【作者】 刘静月；

【导师】 符州；

【作者基本信息】 重庆医科大学 ， 儿科学， 2013， 博士

【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。更多还原

【Abstract】 PART ONETHE EXPESSION OF GLUCOCORTICOID-INDUCEDLEUCINE ZIPPER(GILZ) IN HUMAN AIRWAY EPITHELIALCELLS9HTEObjective: To explore the expression of glucocorticoid-induced leucinezipper in human airway epithelial cells9HTE treated with glucocorticoiddexamethasone.Methods: RT-PCR and western Blot detected the expressions of GILZmRNA and protein treated with DEX for6h,12h and24h.Immunofluorescence assay located GILZ protein.Results: The expressions of GILZ mRNA and protein were low in9HTEcells under normal circumstances, but were significantly changed. Itshowed that the expressions of GILZ mRNA and protein increasedobviously after DEX treated for6h, and continued to maintain a high level until24h. We also observed GILZ protein was located in the cytoplasm of9HTE cells.Conclusion: DEX quickly and significantly induced the expressions ofGILZ mRNA and protein in airway epithelial cells9HTE. PART TWOTHE SCREENING AND IDENTIFICATION OF GILZSILENCING BY SMALL INTERFERING RNA(si-RNA)Objective: To screen the best si-RNA for silencing GILZ from three GILZsiRNAs that were designed and synthesized by si-RNA technology, and usefor subsequent experiments.Methods: We designed and synthesized three GILZ siRNAs: GILZ1si-RNA, GILZ2si-RNA, GILZ3si-RNA. Then they were transfected intohuman airway epithelial cells9HTE via lipofectamine2000.9HTE cellswere collected after were silenced for48h, and we screened and identifiedthe GILZ si-RNA that had the best silencing effect by Realtime-PCR,Western Blot and immunofluorescence assays.Results: We screened that GILZ3si-RNA was the best GILZ si-RNA after GILZ1si-RNA, GILZ2si-RNA, GILZ3si-RNA were transfected into9HTEcells. The average silencing efficiency of GILZ gene was up to55.8%byRealtime-PCR, and the silencing efficiency of GILZ protein was significantby Western Blot and immunofluorescence.Conclusion: We successfully synthesized and identified to obtain a GILZsi-RNA for best silencing effect by si-RNA technology, and it was the keyof subsequent experiments. PART THREETHE STUDY OF GLUCOCORTICOIDS INHIBITING humanAIRWAY EPITHELIAL CELLS REPAIR WAS MEDIARED BYGILZObjective: To explore the influence of GCs mediated by GILZ onMAPK-ERK signaling pathway, proliferation and migration, and clarify theinhibitory effect on airway epithelial cells repair.Methods: We collected cells after non-specific si-RNA and GILZ si-RNAwere transfected into human airway epithelial cells9HTE. Then wedetected the expressions of phosphorylated and total proteins of Raf-1, Mek1/2, Erk1/2(components of the MAPK-ERK signaling pathway) in9HTE cells. MTT and CFSE assays were used to detect the cellproliferation. The cell migration was tested by wound-healing and transwellassays.Results: DEX inhibited phosphorylated protein expressions of Raf-1,Mek1/2, Erk1/2of the MAPK-ERK signaling pathway in human airwayepithelial cells9HTE, and had no significant effect on total proteinexpression. It showed that DEX inhibited the activation of MAPK-ERKsignaling pathway. We also observed that DEX inhibited the cellproliferation and migration. After GILZ si-RNA was transfected into9HTEcells to silence the expression of GILZ, the inhibitory effect onMAPK-ERK signaling pathway, proliferation and migration weresignificantly reduced.Conclusion: DEX could inhibit the activation of MAPK-ERK signalingpathway, proliferation and migration, thereby suppressing the repair ofairway epithelial cells. The inhibitory effect of DEX was mainly mediatedby GILZ. PART FOURTHE INTERVENTION STUDY OF VITAMIN A ONGLUCOCORTICOIDS INHIBITING AIRWAY EPITHELIALCELLS REPAIRObjective: To explore the effect of VitA on GCs inhibiting human airwayepithelial cells repair.Methods: ELISA assay was used to detect the EGF expression of culturesupernatant in human airway epithelialcells9HTE that were treated byDEX and ATRA for24h, and immunofluorescence and Western Blot assaystested the EGFR expressions of phosphorylated and total proteins. Then wedetected the expressions of phosphorylated and total proteins of Raf-1,Mek1/2, Erk1/2(components of the MAPK-ERK signaling pathway) in9HTE cells. MTT assay was used to detect the cell proliferation. The cellmigration was tested by wound-healing and transwell assays.Results: ATRA had no significant effect on the secretion of EGF in9HTE,but promoted the EGFR expressions of phosphorylated and total proteins.ATRA also increased the phosphorylation of Raf-1, Mek1/2, Erk1/2toreduce the inhibitory effect of DEX on MAPK-ERK signaling pathway. Inthe early stage ATRA did not influence the cell proliferation, butsignificantly increased the cell migration.Conclusion: ATRA could induce the phosphorylated protein expression ofEGFR to activate EGFR signaling pathway, then activated the downstream MAPK-ERK signaling pathway, and promoted the cell migration, therebyreducing the side-effect of DEX on inhibiting airway epithelial cells repair.更多还原