本文選題：地塞米松 + GILZ　； 參考：《重慶醫(yī)科大學(xué)》2013年博士論文

【摘要】：第一部分糖皮質(zhì)激素誘導(dǎo)的亮氨酸拉鏈蛋白（GILZ）在人氣道上皮細(xì)胞9HTE中的表達(dá) 目的：明確糖皮質(zhì)激素（GCs）地塞米松（DEX）誘導(dǎo)GILZ在人氣道上皮細(xì)胞9HTE中的表達(dá)。 方法：RT-PCR及Western Blot法檢測(cè)DEX在不同時(shí)間點(diǎn)6小時(shí)、12小時(shí)及24小時(shí)作用后GILZ mRNA及蛋白的表達(dá)情況，同時(shí)細(xì)胞免疫熒光法檢測(cè)GILZ蛋白的定位。 結(jié)果：正常情況下，GILZ mRNA及蛋白在人氣道上皮細(xì)胞9HTE的表達(dá)較低，而在DEX作用下GILZ mRNA及蛋白的表達(dá)均出現(xiàn)明顯改變，6小時(shí)即明顯增高，24小時(shí)仍持續(xù)高表達(dá)，同時(shí)觀察到GILZ蛋白在9HTE細(xì)胞中主要定位在細(xì)胞質(zhì)。 結(jié)論：DEX能夠快速并明顯誘導(dǎo)人氣道上皮細(xì)胞9HTE中GILZmRNA及蛋白的表達(dá)。 第二部分小干擾RNA（si-RNA）沉默GILZ的篩選及鑒定 目的：通過(guò)si-RNA技術(shù)設(shè)計(jì)合成三條GILZ siRNAs并篩選出GILZ沉默效果最佳的一條si-RNA用于后續(xù)實(shí)驗(yàn)。 方法：設(shè)計(jì)并合成三條GILZ siRNAs：GILZ1si-RNA、GILZ2si-RNA及GILZ3si-RNA，通過(guò)脂質(zhì)體2000分別轉(zhuǎn)染進(jìn)人氣道上皮細(xì)胞9HTE，于沉默48小時(shí)后，收集細(xì)胞用Realtime-PCR、Western Blot及細(xì)胞免疫熒光法篩選并鑒定出沉默效果最佳的一條GILZ si-RNA。 結(jié)果：通過(guò)對(duì)GILZ1si-RNA、GILZ2si-RNA及GILZ3si-RNA三條GILZ siRNAs的轉(zhuǎn)染，篩選出GILZ3si-RNA為最佳的一條GILZsi-RNA，Realtime-PCR檢測(cè)GILZ基因沉默效率平均可達(dá)55.8%，而通過(guò)Western Blot及細(xì)胞免疫熒光的檢測(cè)發(fā)現(xiàn)其蛋白沉默效果顯著。 結(jié)論：通過(guò)si-RNA技術(shù)，成功合成鑒定獲得一條沉默效果最佳的GILZ si-RNA，此為用于后續(xù)實(shí)驗(yàn)的關(guān)鍵。 第三部分GILZ介導(dǎo)糖皮質(zhì)激素抑制氣道上皮細(xì)胞修復(fù)的研究 目的：探討GILZ介導(dǎo)GCs對(duì)MAPK-ERK信號(hào)通路、增殖及遷移的影響，明確其對(duì)氣道上皮細(xì)胞修復(fù)的抑制作用。 方法：在non-specific si-RNA及GILZ si-RNA轉(zhuǎn)染48小時(shí)后收集細(xì)胞，Western Blot法檢測(cè)人氣道上皮細(xì)胞9HTE中Raf-1、Mek1/2、Erk1/2（MAPK-ERK信號(hào)通路因子）磷酸化蛋白及其總蛋白的表達(dá)，MTT、CFSE標(biāo)記法檢測(cè)細(xì)胞增殖情況，細(xì)胞劃痕及transwell法檢測(cè) 細(xì)胞遷移情況。 