【作者】 谢江平；

【导师】 丁国善；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 目的:GILZ具有炎症抑制、上皮细胞钠通道的调节等多项作用,目前认为是GCs发挥作用的重要介质。siRNA自被发现就誉为基因研究史上的革命性突破,其对靶mRNA的抑制作用具有特异性和高效性的特点,是特异性抑制基因表达的强效方法。本实验通过体外条件培养获得足够数量和纯度的小鼠腹腔巨噬细胞,应用已构建的编码小鼠GILZ基因的质粒载体和GILZ特异性siRNA体外转染小鼠腹腔巨噬细胞,检测GILZ基因的mRNA和蛋白表达水平,建立GILZ的高表达和沉默模型,为下一步研究巨噬细胞中GILZ基因特性奠定实验基础。方法:体外培养获得足够数量和纯度的小鼠腹腔巨噬细胞,法国Polyplus-transfection公司的INTERFERin^TM和jetPEI^TM转染试剂分别将siGILZ和GILZ转染至小鼠腹腔巨噬细胞中,干扰试验分为空白对照组、无关序列siC组、siGILZ组,分别取处理后1小时、3小时、6小时、12小时和24小时作为观察点。GILZ基因转染试验分为空白对照组、空质粒AAV组、GILZ组,也分别取处理后1小时、3小时、6小时、12小时和24小时作为观察点。RealtimePCR法检测各处理组细胞GILZ的mRNA水平的表达,Western-blot检测各处理组细胞GILZ的蛋白水平的表达。结果:干扰试验中与对照组和无关序列siC组相比较,siGILZ组明显降低6小时后GILZ的mRNA水平和蛋白水平的表达（p<0.05）;GILZ基因转染试验中,与对照组和空质粒AAV组相比较,GILZ组明显升高GILZ的mRNA水平和蛋白水平的表达（p<0.05）,其中6h组表达最高。结论:GILZ的高表达和沉默模型成功构建,可以转染试剂处理6小时作为GILZ干扰和转染的处理时间。目的:巨噬细胞是固有免疫系统中重要的细胞组成部分,具有包括吞噬作用、抗原提呈及分泌细胞因子等功能,在不同微环境中,巨噬细胞主要分为M1型和M2型,M1型巨噬细胞可直接吞噬和杀伤病原微生物和肿瘤细胞、分泌促炎性细胞因子,专职呈递抗原参与正向免疫应答;M2型巨噬细胞表现为较低的抗原呈递能力,并通过分泌抑制性细胞因子IL-10和TGF-β等下调免疫应答。本实验试图探讨GILZ能否通过促进腹腔巨噬细胞转变为M2型巨噬细胞而发挥负向调控作用。方法:以小鼠腹腔巨噬细胞为研究对象,试验分为空白对照组、DEX组、GILZ处理组、siGILZ处理组。RealtimePCR法检测各处理组M1和M2标志性分子表达情况、ELISA法进行细胞因子的检测,流式检测处理后巨噬细胞的吞噬能力,Western-blot检测各组TLR4信号通路分子的表达。结果:与对照组相比较,Dex组与GILZ处理组高表达M2标志性分子Arginase-1、FIZZ-1和Ym-1,低表达M1标志性分子i-NOS;抑制促炎症因子IL-1β、IL-6、TNF-α及IL-12的表达,而提高抗炎症因子IL-10的表达,抑制腹腔巨噬细胞的吞噬能力,下调TLR4、IRAK1、IRAK2和TRAF6等TLR4信号通路分子的表达;而siGILZ却低表达M2标志性分子Arginase-1、FIZZ-1和Ym-1,高表达M1标志性分子i-NOS;促进促炎症因子IL-1β、IL-6、TNF-α及IL-12的表达,而抑制抗炎症因子IL-10的表达,提高腹腔巨噬细胞的吞噬能力,上调TLR4、IRAK1、IRAK2和TRAF6等TLR4信号通路分子的表达（p<0.05）。结论:GILZ促进M2型巨噬细胞的产生,抑制促炎症因子的表达,而促进抗炎症因子IL-10的表达,并可能是通过抑制TLR4信号通路分子的机制。目的:肝脏缺血再灌注损伤是肝脏外科疾病中常见的病理生理过程,地塞米松通过直接抑制中性粒细胞的氢过氧化物生成和中性粒细胞游出,阻止磷脂酶A2和花生四烯酸代谢,从而抑制中性粒细胞介导的缺血再灌注损伤。本实验探讨GILZ是否可通过促进腹腔巨噬细胞转变为M2型巨噬细胞来降低肝脏缺血再灌注损伤。方法:通过invivojetPEI^TM转染试剂构建GILZ体内转染于小鼠肝脏缺血再灌注模型,试验分为空白对照组、Dex组、AAV空质粒处理组、GILZ处理组,取再灌注4小时作为观察点。检测ALT和AST情况、HE染色看病理学变化,ELISA法进行细胞因子的检测。结果:与对照组和AAV空质粒处理组比较,Dex组和GILZ处理组明显降低ALT和AST的表达（p<0.05）,较明显减少小鼠肝脏缺血再灌注的组织损伤,抑制促炎症因子IL-1β、IL-6、TNF-α及IL-12的表达,而提高抗炎症因子IL-10的表达（p<0.05）。结论:本实验从体内研究环境发现GILZ通过诱导M2型巨噬细胞产生,保护缺血再灌注肝脏的肝功能的作用,降低炎症因子的表达,进而保护缺血再灌注的肝脏的结构相对完整。更多还原

【Abstract】 Objective: GILZ have ability of anti-inflammation and regulation of epithelialsodium channels and many other effects.It’s a new important mediator ofglucocorticoid action.SiRNA was looked as a revolutionary breakthrough in thehistory of genetic research, has the specificity and efficiency of the characteristics ininhibition of target mRNA . SiRNA technique is a powerful ways to inhibit geneexpression specifically. The present study, GILZ and siGILZ plasmid vector that hasbeen constructed transfect to the mouse peritoneal macrophages with sufficientquantity and purity in vitro , and detect expression of GILZ mRNA and proteinlevels.The model of high expression and silence of GILZ has been established forfurther study of the genetic characteristics of GILZ in macrophages. METHODS:Cultured sufficient quantity and purity of mouse peritoneal macrophages, siGILZ andGILZ transfected into mouse peritoneal macrophages by the French companyPolyplus-transfection’s transfection reagents INTERFERin^TM and jetPEI^TMrespectively. The interference test is divided into a blank Control group, siC group andsiGILZ group and 1 hour, 3 hours, 6 hours, 12 hours and 24 hours were taken as theobservation point. GILZ gene transfer trials were divided into control group, emptyvector AAV group and GILZ group, were also taken after 1 hour, 3 hours, 6 hours, 12hours and 24 hours for observation. Realtime PCR detected the mRNA expressionlevels of GILZ in each treatment group, Western-blot detected expression of theprotein GILZ of cells in each treatment group. Results: Compared with the controlgroup and siC group, siGILZ significantly decreased the mRNA level and proteinlevels of GILZ after transfected for 6 hours （p <0.05）; GILZ gene transferexperiments, compared with the control group and empty plasmid AAV group, GILZwas