PLTP在香烟诱导A549细胞合成白介素-8中的作用

发布时间：2018-07-21 19:46

【摘要】：目的:香烟烟雾暴露是导致肺气道损伤的重要危险因素之一,而肺泡表面活性物质主要成分脂蛋白代谢紊乱与肺损伤的发生发展密切相关,香烟的吸入能使肺部作为表面活性物质重要成分的PLTP表达增加。然而PLTP与香烟烟雾所引起的肺损伤之间关系方面的研究尚未见报道。因此本研究主要探讨PLTP对香烟烟雾诱导人肺泡上皮细胞A549细胞合成IL-8的影响。方法:本实验采取整体实验及离体实验,第一部分采用SD大鼠连续三天被动吸烟诱导肺损伤模型,肺组织切片经HE染色观察被动吸烟所造成的肺损伤情况,免疫组化检测PLTP及IL-8表达情况,计算BALF中白细胞总数,分类计数中性粒细胞、巨噬细胞、淋巴细胞。第二部分探讨不同浓度CSE对人肺泡II型上皮细胞A549细胞增殖情况的影响,并探讨CSE诱导A549细胞合成IL-8的浓度及时间依赖效应。在检测CSE对A549细胞增殖情况影响的实验中,体外培养人肺泡II型上皮A549细胞株24h,加入0.125%、0.25%、0.5%、1.0%、2.0%、4.0%浓度CSE共培养24h,经MTT法检测A549细胞增殖情况;然后进入浓度效应实验,实验组加入0.25%、0.5%、1.0%浓度CSE刺激,孵育24小时后分别收集细胞及细胞培养上清液,经RT-PCR及ELISA分别检测IL-8 mRNA和IL-8蛋白表达情况,提取细胞总蛋白,通过Western blot测定pltp蛋白表达情况;在时间效应实验中,于实验组加入1.0%浓度cse,分别在6、12、24、48小时收集细胞培养上清液,经elisa法检测il-8含量。第三部分重点探讨pltp在香烟诱导a549细胞分泌il-8中的作用。实验组给予pltpsirna预处理,经cse刺激后收集上清液检测il-8蛋白表达情况,提取细胞总rna通过rt-pcr测定pltp及il-8mrna表达情况,提取细胞总蛋白通过westernblot测定pltp及il-8蛋白表达情况。结果:在第一部分实验中,3天香烟暴露之后烟熏组大鼠肺组织充血水肿;he染色显示正常肺组织肺泡结构完整正常,无炎症细胞浸润,而烟熏组血管腔充血,肺间质炎症细胞浸润,肺泡腔扩大断裂,肺泡融合;ihc结果显示,与对照组比较,烟熏组肺上皮细胞pltp和il-8表达升高;在balf中,烟熏组白细胞总数显著高于对照组,并且中性粒细胞、巨噬细胞、淋巴细胞计数均明显高于空白对照组。在第二部分实验中,在同一个时间点,a549细胞经cse刺激后合成的il-8均明显高于对照组,随着cse刺激浓度的增加,a549细胞合成il-8亦随之增加,当cse作用浓度为1.0%时,il-8的蛋白合成比对照组增加近2倍;而在时间依赖效应实验中,cse作用6小时后,细胞上清液中就可以检测到il-8的表达,a549细胞合成il-8的高峰出现在24小时。在第三部分实验中,沉默pltp基因不仅能在基础水平而且还能在cse共同作用a549细胞情况下促进il-8的合成。结论:香烟烟雾能够导致肺部急性炎症反应,并且可诱发肺组织合成pltp及il-8;cse可诱导人肺泡上皮细胞合成pltp及il-8,而PLTP基因缺失促进香烟诱导A549细胞合成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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563

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