結(jié)果：DEX抑制了人氣道上皮細(xì)胞9HTE中MAPK-ERK信號(hào)通路Raf-1、Mek1/2、Erk1/2磷酸化蛋白的表達(dá)，而對(duì)總蛋白的表達(dá)無(wú)明 顯影響，即抑制了MAPK-ERK信號(hào)通路的激活，同時(shí)也觀察到DEX抑制了細(xì)胞的增殖和遷移。而GILZ si-RNA轉(zhuǎn)染進(jìn)人氣道上皮細(xì)胞9HTE，GILZ的表達(dá)被抑制后，，DEX對(duì)氣道上皮細(xì)胞MAPK-ERK信號(hào)通路、增殖及遷移的抑制作用均明顯減輕。 結(jié)論：DEX能夠抑制MAPK-ERK信號(hào)通路的激活、增殖及遷移，從而抑制了氣道上皮細(xì)胞的修復(fù)作用，而DEX的這一抑制作用主要是通過(guò)GILZ介導(dǎo)的。 第四部分維生素A對(duì)糖皮質(zhì)激素抑制氣道上皮細(xì)胞修復(fù)的干預(yù)研究 目的：明確維生素A（VitA）在人氣道上皮細(xì)胞9HTE中對(duì)GCs抑制氣道上皮細(xì)胞修復(fù)的影響。 方法：通過(guò)DEX及全反式維甲酸（ATRA）2干預(yù)24h后，ELISA法檢測(cè)人氣道上皮細(xì)胞9HTE培養(yǎng)上清液中EGF的表達(dá)，細(xì)胞免疫熒光及Western Blot法檢測(cè)EGFR及磷酸化EGFR的表達(dá)；同時(shí)WesternBlot檢測(cè)人氣道上皮細(xì)胞9HTE中Raf-1、Mek1/2、Erk1/2（MAPK-ERK信號(hào)通路因子）磷酸化蛋白及其總蛋白的表達(dá)，MTT法檢測(cè)細(xì)胞增殖情況，細(xì)胞劃痕及transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移情況。 結(jié)果：ATRA對(duì)人氣道上皮細(xì)胞9HTE EGF的分泌無(wú)明顯影響，但對(duì)EGFR及其磷酸化的蛋白的表達(dá)有促進(jìn)作用。ATRA同時(shí)也增加了MAPK-ERK信號(hào)通路Raf-1、Mek1/2、Erk1/2磷酸化蛋白的表達(dá)從而減輕了DEX對(duì)MAPK-ERK信號(hào)通路激活的抑制作用；ATRA在早期對(duì)細(xì)胞增殖無(wú)明顯影響，但明顯促進(jìn)了9HTE細(xì)胞的遷移。 結(jié)論：ATRA能夠誘導(dǎo)EGFR磷酸化蛋白的表達(dá)從而激活EGFR信號(hào)通路，這也激活了下游的MAPK-ERK信號(hào)通路并促進(jìn)了9HTE細(xì)胞的遷移，從而降低了DEX抑制氣道上皮細(xì)胞修復(fù)的副效應(yīng)。
[Abstract]:Expression of first part of glucocorticoid - induced leucine zipper protein ( GILZ ) in human airway epithelial cells 9HTE

