【摘要】：目的:香煙煙霧暴露是導(dǎo)致肺氣道損傷的重要危險(xiǎn)因素之一,而肺泡表面活性物質(zhì)主要成分脂蛋白代謝紊亂與肺損傷的發(fā)生發(fā)展密切相關(guān),香煙的吸入能使肺部作為表面活性物質(zhì)重要成分的PLTP表達(dá)增加。然而PLTP與香煙煙霧所引起的肺損傷之間關(guān)系方面的研究尚未見報(bào)道。因此本研究主要探討PLTP對(duì)香煙煙霧誘導(dǎo)人肺泡上皮細(xì)胞A549細(xì)胞合成IL-8的影響。方法:本實(shí)驗(yàn)采取整體實(shí)驗(yàn)及離體實(shí)驗(yàn),第一部分采用SD大鼠連續(xù)三天被動(dòng)吸煙誘導(dǎo)肺損傷模型,肺組織切片經(jīng)HE染色觀察被動(dòng)吸煙所造成的肺損傷情況,免疫組化檢測PLTP及IL-8表達(dá)情況,計(jì)算BALF中白細(xì)胞總數(shù),分類計(jì)數(shù)中性粒細(xì)胞、巨噬細(xì)胞、淋巴細(xì)胞。第二部分探討不同濃度CSE對(duì)人肺泡II型上皮細(xì)胞A549細(xì)胞增殖情況的影響,并探討CSE誘導(dǎo)A549細(xì)胞合成IL-8的濃度及時(shí)間依賴效應(yīng)。在檢測CSE對(duì)A549細(xì)胞增殖情況影響的實(shí)驗(yàn)中,體外培養(yǎng)人肺泡II型上皮A549細(xì)胞株24h,加入0.125%、0.25%、0.5%、1.0%、2.0%、4.0%濃度CSE共培養(yǎng)24h,經(jīng)MTT法檢測A549細(xì)胞增殖情況;然后進(jìn)入濃度效應(yīng)實(shí)驗(yàn),實(shí)驗(yàn)組加入0.25%、0.5%、1.0%濃度CSE刺激,孵育24小時(shí)后分別收集細(xì)胞及細(xì)胞培養(yǎng)上清液,經(jīng)RT-PCR及ELISA分別檢測IL-8 mRNA和IL-8蛋白表達(dá)情況,提取細(xì)胞總蛋白,通過Western blot測定pltp蛋白表達(dá)情況;在時(shí)間效應(yīng)實(shí)驗(yàn)中,于實(shí)驗(yàn)組加入1.0%濃度cse,分別在6、12、24、48小時(shí)收集細(xì)胞培養(yǎng)上清液,經(jīng)elisa法檢測il-8含量。第三部分重點(diǎn)探討pltp在香煙誘導(dǎo)a549細(xì)胞分泌il-8中的作用。實(shí)驗(yàn)組給予pltpsirna預(yù)處理,經(jīng)cse刺激后收集上清液檢測il-8蛋白表達(dá)情況,提取細(xì)胞總rna通過rt-pcr測定pltp及il-8mrna表達(dá)情況,提取細(xì)胞總蛋白通過westernblot測定pltp及il-8蛋白表達(dá)情況。結(jié)果:在第一部分實(shí)驗(yàn)中,3天香煙暴露之后煙熏組大鼠肺組織充血水腫;he染色顯示正常肺組織肺泡結(jié)構(gòu)完整正常,無炎癥細(xì)胞浸潤,而煙熏組血管腔充血,肺間質(zhì)炎癥細(xì)胞浸潤,肺泡腔擴(kuò)大斷裂,肺泡融合;ihc結(jié)果顯示,與對(duì)照組比較,煙熏組肺上皮細(xì)胞pltp和il-8表達(dá)升高;在balf中,煙熏組白細(xì)胞總數(shù)顯著高于對(duì)照組,并且中性粒細(xì)胞、巨噬細(xì)胞、淋巴細(xì)胞計(jì)數(shù)均明顯高于空白對(duì)照組。在第二部分實(shí)驗(yàn)中,在同一個(gè)時(shí)間點(diǎn),a549細(xì)胞經(jīng)cse刺激后合成的il-8均明顯高于對(duì)照組,隨著cse刺激濃度的增加,a549細(xì)胞合成il-8亦隨之增加,當(dāng)cse作用濃度為1.0%時(shí),il-8的蛋白合成比對(duì)照組增加近2倍;而在時(shí)間依賴效應(yīng)實(shí)驗(yàn)中,cse作用6小時(shí)后,細(xì)胞上清液中就可以檢測到il-8的表達(dá),a549細(xì)胞合成il-8的高峰出現(xiàn)在24小時(shí)。在第三部分實(shí)驗(yàn)中,沉默pltp基因不僅能在基礎(chǔ)水平而且還能在cse共同作用a549細(xì)胞情況下促進(jìn)il-8的合成。結(jié)論:香煙煙霧能夠?qū)е路尾考毙匝装Y反應(yīng),并且可誘發(fā)肺組織合成pltp及il-8;cse可誘導(dǎo)人肺泡上皮細(xì)胞合成pltp及il-8,而PLTP基因缺失促進(jìn)香煙誘導(dǎo)A549細(xì)胞合成IL-8。
[Abstract]:Objective: cigarette smoke exposure is one of the important risk factors leading to pulmonary duct injury, and the disorder of lipoprotein metabolism, the main component of pulmonary surfactant, is closely related to the occurrence and development of lung injury. Cigarette inhalation can increase the expression of PLTP, which is an important component of surfactant in the lungs. However, the relationship between PLTP and lung injury caused by cigarette smoke has not been reported. Therefore, the effect of PLTP on the production of IL-8 in human alveolar epithelial cells A549 cells induced by cigarette smoke was investigated. Methods: the whole experiment and in vitro experiment were carried out. In the first part, the lung injury model was induced by passive smoking for three consecutive days in SD rats. The lung tissue sections were stained with HE staining to observe the lung injury caused by passive smoking. The expression of PLTP and IL-8 in BALF was determined by immunohistochemistry, and the neutrophils, macrophages and lymphocytes were classified and counted. The second part was to investigate the effect of CSE at different concentrations on the proliferation of human alveolar type II epithelial cell line A549, and to explore