本文關(guān)鍵詞：尼古丁在吸煙導(dǎo)致氣道慢性炎癥中的獨(dú)特效應(yīng)　出處：《重慶醫(yī)科大學(xué)》2010年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 吸煙 尼古丁 炎性因子 黏蛋白類(lèi) 氣道上皮細(xì)胞

【摘要】： 目的:比較單純尼古丁與煙草抽提物對(duì)培養(yǎng)氣道上皮細(xì)胞的效應(yīng)特點(diǎn),并進(jìn)一步了解經(jīng)香煙和尼古丁誘導(dǎo)后的改變規(guī)律,以及相關(guān)炎性因子表達(dá)的誘導(dǎo)效應(yīng)差異,明確尼古丁在致炎抗炎及黏液高分泌方面的功過(guò)地位;同時(shí)明確尼古丁作用的膜受體在正常氣道的分布規(guī)律及表型;并進(jìn)一步了解尼古丁可能存在的抗炎效應(yīng)的分子機(jī)制及相關(guān)信號(hào)轉(zhuǎn)導(dǎo)途經(jīng)。為香煙成份改良提供努力方向,為尼古丁替代療法提供理論依據(jù),同時(shí)幫助完善對(duì)尼古丁外周作用的認(rèn)識(shí),有助于進(jìn)一步拓展尼古丁的有益使用范圍。 方法:(1)體外培養(yǎng)人氣道上皮細(xì)胞系HBE16細(xì)胞,予以不同濃度的香煙氯仿抽提物(CE)、尼古丁刺激,選取最佳作用濃度。觀察刺激前后細(xì)胞的生長(zhǎng)和增殖情況,采用ELISA技術(shù)測(cè)定各組細(xì)胞經(jīng)CE、尼古丁、脂多糖(LPS)刺激后培養(yǎng)上清中前炎性因子腫瘤壞死因子(TNF)-α、白介素(IL)-8、IL-6及黏蛋白(MUC)5AC蛋白相對(duì)含量,real-time PCR檢測(cè)細(xì)胞中上述因子的轉(zhuǎn)錄水平變化情況,免疫熒光化學(xué)法觀察細(xì)胞中MUC5AC的表達(dá)情況。(2)以人腦膠質(zhì)瘤細(xì)胞株U251細(xì)胞為陽(yáng)性對(duì)照,分別設(shè)立CE組、尼古丁組,采用Western印跡分析及real-time PCR檢測(cè)各細(xì)胞組中煙堿型乙酰膽堿受體(nAChRs)各亞型的表達(dá)情況,并采用相關(guān)亞型特異性siRNA轉(zhuǎn)染細(xì)胞,ELISA檢測(cè)轉(zhuǎn)染前后細(xì)胞培養(yǎng)上清中TNF-α、IL-8、IL-6及MUC5AC蛋白水平的變化,real-time PCR檢測(cè)上述因子在基因轉(zhuǎn)錄水平的變化情況。(3)進(jìn)一步將細(xì)胞分為CE組、LPS組、尼古丁+CE組、尼古丁+LPS組,尼古丁+CE+α-BTX組,尼古丁+LPS+α-BTX組,以Western印跡分析各處理組細(xì)胞中磷酸化I-κBα、I-κBα蛋白的表達(dá)情況、細(xì)胞核中核轉(zhuǎn)錄因子(NF)-κBp65的蛋白含量;并采用pNF-κB-Luc熒光素酶報(bào)告基因質(zhì)粒轉(zhuǎn)染法以檢測(cè)各組中NF-κB的活性;檢測(cè)加入NF-κB抑制劑PDTC后對(duì)細(xì)胞TNF-α、IL-8、IL-6的蛋白及基因表達(dá)水平的影響情況;同時(shí)檢測(cè)了各組細(xì)胞中細(xì)胞外信號(hào)激酶1/2(ERK1/2)、c-Jun氨基末端激酶(JNK)及p38絲裂原活化蛋白激酶(MAPK)的磷酸化蛋白表達(dá)情況,并觀察細(xì)胞經(jīng)單純CE及尼古丁刺激后不同時(shí)點(diǎn)上述MAPK信號(hào)因子的表達(dá)及變化情況。 結(jié)果:(1)分別選取100μg/mL的CE及20μM的尼古丁為最適作用濃度,LPS的作用濃度為10μg/mL,分為不同組孵育細(xì)胞。檢測(cè)到CE、LPS刺激后細(xì)胞中TNF-α、IL-8、IL-6、MUC5ACmRNA相對(duì)含量及培養(yǎng)液上清中TNF-α、IL-8、IL-6、MUC5AC蛋白相對(duì)含量均有明顯增加,與對(duì)照組相比,差別有統(tǒng)計(jì)學(xué)意義;而給予尼古丁孵育的細(xì)胞上述指標(biāo)與正常對(duì)照組相比,并未見(jiàn)明顯升高,差別無(wú)統(tǒng)計(jì)學(xué)意義;免疫熒光檢測(cè)到MUC5AC在胞漿中的表達(dá)也較CE、LPS組弱;當(dāng)尼古丁分別與CE、LPS共同作用細(xì)胞后,檢測(cè)到兩組細(xì)胞培養(yǎng)上清液中TNF-α、IL-8、IL-6蛋白及細(xì)胞中mRNA的相對(duì)含量較CE、LPS單獨(dú)孵育組均有顯著下降,P0.05;尼古丁與CE、尼古丁與LPS共同孵育組中上清MUC5AC的蛋白分泌水平也有明顯下降,P0.05,差別有統(tǒng)計(jì)學(xué)意義,但兩組細(xì)胞中MUC5AC mRNA表達(dá)與單獨(dú)CE組、LPS組相比,并未有明顯下降。