糖皮质激素抑制气道上皮细胞修复的机制及干预研究

发布时间：2018-05-03 02:16

本文选题：地塞米松 + GILZ　；参考：《重庆医科大学》2013年博士论文

【摘要】：第一部分糖皮质激素诱导的亮氨酸拉链蛋白（GILZ）在人气道上皮细胞9HTE中的表达目的：明确糖皮质激素（GCs）地塞米松（DEX）诱导GILZ在人气道上皮细胞9HTE中的表达。方法：RT-PCR及Western Blot法检测DEX在不同时间点6小时、12小时及24小时作用后GILZ mRNA及蛋白的表达情况，同时细胞免疫荧光法检测GILZ蛋白的定位。结果：正常情况下，GILZ mRNA及蛋白在人气道上皮细胞9HTE的表达较低，而在DEX作用下GILZ mRNA及蛋白的表达均出现明显改变，6小时即明显增高，24小时仍持续高表达，同时观察到GILZ蛋白在9HTE细胞中主要定位在细胞质。结论：DEX能够快速并明显诱导人气道上皮细胞9HTE中GILZmRNA及蛋白的表达。第二部分小干扰RNA（si-RNA）沉默GILZ的筛选及鉴定目的：通过si-RNA技术设计合成三条GILZ siRNAs并筛选出GILZ沉默效果最佳的一条si-RNA用于后续实验。方法：设计并合成三条GILZ siRNAs：GILZ1si-RNA、GILZ2si-RNA及GILZ3si-RNA，通过脂质体2000分别转染进人气道上皮细胞9HTE，于沉默48小时后，收集细胞用Realtime-PCR、Western Blot及细胞免疫荧光法筛选并鉴定出沉默效果最佳的一条GILZ si-RNA。结果：通过对GILZ1si-RNA、GILZ2si-RNA及GILZ3si-RNA三条GILZ siRNAs的转染，筛选出GILZ3si-RNA为最佳的一条GILZsi-RNA，Realtime-PCR检测GILZ基因沉默效率平均可达55.8%，而通过Western Blot及细胞免疫荧光的检测发现其蛋白沉默效果显著。结论：通过si-RNA技术，成功合成鉴定获得一条沉默效果最佳的GILZ si-RNA，此为用于后续实验的关键。第三部分GILZ介导糖皮质激素抑制气道上皮细胞修复的研究目的：探讨GILZ介导GCs对MAPK-ERK信号通路、增殖及迁移的影响，明确其对气道上皮细胞修复的抑制作用。方法：在non-specific si-RNA及GILZ si-RNA转染48小时后收集细胞，Western Blot法检测人气道上皮细胞9HTE中Raf-1、Mek1/2、Erk1/2（MAPK-ERK信号通路因子）磷酸化蛋白及其总蛋白的表达，MTT、CFSE标记法检测细胞增殖情况，细胞划痕及transwell法检测细胞迁移情况。结果：DEX抑制了人气道上皮细胞9HTE中MAPK-ERK信号通路Raf-1、Mek1/2、Erk1/2磷酸化蛋白的表达，而对总蛋白的表达无明显影响，即抑制了MAPK-ERK信号通路的激活，同时也观察到DEX抑制了细胞的增殖和迁移。而GILZ si-RNA转染进人气道上皮细胞9HTE，GILZ的表达被抑制后，，DEX对气道上皮细胞MAPK-ERK信号通路、增殖及迁移的抑制作用均明显减轻。结论：DEX能够抑制MAPK-ERK信号通路的激活、增殖及迁移，从而抑制了气道上皮细胞的修复作用，而DEX的这一抑制作用主要是通过GILZ介导的。第四部分维生素A对糖皮质激素抑制气道上皮细胞修复的干预研究目的：明确维生素A（VitA）在人气道上皮细胞9HTE中对GCs抑制气道上皮细胞修复的影响。方法：通过DEX及全反式维甲酸（ATRA）2干预24h后，ELISA法检测人气道上皮细胞9HTE培养上清液中EGF的表达，细胞免疫荧光及Western Blot法检测EGFR及磷酸化EGFR的表达；同时WesternBlot检测人气道上皮细胞9HTE中Raf-1、Mek1/2、Erk1/2（MAPK-ERK信号通路因子）磷酸化蛋白及其总蛋白的表达，MTT法检测细胞增殖情况，细胞划痕及transwell实验检测细胞迁移情况。结果：ATRA对人气道上皮细胞9HTE EGF的分泌无明显影响，但对EGFR及其磷酸化的蛋白的表达有促进作用。ATRA同时也增加了MAPK-ERK信号通路Raf-1、Mek1/2、Erk1/2磷酸化蛋白的表达从而减轻了DEX对MAPK-ERK信号通路激活的抑制作用；ATRA在早期对细胞增殖无明显影响，但明显促进了9HTE细胞的迁移。结论：ATRA能够诱导EGFR磷酸化蛋白的表达从而激活EGFR信号通路，这也激活了下游的MAPK-ERK信号通路并促进了9HTE细胞的迁移，从而降低了DEX抑制气道上皮细胞修复的副效应。
[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 .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.6

【参考文献】