PM2.5对哮喘大鼠气道炎症的影响及机制研究

发布时间：2018-03-01 17:25

本文关键词： PM2.5 哮喘炎症反应 ICAM-1 NF-κB　出处：《天津医科大学》2015年博士论文　论文类型：学位论文

【摘要】：目的:研究细颗粒物PM2.5对哮喘模型大鼠气道炎症的影响,观察PM2.5对NF-κB和ICAM-1表达的调控,进而探讨PM2.5促进炎症的可能机制。方法:1.使用PM采样器收集空气中PM2.5样本,样本洗脱后过滤,真空冷冻干燥后,用磷酸盐缓冲溶液配制PM2.5悬液。2.50只Wistar大鼠随机分组,共计5组:PM2.5高剂量组(0.24mg/次)、PM2.5中剂量组(0.16mg/次)、PM2.5低剂量组(0.08mg/次)、生理盐水对照组和正常对照组。模型大鼠予卵蛋白皮下和腹腔注射两次致敏,14天后再行卵蛋白雾化吸入连续14天,建立大鼠过敏性哮喘模型。从雾化开始第1天,气管注入PM2.5悬液,每三天一次,共计5次,观察各组大鼠哮喘发作表现。雾化结束2天后取材,进行外周血白细胞计数和BALF嗜酸性粒细胞(EOS)计数,HE染色观察气道病理改变,ELISA试剂盒检测血清总Ig E。3.应用MTS法观察PM2.5对RAW264.7细胞增殖的影响。RAW264.7细胞接种于培养板,8个复孔。分别加入PM2.5悬液终浓度为3.2、1.6、0.8、0.4、0.2mg/ml,培养24h后,4个复孔MTS法检测OD值观察不同浓度PM2.5对RAW264.7细胞增殖的影响。同时,提取另外4个复孔培养液中的上清液,用ELISA方法检测IL-1β和TNF-α浓度。ELISA方法检测PM2.5对哮喘大鼠血清和BALF中IL-1β和TNF-α浓度的影响。4.应用RT-PCR(实时荧光定量PCR)和Western Blot方法,观察PM2.5体外浓度为3.2、1.6、0.8mg/ml时对RAW264.7细胞ICAM-1和NF-κB的m RNA和蛋白表达的影响。应用实时荧光定量PCR和Western Blot方法检测哮喘模型大鼠肺组织和脾组织,观察PM2.5的体内作用对ICAM-1和NF-κB的m RNA和蛋白表达的影响。结果:1.PM2.5收集共计2.5g,磷酸盐缓冲溶液配置悬液100mg/ml,-20℃冷冻保存备用。2.成功建立大鼠哮喘模型。3.PM2.5高、中、低剂量(0.24、0.16、0.08mg/次)能够加重哮喘大鼠的哮喘发作症状,PM2.5高剂量和中剂量能够明显增加外周血白细胞总数和BALF中EOS数量,与生理盐水对照组相比有统计学意义(P0.05),PM2.5高、中剂量组之间比较,没有统计学意义(P0.05);PM2.5高、中、低剂量能够增加气道内EOS、中性粒细胞和巨噬细胞等炎性细胞浸润,促进杯状细胞增生,促进小支气管粘膜水肿等病理改变。PM2.5高剂量和中剂量能够增加哮喘大鼠血清中总Ig E含量,与生理盐水对照组相比有统计学意义(P0.05),高、中剂量组之间比较,没有统计学意义(P0.05)。4.PM2.5体外浓度为3.2-0.8 mg/ml能够抑制RAW264.7细胞增殖,与对照组相比有统计学意义(P0.05),抑制率为43.06%-22.89%。PM2.5体外浓度为3.2-0.4 mg/ml能够促进RAW264.7细胞IL-1β和TNF-α的产生,与生理盐水对照组相比有统计学意义(P0.05);PM2.5高剂量和中剂量能够增加哮喘大鼠血清和BALF中IL-1β和TNF-α浓度,与生理盐水对照组相比有统计学意义(P0.05)。5.PM2.5体外浓度为3.2-0.8mg/ml能够上调RAW264.7细胞ICAM-1和NF-κB的m RNA和蛋白表达的影响,与对照组相比有统计学意义(P0.05);PM2.5高剂量和中剂量能够增加哮喘大鼠肺组织ICAM-1和NF-κB的m RNA和蛋白表达,与生理盐水对照组相比有统计学意义(P0.05);高剂量组NF-κB蛋白表达高于低剂量组,差别有统计学意义(P0.05);然而,PM2.5高、中、低剂量对哮喘大鼠脾组织ICAM-1和NF-κB的m RNA和蛋白表达没有明显影响,与生理盐水对照组相比无统计学意义(P0.05)。结论:1.PM2.5使过敏性哮喘大鼠的气道病理改变加重、外周血白细胞和BALF中EOS增高,表明PM2.5可加重哮喘气道的病理状态,促进炎症过程。2.PM2.5体外抑制RAW264.7细胞增殖,促进RAW264.7细胞IL-1β和TNF-α的产生,以及增加哮喘大鼠血清和BALF中IL-1β和TNF-α浓度,提示PM2.5对炎症细胞因子的分泌具有促进作用。3.PM2.5对过敏性哮喘的促炎症机制之一可能是通过上调NF-κB,增加气道炎症因子的分泌,进而上调气道ICAM-1的表达,促进炎细胞浸润而发挥作用。4.实验结果提示,PM2.5浓度在100ug/m3时即可加重哮喘症状及气道病理损伤,浓度在200ug/m3以上时可以使外周血白细胞计数、BALF中EOS计数和血清总Ig E水平明显升高,细胞因子IL-1β和TNF-α分泌增加,使肺组织NF-κB和ICAM-1表达增加;而PM2.5浓度在300ug/m3以内时不足以引起机体整体免疫系统NF-κB和ICAM-1表达的上升。
[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.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.6

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