(2)培養(yǎng)的人HBE16氣道上皮細(xì)胞中,α1、α5、α7、β2 nAChR mRNA均有明顯表達(dá),尼古丁刺激組中上述指標(biāo)的表達(dá)高于正常對(duì)照組及CE刺激組;正常組HBE16細(xì)胞、經(jīng)CE及尼古丁刺激后的HBE16細(xì)胞,均未見(jiàn)α2、α4、β1 nAChR mRNA表達(dá);經(jīng)尼古丁刺激后,見(jiàn)少量α3 nAChR mRNA表達(dá)。細(xì)胞中α1、α5、α7、β2 nAChR蛋白均有明顯表達(dá),尼古丁刺激組表達(dá)的量多于未予刺激的HBE16細(xì)胞組;未予尼古丁刺激的HBE16細(xì)胞組見(jiàn)微弱的α2、α3、α4蛋白表達(dá),而經(jīng)尼古丁刺激后,上述的蛋白表達(dá)并不明顯,兩組細(xì)胞均未見(jiàn)有明確的β1蛋白表達(dá)。細(xì)胞轉(zhuǎn)染α1 nAchR siRNA、α5nAchR siRNA后再予以尼古丁及LPS處理,檢測(cè)到培養(yǎng)上清中TNF-α、IL-8、IL-6蛋白相對(duì)含量及細(xì)胞中mRNA表達(dá)水平與尼古丁及LPS共同孵育組中相比,差別無(wú)統(tǒng)計(jì)學(xué)意義,而α7 nAchR siRNA轉(zhuǎn)染的細(xì)胞組經(jīng)尼古丁及LPS刺激后,培養(yǎng)上清中上述蛋白的相對(duì)含量及細(xì)胞中mRNA相對(duì)含量與LPS、尼古丁共同刺激組相比,有明顯升高。(3)CE及LPS單獨(dú)刺激后,磷酸化-I-κBα蛋白及細(xì)胞核中NF-κBp65蛋白均有顯著增加,與正常對(duì)照組相比,P0.01;加入尼古丁與CE及LPS共同孵育的細(xì)胞組中,磷酸化-I-κBα及NF-κBp65均出現(xiàn)明顯降低,與單獨(dú)CE、LPS孵育組相比,差別有統(tǒng)計(jì)學(xué)意義,P0.01;I-κBα蛋白的含量未見(jiàn)明顯變化;尼古丁+CE+α-BTX組、尼古丁+LPS+α-BTX組中磷酸化-I-κBα蛋白及NF-κBp65蛋白的含量與尼古丁+CE組、尼古丁+LPS組相比有明顯增加。轉(zhuǎn)染熒光素酶報(bào)告質(zhì)粒pNF-kB-luc后觀察到,尼古丁+CE組、尼古丁+LPS組中的熒光強(qiáng)度值小于CE組、LPS組,差別有統(tǒng)計(jì)學(xué)意義,P0.01,尼古丁+CE+α-BTX組、尼古丁+LPS+α-BTX組中的熒光強(qiáng)度值出現(xiàn)增加,與尼古丁+CE組、尼古丁+LPS組相比,P0.01。進(jìn)一步PDTC預(yù)處理細(xì)胞30min后,再分別予以CE、LPS刺激,細(xì)胞培養(yǎng)上清中TNF-α、IL-8、IL-6蛋白相對(duì)含量及細(xì)胞mRAN水平較與單純CE刺激組、單純LPS刺激組相比均有明顯下降,P0.01;尼古丁+CE組、尼古丁+LPS組中上述因子的蛋白相對(duì)含量及mRNA相對(duì)含量也較單純CE組、單純LPS組有明顯下降;將細(xì)胞以PDTC預(yù)處理后,再分別施以尼古丁+CE、尼古丁+LPS刺激,細(xì)胞培養(yǎng)上清中的TNF-α、IL-8、IL-6蛋白相對(duì)含量下降更為明顯,與單純CE刺激組、單純LPS刺激組相比,P0.01。細(xì)胞經(jīng)CE及LPS刺激后,可觀察到細(xì)胞中磷酸化p38 MAPK、ERK1/2、JNK蛋白表達(dá)增多,與對(duì)照組相比,P0.01;加入尼古丁孵育的細(xì)胞,再分別經(jīng)CE及LPS刺激,檢測(cè)到細(xì)胞中磷酸化ERK1/2蛋白的相對(duì)含量顯著降低,分別為0.34±0.07、0.39±0.08,與單純CE組(0.74±0.12)、LPS組(0.79±0.13)相比,差別有統(tǒng)計(jì)學(xué)意義,P0.01;而p38及JNK磷酸化蛋白未見(jiàn)有明顯減少。