尼古丁对人气道上皮基因组和蛋白组学的影响

发布时间：2018-05-08 12:46

本文选题：尼古丁 + 人气道上皮细胞　；参考：《广州医学院》2012年硕士论文

【摘要】：目的吸烟是引起慢性阻塞性肺疾病（chronic obstructive pulmonarydisease,COPD）的主要原因。气道重塑是COPD不可逆性气流受限的主要原因。一般认为，COPD气道重塑的机制与炎症细胞活化、氧化抗氧化失衡、蛋白酶抗蛋白酶失衡、细胞凋亡等有关。最近，越来越多的研究表明上皮间质转分化（epithelial mesenchymal transdifferentiation,EMT）参与了气道重塑的发生。气道上皮作为抵御外来损伤因素的第一道防线，吸入的烟草烟雾进入人体最先直接接触人体的气道。尼古丁是香烟烟雾中的主要有害成分。尼古丁对气道上皮的直接影响目前还不清楚。本研究首次采用基因芯片技术和双向凝胶电泳技术观察尼古丁刺激人气道上皮细胞后差异基因和蛋白的表达情况，为进一步阐明气道重塑机制提供线索。方法一、体外培养原代人小气道上皮细胞，待原代人气道上皮细胞生长至80%融合时，换用无生长因子的基础培养基培养24小时，使细胞同步于静止期。将细胞随机分成两组，每组再分成两个平行组，即尼古丁刺激4小时组和空白对照组，，尼古丁刺激48小时组和空白对照组。实验组加入尼古丁（1×10-5M），对照组加入PBS，分别孵育4小时和48小时后吸走，加入Trizol试剂，收集细胞用于基因芯片检测。二、根据基因芯片结果，选取部分表达差异基因用荧光定量PCR方法进行验证。三、体外培养人支气管上皮细胞16HBE,待16HBE细胞生长至80%融合时，换含1%的胎牛血清的DMEM培养基培养24小时，使细胞同步于静止期。采用随机化的原则进行分组：（1）对照组，加入PBS；（2）实验组，加入尼古丁（终浓度1×10~（-5M）），孵育72小时后加入裂解液收集细胞用于双向凝胶电泳。通过双向凝胶电泳实验分离总蛋白，差异表达蛋白质点应用基质辅助激光解析电离飞行时间质谱获取肽质量指纹图谱并通过NCBI数据库鉴定。结果一、基因芯片结果（1）尼古丁处理4h后，表达上调的基因有63个，表达下调的基因有44个，主要涉及细胞对外界刺激的应激反应有关基因。（2）尼古丁处理48h后，表达上调的基因有860个，表达下调的基因有582个。对差异基因进行Gene GO分析和Pathway分析，发现差异基因涉及到胚胎发育、细胞极性维持、细胞与细胞黏附等与细胞转分化相关的重要生理过程和信号转导通路。（3）进一步对个别基因进行分析，发现尼古丁可以下调上皮角蛋白（CK）、上皮黏蛋白（MUC）等具有上皮特征的基因；上调纤维连接蛋白1（FN1）、N钙粘蛋白（N-cadherin）等具有间质细胞特征的基因。二、荧光定量PCR结果荧光定量PCR验证的基因差异表达与基因芯片结果变化趋势一致。三、双向凝胶电泳结果双向凝胶电泳图谱中找到23个表达量有明显差异的蛋白质点，质谱鉴定出19种蛋白质。其中与细胞骨架相关的蛋白明显下调，如微管蛋白、γ_1-肌动蛋白等。结论不论从基因组水平还是从蛋白组水平，均发现一系列与EMT发生过程有关的基因或蛋白的改变。
[Abstract]:objective
Smoking is the main cause of chronic obstructive pulmonary disease (chronic obstructive pulmonarydisease, COPD). Airway remodeling is the main reason for the irreversible airflow limitation of COPD. It is generally believed that the mechanism of COPD airway remodeling is related to inflammatory cell activation, oxidation antioxidant imbalance, egg white enzyme anti protease imbalance, cell apoptosis and so on. The more studies have shown that epithelial mesenchymal transdifferentiation (EMT) is involved in the development of airway remodeling. The airway epithelium is the first line of defense against foreign injury factors, and inhaled tobacco smoke enters the body first directly to the human body's airway. Nicotine is the major harmful effect of cigarette smoke. The direct effect of nicotine on the airway epithelium is not clear. This study is the first to observe the expression of different genes and proteins after nicotine stimulation of human airway epithelial cells by gene chip technology and two-dimensional gel electrophoresis, which provides clues for further clarifying the mechanism of airway remodeling.
Method
First, the primary cultured human small airway epithelial cells were cultured in vitro. When the primary human airway epithelial cells grew to 80% fusion, the cells were cultured for 24 hours with no growth factor, and the cells were synchronized to the stationary phase. The cells were randomly divided into two groups, each group was then divided into two parallel groups, that is, nicotine stimulation for 4 hours and blank control, nicotine spines. The 48 hour group and the blank control group were stimulated. The experimental group was added to nicotine (1 x 10-5M), and the control group was added to PBS. After incubation for 4 hours and 48 hours respectively, the group was added to the Trizol reagent, and the cells were collected for gene chip detection.
Two, according to the results of gene chip, some differentially expressed genes were selected and validated by fluorescence quantitative PCR.
Three, the human bronchial epithelial cell 16HBE was cultured in vitro. When 16HBE cells grew to 80% fusion, the DMEM medium containing 1% fetal bovine serum was cultured for 24 hours, and the cells were synchronized at the stationary phase. (1) the control group, adding PBS, (2) the experimental group, added nicotine (final concentration 1 * 10~ (-5M)), and incubated for 72 hours after incubation. The cells in the lysate were collected for two-dimensional gel electrophoresis. The total protein was separated by two-dimensional gel electrophoresis. The differential protein points were used to obtain peptide mass fingerprints by matrix assisted laser analytical ionization time of flight mass spectrometry and identified by NCBI database.
Result
1. Results of gene chip
(1) after nicotine treatment of 4h, 63 genes were up-regulated, and 44 genes were down regulated.
(2) after nicotine was treated with 48h, there were 860 up-regulated genes and 582 down-regulated genes. The difference genes were analyzed by Gene GO and Pathway analysis. The difference genes involved important physiological processes and signal transduction pathways related to embryo development, cell polarity maintenance, cell adhesion and cell adhesion.
(3) further analysis of individual genes shows that nicotine can down regulate epithelial keratin (CK), epithelial mucin (MUC) and other epithelial genes; up - regulation of fibronectin 1 (FN1), N cadherin (N-cadherin), and so on, which have the characteristics of interstitial cells.
Two, fluorescent quantitative PCR results showed that the differential expression of genes verified by fluorescent quantitative PCR was consistent with the results of gene chip.
Three, in bi-directional gel electrophoresis, 23 proteins with distinct differences were found in bi-directional gel electrophoresis, and 19 proteins were identified by mass spectrometry. The proteins associated with cytoskeleton were obviously downregulated, such as microtubulin, gamma _1- actin and so on.
conclusion
A series of changes in gene or protein related to the occurrence of EMT were found at both genomic level and proteome level.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9

【参考文献】