【作者】 赖宇；

【导师】 张琦；

【作者基本信息】 成都中医药大学 ， 中医临床基础， 2014， 博士

【摘要】 目的：利用基因芯片技术,分析温阳消饮法对胸腔积液大鼠胸膜基因表达的调控作用。方法：48只雄性SD大鼠适应性饲养3天后,随机分为A组(40只)与空白对照组(8只)。A组动物以戊巴比妥钠麻醉后,采用1%λ-角叉菜胶溶液进行右侧胸膜腔注射。24h后,运用CR数字成像系统观察胸部X光片,筛选出胸腔积液明显的大鼠,作为造模成功的动物备用。将造模成功的动物随机分为：温阳消饮组(10只),每日1次温阳消饮法代表方水煎液灌胃(5ml/kg);模型组(9只),每日1次生理盐水灌胃(5ml/kg)。空白对照组(8只),每日1次生理盐水灌胃(5ml/kg)。连续给药4天后,动物进行安乐死。每组取3只动物的右侧脏层和壁层胸膜,利用Agilent单通道表达谱芯片对胸膜组织基因表达情况进行分析。结果：(1)壁层胸膜组织模型组与空白对照组、温阳消饮组与模型组差异表达基因涉及多个生物学过程和信号通路。在造模因素作用下,基因Fgd4、Mef2a、Fhl3、Dyrk2、Slc24a6、 Pbrm1、LOC498368、Tp53i11、Rcan3、Hapln4、Abil和Hlx表达上调,在中药干预下表达下调；Nme7、Pold2、Klk1l和Fkbp5在造模条件下表达下调,用药后上调。(2)脏层胸膜组织模型组与空白对照组、温阳消饮组与模型组差异表达基因涉及多个生物学过程和信号通路。在造模因素作用下,基因Rhoa、RGD1306349、Nsd1、Figf、Brd4、 Crebbp、Sos2、Irf2、Brd2、Tspyl2、Eif4g2、Lcor、Isy1、Sf3a3、Msl2l1、Myo9a、 Papolg、Vasp、Pdcd7、Zbtb11、RGD1559904、Csnklg1、Leng8、Ddx10、Arhgef3、 Smg6、Zfat、Tm9sf3、Otud5和Chd6表达上调,在温阳消饮法代表方干预下表达下调；Ksr1、Slc25a27、Ptrf、Efemp1、RGD1563319、Cyb5r3、Gpr135、Polr3gl、 Kcna5、Ugp2、Acad11和Btbdll在造模条件下表达下调,用温阳消饮法代表方治疗后上调。结论：前期研究发现,温阳消饮法有助于λ-角叉菜胶诱导的豚鼠胸腔积液的吸收。本实验表明,其机制可能与苓桂术甘汤合葶苈大枣泻肺汤给药调控多个生物学过程及信号通路有关。本研究从基因水平揭示了温阳消饮法的部分机制：①温阳消饮法可下调壁层胸膜炎症促进基因Mef2a、凋亡诱导基因Dyrk2和Tp53i11表达,改善炎症效应。②温阳消饮法可下调脏层胸膜炎症促进基因Rhoa、Figf、 Brd4、Crebbp和Brd2表达,上调炎症抑制基因Ksr1和Slc25a127表达,从而降低炎性损害。此外,Fgd4、Fh13和Nme7等基因的表达变化与本研究的关系有待进一步确认。更多还原

【Abstract】 Objective:Genechip technology was used to measure changes in RNA expression, affected by therapeutic method of warming Yang to relieve fluid retention, of pleura in rat with X-carrageenan-induced pleural effusion.Methods:Male Sprague-Dawley rats were allowed to acclimate for3days before use, and then were randomly divided into group A and control group. Rats in group A were anesthetized with pentobarbital sodium, followed by the intrapleural injection of1%λ-carrageenan solution. At24h after the injection with carrageenan, the chest of rats was x-rayed, and the animals with a significant pleural effusion were determined by computed radiography. These valid rats were further divided into treatment group with intragastric administration of Chinese herb decoction (5ml/kg,1time per day) as well as model group with saline (5ml/kg,1time per day). Rats in control group were given saline (5ml/kg,1time per day). Four days later, the animals were euthanized by bleeding from the abdominal aorta under deep anesthesia with chloral hydrate. The parietal and visceral pleura of three rats from each group were obtained and the differential gene expression was analyzed using Agilent genechip.Results:In the parietal and visceral pleura, the differential gene expression between model and control groups as well as between treatment and model groups was associated with several types of biological processes and signaling pathways. In the parietal pleura, the therapy of warming Yang to relieve fluid retention could conversely regulate differential expression of16genes caused by X-carrageenan, including Fgd4, Mef2a, Fhl3, Dyrk2, Slc24a6, Nme7, Pbrml, LOC498368, Tp53i11, Rcan3, Hapln4, Abil, Hlx, Pold2, Klk11and Fkbp5. Moreover, in the visceral pleura, this treatment could conversely regulate differential expression of42genes affected by λ-carrageenan, including Rhoa, RGD1306349, Nsdl, Figf, Brd4, Crebbp, Sos2, Irf2, Brd2, Tspyl2, Eif4g2, Lcor, Ksrl, Slc25a27, Ptrf, Efempl, Isyl, Sf3a3, Msl2l1, Myo9a, Papolg, Vasp, Pdcd7, Zbtbll, RGD1559904, Csnklgl, Leng8, Ddx10, Arhgef3, Smg6, Zfat, Tm9sf3, Otud5, Chd6, RGD1563319, Cyb5r3, Gpr135, Polr3gl, Kcna5, Ugp2, Acadll and Btbdll.Conclusion:In our previous study with a Guinea Pig model of hydrothorax, we demonstrated that the therapeutic method of warming Yang to relieve fluid retention can facilitate recovering from pleural effusion. Our current study found that this therapeutic effect might be related to multiple biological processes and signaling pathways. The results also indicated that this therapy can down-regulate Mef2a, Dyrk2and Tp53i11expressed in parietal pleura and then reduce inflammation. Moreover, in visceral pleura it can down-regulate Rhoa, Figf, Brd4, Crebbp and Brd2, and up-regulate Ksrl and Slc25a27, which was associated with alleviative inflammatory injury.更多还原