胞總數(shù)和BALF中EOS數(shù)量,與生理鹽水對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05),PM2.5高、中劑量組之間比較,沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05);PM2.5高、中、低劑量能夠增加氣道內(nèi)EOS、中性粒細(xì)胞和巨噬細(xì)胞等炎性細(xì)胞浸潤(rùn),促進(jìn)杯狀細(xì)胞增生,促進(jìn)小支氣管粘膜水腫等病理改變。PM2.5高劑量和中劑量能夠增加哮喘大鼠血清中總Ig E含量,與生理鹽水對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05),高、中劑量組之間比較,沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05)。4.PM2.5體外濃度為3.2-0.8 mg/ml能夠抑制RAW264.7細(xì)胞增殖,與對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05),抑制率為43.06%-22.89%。PM2.5體外濃度為3.2-0.4 mg/ml能夠促進(jìn)RAW264.7細(xì)胞IL-1β和TNF-α的產(chǎn)生,與生理鹽水對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05);PM2.5高劑量和中劑量能夠增加哮喘大鼠血清和BALF中IL-1β和TNF-α濃度,與生理鹽水對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05)。5.PM2.5體外濃度為3.2-0.8mg/ml能夠上調(diào)RAW264.7細(xì)胞ICAM-1和NF-κB的m RNA和蛋白表達(dá)的影響,與對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05);PM2.5高劑量和中劑量能夠增加哮喘大鼠肺組織ICAM-1和NF-κB的m RNA和蛋白表達(dá),與生理鹽水對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05);高劑量組NF-κB蛋白表達(dá)高于低劑量組,差別有統(tǒng)計(jì)學(xué)意義(P0.05);然而,PM2.5高、中、低劑量對(duì)哮喘大鼠脾組織ICAM-1和NF-κB的m RNA和蛋白表達(dá)沒(méi)有明顯影響,與生理鹽水對(duì)照組相比無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.PM2.5使過(guò)敏性哮喘大鼠的氣道病理改變加重、外周血白細(xì)胞和BALF中EOS增高,表明PM2.5可加重哮喘氣道的病理狀態(tài),促進(jìn)炎癥過(guò)程。2.PM2.5體外抑制RAW264.7細(xì)胞增殖,促進(jìn)RAW264.7細(xì)胞IL-1β和TNF-α的產(chǎn)生,以及增加哮喘大鼠血清和BALF中IL-1β和TNF-α濃度,提示PM2.5對(duì)炎癥細(xì)胞因子的分泌具有促進(jìn)作用。3.PM2.5對(duì)過(guò)敏性哮喘的促炎癥機(jī)制之一可能是通過(guò)上調(diào)NF-κB,增加氣道炎癥因子的分泌,進(jìn)而上調(diào)氣道ICAM-1的表達(dá),促進(jìn)炎細(xì)胞浸潤(rùn)而發(fā)揮作用。4.實(shí)驗(yàn)結(jié)果提示,PM2.5濃度在100ug/m3時(shí)即可加重哮喘癥狀及氣道病理?yè)p傷,濃度在200ug/m3以上時(shí)可以使外周血白細(xì)胞計(jì)數(shù)、BALF中EOS計(jì)數(shù)和血清總Ig E水平明顯升高,細(xì)胞因子IL-1β和TNF-α分泌增加,使肺組織NF-κB和ICAM-1表達(dá)增加;而PM2.5濃度在300ug/m3以內(nèi)時(shí)不足以引起機(jī)體整體免疫系統(tǒng)NF-κB和ICAM-1表達(dá)的上升。
[Abstract]:Objective: To study the effect of fine particles PM2.5 on the airway inflammation of asthmatic rats, observe the PM2.5 regulation on the expression of NF- kappa B and ICAM-1, and to explore the possible mechanism of PM2.5 in promoting inflammation. Methods: 1. using the PM sampler to collect PM2.5 samples in the air sample, eluting after filtration, vacuum freeze drying, prepared PM2.5 suspension liquid.2.50 Wistar rats were randomly divided into phosphate buffer solution, a total of 5 groups: high dose of PM2.5 group (0.24mg/), middle dose of PM2.5 group (0.16mg/), PM2.5 low dose group (0.08mg/), saline control group and normal control group. Rats received subcutaneous and intraperitoneal injection of two egg protein sensitization, and ovalbumin inhalation for 14 days and 14 days later, the establishment of allergic asthma model of rats. The atomization began on the first day, intratracheal instillation of PM2.5 suspension, once every three days, a total of 5 times, observe the asthma rats attack. 2 days after the end of the atomization Material, counting and BALF peripheral white blood cells, eosinophil count (EOS), airway pathological changes were observed by HE staining, ELISA kit to detect serum total Ig E.3. MTS method was used to observe the influence of PM2.5 on the proliferation of RAW264.7 cells and.RAW264.7 cells were seeded in culture plates, 8 holes. Don't join the PM2.5 suspension the final concentration of 3.2,1.6,0.8,0.4,0.2mg/ml, 24h after culture, 4 wells measured the OD value of MTS method to observe the effect of different concentrations of PM2.5 on the proliferation of RAW264.7 cells. At the same time, the other 4 extraction holes in the supernatant liquid, using ELISA method for detection of IL-1 beta and TNF- alpha concentration.ELISA method to detect the effect of PM2.5 on IL-1 in serum and BALF in asthma rat alpha beta and TNF- concentration of.4. RT-PCR (Application of real time fluorescence quantitative PCR) and Western Blot method, PM2.5 was observed in vitro concentration effect on the expression of M RNA and protein in RAW264.7 cells ICAM-1 and NF- kappa B at 3.2,1.6,0.8mg/ml. Using real-time quantitative PCR method for the detection of Blot and Western in lung tissue of asthmatic rats and spleen tissue expression