加入α7 nAChR特異性抑制劑α-BTX的尼古丁+CE及尼古丁+LPS組中,磷酸化ERK1/2蛋白的相對(duì)含量分別為0.59±0.11,0.63±0.13,與未加α-BTX刺激的尼古丁+CE組、尼古丁+ LPS組相比,差別有統(tǒng)計(jì)學(xué)意義,P0.01;但同樣對(duì)p38及JNK磷酸化蛋白的影響不大。進(jìn)一步觀察到HBE16細(xì)胞經(jīng)CE處理后,p38MAPK、pERK1/2及pJNK蛋白的表達(dá)隨時(shí)間延長(zhǎng)而增強(qiáng),6h時(shí)間點(diǎn)的表達(dá)水平明顯高于30min時(shí)的表達(dá)水平,P0.01;經(jīng)尼古丁孵育的細(xì)胞,p38MAPK在作用30min后有表達(dá),隨著時(shí)間延長(zhǎng)表達(dá)逐漸降低,pERK1/2在各個(gè)時(shí)間點(diǎn)均未見(jiàn)明顯表達(dá),pJNK在第30min開(kāi)始表達(dá),2h作用表達(dá)最強(qiáng),在第6h表達(dá)開(kāi)始降低,與2h時(shí)間點(diǎn)的表達(dá)量相比,差別有統(tǒng)計(jì)學(xué)意義,P0.01。 結(jié)論:(1)尼古丁在香煙所致氣道黏液高分泌環(huán)節(jié)中并無(wú)過(guò)多正向效應(yīng),相反,其可能通過(guò)減少該過(guò)程中致炎因子的產(chǎn)生,抑制后續(xù)過(guò)度炎癥反應(yīng)。(2)尼古丁能減少前炎性因子及重要炎性趨化因子TNF-α、IL-8、IL-6蛋白等的產(chǎn)生,具有一定的抗炎功能,且證實(shí)系通過(guò)與α7 nAChR結(jié)合起降低I-κBα磷酸化的作用,從而抑制核轉(zhuǎn)錄因子NF-κB核轉(zhuǎn)位,發(fā)揮抑制上述因子表達(dá)的效應(yīng);該過(guò)程也可能系尼古丁通過(guò)減少ERK1/2活化水平而實(shí)現(xiàn)。(3)尼古丁不增加黏蛋白MUC5AC的基因轉(zhuǎn)錄及合成,且能使炎性刺激所生成的多余黏液外分泌相對(duì)減少,從而維持氣道在慢性炎性刺激下黏液的高潴留狀態(tài),形成氣道黏液生成與高分泌的相對(duì)穩(wěn)態(tài)。
[Abstract]:Objective: To compare effect of nicotine and tobacco extract on cultured airway epithelial cells, and further understand the changes of cigarettes and nicotine induced, and the expression of inflammatory factors induced by different effects, clear nicotine in inflammation and mucus secretion of anti-inflammatory and high status; and clear the distribution and phenotype of membrane effect of nicotine receptors in the normal airway; and further understanding of the molecular mechanisms underlying the anti-inflammatory effects of nicotine may exist and the related signal transduction pathway. To provide direction for improvement of cigarette components, to provide a theoretical basis for nicotine replacement therapy, while helping to improve the understanding of the peripheral effects of nicotine, help to further expand the scope of beneficial use of nicotine.
