本文選題：尼古丁 + 人氣道上皮細胞　； 參考：《廣州醫(yī)學院》2012年碩士論文

【摘要】：目的 吸煙是引起慢性阻塞性肺疾�。╟hronic obstructive pulmonarydisease,COPD）的主要原因。氣道重塑是COPD不可逆性氣流受限的主要原因。一般認為，COPD氣道重塑的機制與炎癥細胞活化、氧化抗氧化失衡、蛋白酶抗蛋白酶失衡、細胞凋亡等有關(guān)。最近，越來越多的研究表明上皮間質(zhì)轉(zhuǎn)分化（epithelial mesenchymal transdifferentiation,EMT）參與了氣道重塑的發(fā)生。氣道上皮作為抵御外來損傷因素的第一道防線，吸入的煙草煙霧進入人體最先直接接觸人體的氣道。尼古丁是香煙煙霧中的主要有害成分。尼古丁對氣道上皮的直接影響目前還不清楚。本研究首次采用基因芯片技術(shù)和雙向凝膠電泳技術(shù)觀察尼古丁刺激人氣道上皮細胞后差異基因和蛋白的表達情況，為進一步闡明氣道重塑機制提供線索。 方法 一、體外培養(yǎng)原代人小氣道上皮細胞，待原代人氣道上皮細胞生長至80%融合時，換用無生長因子的基礎(chǔ)培養(yǎng)基培養(yǎng)24小時，使細胞同步于靜止期。將細胞隨機分成兩組，每組再分成兩個平行組，即尼古丁刺激4小時組和空白對照組，，尼古丁刺激48小時組和空白對照組。實驗組加入尼古�。�1×10-5M），對照組加入PBS，分別孵育4小時和48小時后吸走，加入Trizol試劑，收集細胞用于基因芯片檢測。 二、根據(jù)基因芯片結(jié)果，選取部分表達差異基因用熒光定量PCR方法進行驗證。 三、體外培養(yǎng)人支氣管上皮細胞16HBE,待16HBE細胞生長至80%融合時，換含1%的胎牛血清的DMEM培養(yǎng)基培養(yǎng)24小時，使細胞同步于靜止期。采用隨機化的原則進行分組：（1）對照組，加入PBS；（2）實驗組，加入尼古�。ńK濃度1×10~（-5M）），孵育72小時后加入裂解液收集細胞用于雙向凝膠電泳。通過雙向凝膠電泳實驗分離總蛋白，差異表達蛋白質(zhì)點應(yīng)用基質(zhì)輔助激光解析電離飛行時間質(zhì)譜獲取肽質(zhì)量指紋圖譜并通過NCBI數(shù)據(jù)庫鑒定。 結(jié)果 一、基因芯片結(jié)果 （1）尼古丁處理4h后，表達上調(diào)的基因有63個，表達下調(diào)的基因有44個，主要涉及細胞對外界刺激的應(yīng)激反應(yīng)有關(guān)基因。 （2）尼古丁處理48h后，表達上調(diào)的基因有860個，表達下調(diào)的基因有582個。對差異基因進行Gene GO分析和Pathway分析，發(fā)現(xiàn)差異基因涉及到胚胎發(fā)育、細胞極性維持、細胞與細胞黏附等與細胞轉(zhuǎn)分化相關(guān)的重要生理過程和信號轉(zhuǎn)導(dǎo)通路。 （3）進一步對個別基因進行分析，發(fā)現(xiàn)尼古丁可以下調(diào)上皮角蛋白（CK）、上皮黏蛋白（MUC）等具有上皮特征的基因；上調(diào)纖維連接蛋白1（FN1）、N鈣粘蛋白（N-cadherin）等具有間質(zhì)細胞特征的基因。 二、熒光定量PCR結(jié)果熒光定量PCR驗證的基因差異表達與基因芯片結(jié)果變化趨勢一致。 三、雙向凝膠電泳結(jié)果雙向凝膠電泳圖譜中找到23個表達量有明顯差異的蛋白質(zhì)點，質(zhì)譜鑒定出19種蛋白質(zhì)。其中與細胞骨架相關(guān)的蛋白明顯下調(diào)，如微管蛋白、γ_1-肌動蛋白等。 結(jié)論 不論從基因組水平還是從蛋白組水平，均發(fā)現(xiàn)一系列與EMT發(fā)生過程有關(guān)的基因或蛋白的改變。
[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.

【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R563.9

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