observed in vivo effects of PM2.5 on ICAM-1 and NF- m RNA and kappa B protein. Results: 1.PM2.5 collected a total of 2.5G, phosphate buffer solution suspension configuration 100mg/ml, -20 C cryopreservation.2. successfully established.3.PM2.5 the rat model of asthma, high, low dose (0.24,0.16,0.08mg/) symptoms can worsen asthma in rats with asthma, PM2.5 high dose and middle dose could significantly increase the number of peripheral white blood cell count and BALF in EOS, were statistically significant compared with control group, normal saline (P0.05), PM2.5, comparison between dose group was not statistically significant (P0.05); PM2.5 high, low dose can increase airway EOS, neutrophils and macrophages and other inflammatory cell infiltration, goblet cells to promote proliferation, promote small bronchial mucosa in water Swollen and pathological changes of.PM2.5 high dose and middle dose could increase the serum total Ig in asthmatic rats E levels were statistically significant compared with control group, normal saline (P0.05), a comparison between high, medium dose group, no statistical significance (P0.05) concentration of 3.2-0.8 mg/ ml.4.PM2.5 in vitro can inhibit the proliferation of RAW264.7 cells, there was the significance compared with the control group (P0.05), 43.06%-22.89%.PM2.5 inhibition in vitro concentration of 3.2-0.4 mg/ml can promote RAW264.7 cell IL-1 and TNF- beta alpha production was statistically significant compared with control group, normal saline (P0.05); PM2.5 high and middle dose of IL-1 can increase the concentration of serum beta and TNF- alpha and BALF in asthmatic rats, have statistical significance compared with control group, normal saline (P0.05) in vitro.5.PM2.5 concentration of 3.2-0.8mg/ml can affect the expression of M RNA and protein were up-regulated in RAW264.7 cells ICAM-1 and NF- kappa B, and the control group Compared with statistical significance (P0.05); high dose and middle dose of PM2.5 can increase the expression of M and RNA protein in lung tissue of ICAM-1 and NF- K B in asthmatic rats, there was statistical significance compared with control group, normal saline (P0.05); the expression of the high dose group of NF- kappa B protein higher than the low dose group, the difference was statistically significant (P0.05); however, PM2.5 high, low dose of M RNA and protein in the spleen tissue of asthmatic rats ICAM-1 and NF- K B expression had no significant effect, no statistical significance compared with the saline control group (P0.05). Conclusion: 1.PM2.5 airway pathological changes of rats with allergic asthma exacerbation, peripheral white blood cells and BALF EOS increased, showed that PM2.5 can aggravate asthma airway pathological state, promote inflammatory processes inhibit the proliferation of RAW264.7 cells.2.PM2.5 in vitro, RAW264.7 cells promote IL-1 beta and TNF- alpha, and IL-1 increased in asthmatic rats and blood BALF beta and TNF- alpha. , suggesting that PM2.5 on inflammatory cytokine secretion of proinflammatory mechanisms can promote the effect of.3.PM2.5 on allergic asthma may be through upregulation of NF- kappa B, increased secretion of inflammatory mediators in the airway, airway and upregulation of the expression of ICAM-1, promote the infiltration of inflammatory cells and play a role in.4. the results suggest that the concentration of PM2.5 can aggravate asthma symptoms and the airway pathological injury in 100ug/m3, the concentration of peripheral white blood cell count is above 200ug/m3, the total Ig EOS count and serum level of E was increased in BALF, IL-1 and TNF- alpha beta cell factor secretion increased, the lung tissue of NF- kappa B and increase the expression of ICAM-1; while the PM2.5 concentration is less than 300ug/m3 is not enough the whole body immune system caused by the increased expression of NF- kappa B and ICAM-1.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R725.6

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相關(guān)期刊論文 前1條

1 李輝;郭家秀;尹華強(qiáng);;PM_(2.5)對(duì)人體健康的影響研究進(jìn)展[J];四川化工;2013年01期

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本文編號(hào)：1552863