Methods: (1) HBE16 cells in human airway epithelial cells cultured in vitro, the cigarette to the chloroform extract of different concentration (CE), nicotine stimulation, selection of optimal concentration. The observation before and after stimulation of cell growth and proliferation, determination of nicotine by CE, cells were used ELISA technology, lipopolysaccharide (LPS) of the tumor in the supernatant of cultured inflammatory necrosis factor stimulation (TNF) - alpha, interleukin (IL) -8, IL-6 and mucin5ac (MUC) the relative content of 5AC protein, changes in transcription of the real-time factor of PCR were detected by immunofluorescence, photochemical method to observe the expression of MUC5AC in cells. (2) in human brain glioma tumor cell line U251 cells were set as positive control, CE group, nicotine group, by Western blot analysis and real-time PCR were detected by groups of nicotinic acetylcholine receptor (nAChRs) expression of the subtypes, and the subtype specific SiRNA transfected cells, and ELISA detection of transfected cell culture supernatant of TNF- alpha, IL-8, changes of IL-6 and MUC5AC protein levels, real-time PCR to detect the changes in gene transcription factor. (3) the cells were divided into CE group, LPS group, +CE group, +LPS group, nicotine, nicotine, nicotine +CE+ alpha -BTX group, -BTX group with nicotine +LPS+ alpha, Western blot analysis of each group of cells in phosphate I- kappa B alpha, expression of I- kappa B alpha protein, cell nuclear transcription factor (NF) - kappa Bp65 protein content; and the use of pNF- kappa B-Luc luciferase reporter gene plasmid transfection method was used to detect NF- kappa B activity detection; adding NF- kappa B inhibitor PDTC on the expression of TNF- alpha, IL-8, gene and protein expression level of IL-6 effect; also detected while cell in extracellular signal kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 mitogen The phosphorylation protein expression of activated protein kinase (MAPK) was observed, and the expression and change of the above MAPK signal factors were observed at different times after CE and nicotine stimulation.
Results: (1) nicotine were selected 100 g/mL CE and 20 M is the most suitable concentration, the concentration of LPS is 10 g/mL, divided into different groups. The cells were incubated to detect CE, TNF- alpha, LPS stimulation in cells after IL-8, IL-6, MUC5ACmRNA relative content and culture of TNF- alpha supernatant, IL-8, IL-6, MUC5AC protein content was increased significantly, compared with the control group, the difference was statistically significant; and given the above indexes nicotine incubation cells compared with normal control group, and there was no obvious increase, the difference was not statistically significant; immunofluorescence detected the expression of MUC5AC in cytoplasm was CE, group LPS and CE were weak; when nicotine, LPS cells, detected two groups of cell culture supernatant of TNF- alpha, IL-8, mRNA and relative content of IL-6 protein in cells with CE, LPS alone incubation group were significantly decreased, P0.05 and CE; nicotine, nicotine and LPS were incubated together Group in the supernatant of MUC5AC protein secretion level was decreased, P0.05, the difference was statistically significant, but the two groups of cells in the MUC5AC expression of mRNA and CE alone group, LPS group, did not significantly decrease. (2) in cultured human airway epithelial cells HBE16, alpha 1, alpha 5, alpha 7 beta. 2 nAChR mRNA showed obvious expression, expression of the index of nicotine stimulation was higher than in the normal control group and CE stimulation group; normal group of HBE16 cells by CE and nicotine stimulated HBE16 cells showed no expression of alpha 2, alpha 4, beta 1 nAChR mRNA; after nicotine stimulation, a few expression of 3 nAChR mRNA. Cells in alpha 1, alpha 5, alpha 7 beta 2, nAChR protein expression was evident in HBE16 cell group nicotine stimulation group was much higher than the expression does not stimulate the cells in HBE16 group; not the weak nicotine stimulation of alpha 2, alpha 3, alpha 4 expression, but after nicotine stimulation, the the expression is not obvious, two There is no clear cell beta 1 protein expression in cells transfected with nAchR siRNA. Alpha 1, alpha 5nAchR after siRNA and LPS treatment to nicotine, detected in culture supernatant of IL-8, TNF- alpha, compared with nicotine and LPS levels were incubated in group expression of mRNA relative content and cell IL-6 protein, no difference statistical significance, and the cell group alpha 7 nAchR siRNA transfected by LPS and nicotine stimulation, LPS and training of the relative content of the relative content of mRNA and the cell protein in the supernatant, nicotine