Objective : To clarify the expression of GILZ induced by dexamethasone ( DEX ) in human airway epithelial cells 9HTE .

Methods : The expression of GILZ mRNA and protein were detected by RT - PCR and Western Blot at 6 hours , 12 hours and 24 hours at different time points , and the localization of GILZ protein was detected by immunofluorescence assay .

Results : In normal condition , the expression of GILZ mRNA and protein in human airway epithelial cells 9HTE was lower , while the expression of GILZ mRNA and protein was significantly increased in the presence of DEX . The expression of GILZ mRNA and protein was significantly increased in 6 hours .

Conclusion : DEX can rapidly and obviously induce the expression of GILZmRNA and protein in human airway epithelial cells 9HTE .

Screening and identification of the second part of small interfering RNA ( si - RNA ) silencing GILZ

Objective : To synthesize three GILZ by si - RNA technique and to screen out the best effect of GILZ silencing in the follow - up experiment .

Methods : GILZ1si - RNA , GILZ2si - RNA and GILZ3si - RNA were designed and synthesized .

Results : GILZ3si - RNA and GILZ3si - RNA were transfected with GILZ3si - RNA , GILZ2si - RNA and GILZ3si - RNA .

Conclusion : The best GILZ - RNA was successfully synthesized by si - RNA technique , which was the key to follow - up experiment .

The third part GILZ - mediated glucocorticoid - mediated inhibition of airway epithelial cells

Objective : To investigate the effect of GILZ on MAPK - ERK signal pathway , proliferation and migration , and to clarify the inhibitory effect of GILZ on airway epithelial cell repair .

Methods : After 48 hours after transfection of non - specific si - RNA and GILZ - RNA , the expression , expression , MTT and CFSE labeling method were used to detect the expression of phosphorylated protein and its total protein in human airway epithelial cells 9HTE . MTT assay and CFSE labeling method were used to detect cell proliferation , cell scratches and transwell method .

Cell migration .

Results : DEX inhibited the expression of MAPK - ERK signaling pathway in human airway epithelial cells 9HTE , and the expression of MAPK - ERK signaling pathway , Mek1 / 2 , Erk1 / 2 phosphorylation protein , was unknown .

The activation of MAPK - ERK signaling pathway was inhibited , while DEX inhibited the proliferation and migration of MAPK - ERK signal pathway . The inhibition of MAPK - ERK signaling pathway , proliferation and migration of DEX to airway epithelial cells was significantly reduced after GILZ - RNA was transfected into human airway epithelial cells 9HTE and GILZ .

Conclusion : DEX can inhibit the activation , proliferation and migration of MAPK - ERK signaling pathway , thereby inhibiting the repair of airway epithelial cells , while DEX is mainly mediated by GILZ .

The Intervention Study of the fourth Part of Vitamin A in the Treatment of Airway Epithelial Cells by Glucocorticoids

Objective : To clarify the effect of vitamin A ( VitA ) on the inhibition of airway epithelial cells in human airway epithelial cells 9HTE .

Methods : Using DEX and all trans retinoic acid ( ATRA ) for 24 h , the expression of EGF in the supernatant of human airway epithelial cells 9HTE was detected by ELISA , and the expression of EGFR and phosphorylated EGFR was detected by immunofluorescence and Western Blot .
At the same time Western Blot was used to detect the expression of phosphorylated protein and its total protein in human airway epithelial cells 9HTE . The expression of phosphorylated protein and its total protein in human airway epithelial cells 9HTE was detected by MTT assay . Cell proliferation was detected by MTT assay , cell scratches and transwell test were used to detect cell migration .

Results : ATRA has no significant effect on the secretion of human airway epithelial cells 9HTE EGF , but also contributes to the expression of EGFR and its phosphorylated proteins . ATRA also increases the expression of MAPK - ERK signaling pathways , the ERK - ERK signaling pathway , the phosphorylation protein , and reduces the inhibitory effect of DEX on MAPK - ERK signaling pathway activation .
ATRA had no significant effect on cell proliferation in the early stage , but it significantly promoted the migration of 9HTE cells .

Conclusion : ATRA can induce the expression of EGFR phosphorylation protein to activate EGFR signaling pathway , which also activates the downstream MAPK - ERK signaling pathway and promotes the migration of 9HTE cells , thus reducing DEX inhibition of airway epithelial cell repair .

【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】：2013
【分類(lèi)號(hào)】：R725.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉靜月;符州;羅征秀;王莉佳;李欣;;糖皮質(zhì)激素誘導(dǎo)人氣道上皮細(xì)胞9HTE_0凋亡的研究[J];免疫學(xué)雜志;2011年09期

本文編號(hào)：1836527