the concentration and time-dependent effect of CSE on the synthesis of IL-8 in A549 cells. In order to detect the effect of CSE on the proliferation of A549 cells, human alveolar type II epithelial A549 cell line was cultured in vitro for 24 h, and 0.125% 0.25% CSE was added to culture for 24 h. The proliferation of A549 cells was detected by MTT assay, and then entered the concentration effect experiment. The experimental group was stimulated with 1.0% CSE and incubated for 24 hours. The expression of IL-8 mRNA and IL-8 protein were detected by RT-PCR and Elisa, and the expression of pltp protein was detected by Western blot. In the time effect experiment, 1.0% cseine was added to the experimental group, and the supernatant of cell culture was collected after 48 hours at 6o 12 and 24 min, and the content of il-8 was detected by elisa method. The third part focuses on the role of pltp in the secretion of il-8 by cigarette-induced A549 cells. The experimental group was pretreated with pltpsirna, the supernatant was collected after cse stimulation to detect the expression of il-8 protein, the total rna was extracted by rt-pcr to detect the expression of pltp and il-8mrna, and the total cell protein was extracted to detect the expression of pltp and il-8 by westernblot. Results: in the first part of the experiment, after 3 days of cigarette exposure, the pulmonary tissue congestion and edema in the smoking group showed that the alveolar structure of the normal lung tissue was intact and normal, no inflammatory cells infiltrated, but the vascular cavity of the smoked group was congested. Pulmonary interstitial inflammatory cells infiltrated, alveolar cavity enlarged and ruptured. The results of alveolar fusion showed that the expression of pltp and il-8 in the lung epithelial cells in the fumigated group was higher than that in the control group, and the total number of white blood cells in the fumigated group was significantly higher than that in the control group. The counts of neutrophils, macrophages and lymphocytes were significantly higher than those in the control group. In the second part of the experiment, the il-8 synthesis of A549 cells stimulated by cse at the same time point was significantly higher than that of the control group, and the synthesis of il-8 was also increased with the increase of cse stimulation concentration. When the concentration of cse was 1.0, the protein synthesis of Isoil-8 was nearly twice as much as that of the control group, and the peak of il-8 synthesis in the cell supernatant was detected in the supernatant of A549 cells at 24 hours after 6 hours of treatment with CSE in the time-dependent effect experiment. In the third part of the experiment, the silencing of pltp gene can promote the synthesis of il-8 not only at the basic level but also in the condition of cse co-acting on A549 cells. Conclusion: cigarette smoke can induce acute pulmonary inflammation and induce lung tissue synthesis of pltp and il-8cse to induce the synthesis of pltp and il-8 in human alveolar epithelial cells, while the deletion of PLTP gene can promote the production of IL-8 in A549 cells induced by cigarette.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R563

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