stimulation groups had significantly increased. (3) CE and LPS alone stimulated the phosphorylation of both NF- kappa Bp65 protein -I- kappa B alpha protein and the nucleus increased significantly, compared with normal control group, P0.01 group and CE cells; adding nicotine and LPS were incubated in the phosphorylation of -I- kappa B alpha and NF- K Bp65 were significantly reduced, and CE alone, LPS incubation group compared, there was statistical difference Significance of P0.01; no significant changes in the content of I- kappa B alpha protein +CE+ alpha -BTX group; nicotine, nicotine +LPS+ alpha -BTX group phosphorylated -I- kappa B alpha kappa Bp65 protein and NF- protein content of nicotine and nicotine in +CE group, compared with the +LPS group increased significantly. Transfection of luciferase reporter plasmid pNF-kB-luc was observed after nicotine in group +CE, the fluorescence intensity of nicotine in the +LPS group was lower than CE group, LPS group, a statistically significant difference between P0.01 and nicotine +CE+ alpha -BTX group, the fluorescence intensity of nicotine +LPS+ alpha in -BTX group increased along with nicotine nicotine compared to +CE group, + LPS group, P0.01. PDTC further pretreatment of cells after 30min. Then respectively CE, LPS stimulation, cell culture supernatant of TNF- alpha, IL-8, IL-6 protein content and cell level of mRAN compared with pure CE stimulation group, simple LPS stimulation group were significantly decreased compared with P0.01 group, +CE; nicotine, the nicotine in the +LPS group The relative content of mRNA and factor protein compared with CE group, LPS group decreased significantly; the cells pre treated with PDTC, then were treated with nicotine +CE +LPS nicotine stimulation, supernatants of TNF- alpha, IL-8, the relative content of IL-6 protein decreased obviously, and the simple CE stimulation group simple, LPS stimulation group compared to P0.01. cells by CE and LPS after stimulation were observed in the phosphorylation of p38 MAPK, ERK1/2, JNK protein expression increased, compared with control group P0.01; adding the cells incubated with nicotine, respectively by CE and LPS stimulation, the relative content of phosphorylated ERK1/2 into cells detection of proteins was significantly decreased, respectively, 0.34 + 0.07,0.39 + 0.08, and CE group (0.74 + 0.12), LPS group (0.79 + 0.13) compared to a statistically significant difference between P0.01 and p38; and the phosphorylation of JNK was decreased significantly. With alpha 7 nAChR specific inhibitors of alpha -BTX Nicotine and nicotine +CE in the +LPS group, the relative content of phosphorylated ERK1/2 protein were 0.59 + 0.11,0.63 + 0.13, +CE group and nicotine without alpha -BTX stimulation, compared with nicotine + LPS group, the difference was statistically significant, P0.01; but the same effect on p38 and phosphorylation of JNK protein is further observed in HBE16. Cells treated with CE, p38MAPK, pERK1/2 and the expression of pJNK protein increased with time, the expression, the expression level of 6h was significantly higher than that of 30min at the time point of P0.01; by nicotine incubation of the cells, the expression of p38MAPK in 30min, with the prolonged expression gradually decreased, pERK1/2 at each time point were there was no obvious expression, pJNK was expressed in 30min, 2h was the strongest, began to decrease in 6h expression, compared with the expression of 2h, the difference was statistically significant, P0.01.
Conclusion: (1) the nicotine in cigarettes caused by airway mucus hypersecretion process does not have too many positive effects, on the contrary, it can reduce the inflammatory cytokines in this process, inhibition of subsequent excessive inflammatory reaction. (2) nicotine can reduce proinflammatory cytokine and chemokine TNF- alpha, IL-8, IL-6 protein production has certain anti-inflammatory function, and confirmed by nAChR and 7 with low I- and alpha kappa B alpha phosphorylation, thereby inhibiting nuclear factor kappa B nuclear translocation of NF-, expression of the inhibitory effect factor; the process may be by reducing the level of nicotine ERK1/2 activation. (3) nicotine does not increase the synthesis of mucin gene transcription and MUC5AC, and can make the inflammatory stimulus generated excess mucus exocrine is relatively reduced, so as to maintain the airway in chronic inflammatory stimulation of mucus high retention of airway mucus, students A relatively steady and hypersecretion.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 David A Scott;Michael Martin;;Exploitation of the nicotinic anti-inflammatory pathway for the treatment of epithelial inflammatory diseases[J];World Journal of Gastroenterology;2006年46期

，

本文編號(hào)：1388350