significantly increased the expression of GILZ in mRNA and protein levels （p<0.05）, in which the highest expression of 6h group. Conclusion: Overexpression andsilence model of GILZ has been builded successfully. Objective: Macrophages are important innate immune cell, it’s functions includingphagocytosis, antigen presentation and secretion of cytokines and other functions.In adifferent microenvironment, macrophages consists of M1 and M2-type model, M1Macrophage can directly engulf and destruct the pathogenic microorganisms andtumor cells, present antigen and secrete proinflammatory cytokines, involved in ppositive immune responserofessionally; M2 macrophages showed lowerantigen-presenting ability, ana secreted the inhibitory Cytokine such as IL-10 andTGF-βthat reduced immune response. This study attempts to explore whether GILZpromoting peritoneal macrophages changed to the M2-type macrophages and playinga role in the negative regulation. Methods: Mouse peritoneal macrophages was thestudyobject, the test divided into control group, DEX group, GILZ treatment groupand siGILZ treatment group. Realtime PCR Detected expression of symbol moleculeof M1 and M2 in each treatment group, ELISA method for detection of cytokines,flow cytometry for macrophage phagocytosis , Western-blot to detect the expression ofTLR4 signaling molecules. Results: Compared with the control group, Dex treatmentgroup and GILZ group express high level of Arginase-1, FIZZ-1 and Ym-1 of M2,low level of i-NOS of M1; inhibition of pro-inflammatory factor IL-1β, IL-6, TNF-αand IL-12 expression, and increased anti-inflammatory factor IL-10 expression,inhibition of macrophage phagocytic capacity, reduced the expression of TLR4signaling pathway molecules such as TLR4, IRAK1, IRAK2 and TRAF6; WhilesiGILZ has the low expression of Arginase-1, FIZZ-1 and Ym-1 of M2, highexpression of M1 molecules i-NOS; to promote pro-inflammatory factorαIL-1β, IL-6,TNF- and IL-12 Expression, and inhibition of anti-inflammatory factor IL-10expression, enhance macrophage phagocytosis , increased TLR4 signaling moleculesexpression such as TLR4, IRAK1, IRAK2 and TRAF6 （p <0.05）. Conclusion: GILZ promote the production of M2 macrophages, inhibit the expression ofproinflammatory factors, the promot of anti-inflammatory factor IL-10 expression,and probably by inhibiting the mechanism of TLR4 signaling molecules. Objective: Hepatic ischemia reperfusion injury is a common pathophysiology of liversurgical diseases. Through the direct inhibition generation of neutrophil hydrogenperoxide and neutrophil swim out ,dexamethasone block the phospholipase A2 andarachidonic acid metabolism, thereby inhibiting neutrophil-mediatedischemia-reperfusion injury. This study investigated whether GILZ reduce the hepaticischemia-reperfusion injury by promoting peritoneal macrophages changes to theM2-type macrophages. Methods: Build GILZ transfection by transfection reagent invivo jetPEI^TM in mouse liver ischemia-reperfusion model in vivo.test divided intocontrol group, Dex group, AAV empty plasmid treated group, GILZ treatment group,taking 4 hours after reperfusion as observation point. ALT and AST test performed,HE staining for pathological changes, the level of cytokines by ELISA. Results:Compared with the control group and the AAV empty plasmid treatment group, Dextreatment group and GILZ group significantly reduced the expression of ALT andAST （p <0.05）, significantly reduced liver ischemia reperfusion injury in pathology,inhibition expression of proinflammatory factors IL-12IL-1β, IL-6 and TNF-α, andincreased anti-inflammatory factor IL-10 expression （p <0.05）. Conclusion: Thisstudy found that GILZ protecting the function of liver in liver ischemia /reperfusion ,reducing the expression of inflammatory cytokines by inducingproduction of M2-type macrophages, thereby protecting Structural integrity of liver inischemia-reperfusion.更多还原