PLTP在香煙誘導(dǎo)A549細胞合成白介素-8中的作用
發(fā)布時間：2018-07-21 19:46
【摘要】：目的:香煙煙霧暴露是導(dǎo)致肺氣道損傷的重要危險因素之一,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道。因此本研究主要探討PLTP對香煙煙霧誘導(dǎo)人肺泡上皮細胞A549細胞合成IL-8的影響。方法:本實驗采取整體實驗及離體實驗,第一部分采用SD大鼠連續(xù)三天被動吸煙誘導(dǎo)肺損傷模型,肺組織切片經(jīng)HE染色觀察被動吸煙所造成的肺損傷情況,免疫組化檢測PLTP及IL-8表達情況,計算BALF中白細胞總數(shù),分類計數(shù)中性粒細胞、巨噬細胞、淋巴細胞。第二部分探討不同濃度CSE對人肺泡II型上皮細胞A549細胞增殖情況的影響,并探討CSE誘導(dǎo)A549細胞合成IL-8的濃度及時間依賴效應(yīng)。在檢測CSE對A549細胞增殖情況影響的實驗中,體外培養(yǎng)人肺泡II型上皮A549細胞株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細胞增殖情況;然后進入濃度效應(yīng)實驗,實驗組加入0.25%、0.5%、1.0%濃度CSE刺激,孵育24小時后分別收集細胞及細胞培養(yǎng)上清液,經(jīng)RT-PCR及ELISA分別檢測IL-8 mRNA和IL-8蛋白表達情況,提取細胞總蛋白,通過Western blot測定pltp蛋白表達情況;在時間效應(yīng)實驗中,于實驗組加入1.0%濃度cse,分別在6、12、24、48小時收集細胞培養(yǎng)上清液,經(jīng)elisa法檢測il-8含量。第三部分重點探討pltp在香煙誘導(dǎo)a549細胞分泌il-8中的作用。實驗組給予pltpsirna預(yù)處理,經(jīng)cse刺激后收集上清液檢測il-8蛋白表達情況,提取細胞總rna通過rt-pcr測定pltp及il-8mrna表達情況,提取細胞總蛋白通過westernblot測定pltp及il-8蛋白表達情況。結(jié)果:在第一部分實驗中,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,無炎癥細胞浸潤,而煙熏組血管腔充血,肺間質(zhì)炎癥細胞浸潤,肺泡腔擴大斷裂,肺泡融合;ihc結(jié)果顯示,與對照組比較,煙熏組肺上皮細胞pltp和il-8表達升高;在balf中,煙熏組白細胞總數(shù)顯著高于對照組,并且中性粒細胞、巨噬細胞、淋巴細胞計數(shù)均明顯高于空白對照組。在第二部分實驗中,在同一個時間點,a549細胞經(jīng)cse刺激后合成的il-8均明顯高于對照組,隨著cse刺激濃度的增加,a549細胞合成il-8亦隨之增加,當cse作用濃度為1.0%時,il-8的蛋白合成比對照組增加近2倍;而在時間依賴效應(yīng)實驗中,cse作用6小時后,細胞上清液中就可以檢測到il-8的表達,a549細胞合成il-8的高峰出現(xiàn)在24小時。在第三部分實驗中,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細胞情況下促進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)人肺泡上皮細胞合成pltp及il-8,而PLTP基因缺失促進香煙誘導(dǎo)A549細胞合成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)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R563
[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)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R563
【相似文獻】
相關(guān)期刊論文 前10條
1 ;三種基因即可使人體細胞癌變[J];中國腫瘤;1999年12期
2 田曉川;線粒體DNA異常與細胞癌變的關(guān)系[J];重慶醫(yī)學(xué);2003年11期
3 小魯;德科學(xué)家觀察細胞彈性診斷癌癥[J];今日科苑;2005年04期
4 編輯部;;免疫細胞癌變信號通路研究獲進展[J];中國醫(yī)藥科學(xué);2012年13期
5 宋艾芝;放射線致細胞癌變的指標[J];日本醫(yī)學(xué)介紹;1992年07期
6 張建明,柯楊;細胞癌變的遺傳模型研究回顧與展望[J];國外醫(yī)學(xué).遺傳學(xué)分冊;1997年01期
7 陳壽東;張從福;;人體細胞癌變及其機理分析[J];新高考(物理化學(xué)生物);2009年10期
8 李欣;;細胞癌變“簡史” “流氓細胞”是怎么發(fā)展成癌癥的?[J];健康管理;2013年09期
9 胡義德,錢桂生,鄭頌國,曹世龍;線粒體DNA與細胞癌變關(guān)系研究進展[J];中國癌癥雜志;2000年01期
10 陸東東;線粒體DNA與細胞癌變關(guān)系的研究[J];陜西醫(yī)學(xué)檢驗;2002年01期
相關(guān)會議論文 前10條
1 朱紅梅;張大志;金生;任紅;張曉艷;湯為學(xué);;腫瘤細胞培養(yǎng)上清液誘導(dǎo)正常細胞癌變及可能機制探討[A];第五屆全國肝臟疾病臨床暨中華肝臟病雜志成立十周年學(xué)術(shù)會議論文匯編[C];2006年
2 時永香;蘇玲;邵明;;《細胞-一個和諧的社會》通識教育核心課程體系[A];“細胞活動 生命活力”——中國細胞生物學(xué)學(xué)會全體會員代表大會暨第十二次學(xué)術(shù)大會論文摘要集[C];2011年
3 高澤紅;于晶;林均岫;丁建華;;細胞癌變代謝產(chǎn)物的光譜檢測分析[A];中國光學(xué)學(xué)會2006年學(xué)術(shù)大會論文摘要集[C];2006年
4 穆云川;劉小孫;鄔一軍;鐘潔;于吉人;;端�？s短是胃粘膜細胞癌變的早期信號分子[A];2009年浙江省外科學(xué)學(xué)術(shù)年會論文匯編[C];2009年
5 趙W，
本文編號：2136663
本文鏈接：http://sikaile.net/yixuelunwen/huxijib/2136663.html
最近更新
教材專著
