DNA微阵列方法鉴别七氟醚和丙泊酚麻醉下诱导的基因表达情况

发布时间：2018-07-18 13:38

【摘要】：冠状动脉旁路移植术(Coronary artery bypass grafting, CABG),是被广泛接受的冠心病的有效治疗方法。根据有无体外循环,CABG可分为体外循环冠状动脉搭桥术(Conventional coronary artery bypass grafting, CCABG)和非体外循环冠状动脉搭桥术(Off-pump coronary artery bypass grafting, OPCABG)。与CCABG相比,OPCABG可降低手术接受者的肾脏功能损害、心肌损伤、大脑损伤、中风、房颤、神经认知以及器官功能障碍等风险。因此,OPCABG已被广泛应用于冠心病的临床手术治疗。然而,OPCABG仍不能避免手术操作本身对于心肌所脏成的损伤以及再灌注损伤。研究指出,某些炎症相关的细胞因子,如白细胞介素6(Interleukin-6),丫干扰素(Interferon-y),高敏感性C-反应蛋白(High sensitivity C-reactive protein)和粒细胞集落刺激因子(Granulocyte colony-stimulating factor)等,OPCABG接受者释放水平与体外循环情况下相似,甚至更高。因此,抑制心肌缺血再灌注性损伤,完善围手术期的管理,对于OPCABG同样必要。研究显示在心肌缺血发生前或发生后采取一些措施,如麻醉剂干预,在一定程度上可以减少心肌缺血所造成的心肌细胞损伤以及后续的再灌注损伤。七氟醚是一种吸入性麻醉剂,通过实施吸入性七氟醚麻醉,可显著减少因心肌缺血而导致的梗死面积,并且通过降低Ca2+负荷,可保护心肌抵抗再灌注损伤。对于CABG接受者心房组织活检的研究表明,七氟醚在缺血性前适应阶段可以下调血小板内皮细胞黏附分子-1(PECAM-1/CD31)的表达水平,同时上调过氧化氢酶的表达。经七氟醚处理后,虽然手术接受者血清中IL-10和IL-1受体拮抗剂水平仍有显著提高,但IL-6和IL-8血清水平得到了显著性抑制。除了吸入性麻醉药外,静脉麻醉剂,如丙泊酚也可以降低冠状动脉旁路移植术接受者心肌再灌注损伤的程度。异丙酚可维持一氧化氮合酶(NOS)以及磷酸肌醇-3-激酶(phosphoinositide-3-kinase)/蛋白激酶B(Akt)的活性,该特性可能是异丙酚减少缺血性再灌注损伤的分子机制。此外,异丙酚具有与自由基清除剂相似的化学结构,而自由基清除剂的释放是七氟醚所诱导的缺血性前适应的必要条件,因此推测,异丙酚对缺血性再灌注同样具有一定的缺血性前适应作用。虽然已有大量研究证实七氟醚和丙泊酚具有有效的心脏保护作用,但其心肌保护作用的潜在机制仍不清楚。本研究利用生物信息学技术,基于基因表达谱芯片数据,首次分析了吸入性七氟醚处理组特异性差异表达基因、静脉注射丙泊酚处理组特异性差异表达基因以及两组共有的差异表达基因,共三组差异表达基因；随后我们对三组差异表达基因进行了功能注释和分析；并探索了三组差异表达基因所在的互作功能模块。以期从更加细致的分子互作角度阐释吸入性七氟醚麻醉和静脉注射丙泊酚麻醉的心肌保护机制,为寻求CABG围手术期更有效的心肌保护方式奠定理论基础。具体研究结果如下： 1从GEO (Gene Expression Omnibus)中下载吸入性七氟醚麻醉和静脉注射丙泊酚麻醉处理的冠状动脉旁路移植术病人心肌组织芯片表达谱数据(编号为GSE4386)。采用R语言中的Affy包对芯片数据进行预处理,并利用multtest包对吸入性七氟醚麻醉组和静脉麻醉剂丙泊酚组所诱导的差异性表达基因进行筛选。从吸入性七氟醚麻醉和静脉麻醉剂丙泊酚组分别筛选得到了879个和290个满足差异阈值(FDR0.05和|logFC|1)的差异表达基因。 2.通过对吸入性七氟醚麻醉和静脉麻醉剂丙泊酚组中所筛选得到的差异表达基因进行比较,得到吸入性七氟醚麻醉诱导下特异性差异表达基因879个,静脉麻醉剂丙泊酚诱导下特异性差异表达基因290个,以及共同诱导的差异表达基因275个。进一步比较三类差异表达基因分别在两个处理组中的表达水平发现,七氟醚处理组特异性差异表达基因仅在七氟醚组表达水平有统计学差异(P=0.01239),在丙泊酚处理组无差异性表达；丙泊酚处理组特异性差异性表达基因仅在丙泊酚组表达水平有统计学差异(P=0.02206)；而两组共有差异表达基因在两个处理组均有差异性表达(P=2.98e-14),即分析结果与差异表达基因筛选结果一致。 3.利用DAVID在线工具对七氟醚组和丙泊酚组特异性差异表达基因进行GO分析,富集这些差异基因参与的关键生物学过程。其中,七氟醚处理组特异性差异表达基因最显著参与伤害应答反应(n=72)；丙泊酚处理组特异性差异表达基因最显著参与对有机物质的应答反应(n=45)。进一步对两组特异性差异表达基因以及共有差异表达基因所注释的基因用BLASTX与COG直系同源簇数据库进行比对分析,三类差异表达基因从cellular_component、molecular_function、biological process三方面获得的COG功能分类很相似。 4.利用软件WebGestalt搜索七氟醚组和丙泊酚组特异性差异表达基因及两组的差异共表达基因所在的互作功能模块,分别筛选到两种麻醉剂共同诱导的、吸入性七氟醚麻醉组和静脉麻醉剂丙泊酚诱导下的特异性差异表达基因存在的显著性的功能模块数量为2个、1个和1个(FDR0.05)。其中,差异共表达基因所在显著性功能模块分别包含10个和18个差异表达基因,如：ATF3、JUND、FOSB、JUNB及TNFAIP3。七氟醚处理组特异性差异表达基因所在显著性功能模块包含两个上调基因,分别为CD93和LRRC32;以及七个下调基因,分别为CPXM1、FGFR2、 FLRT3、IL17RD、IRS1、IL7和CSFIR。丙泊酚处理组特异性差异表达基因所在显著性功能模块共包含8个上调基因,分别为：CISH、PTPN1、SOCS1、IL15RA、 RCAN1、IL6R、DUSP4和STAT3。对功能模块中各基因利用EASE再次进行GO功能富集分析,其所参与的主要功能涉及转录调控、细胞过程调控等,与差异表达基因的功能注释非常相似。本研究从分子生物学角度系统地阐述了七氟醚和丙泊酚在CABG中的心肌保护作用。结果显示,吸入性麻醉剂七氟醚和静脉麻醉剂丙泊酚在保护心肌作用中具有协同的作用,因此,我们推测,吸入性麻醉剂七氟醚和静脉麻醉剂丙泊酚两种方式的静吸复合麻醉方式,在冠状动脉旁路移植手术中可能会具有更好保护心肌细胞作用。我们仍需进一步的分子生物学体内和体外实验论证该结论。课题创新性与意义研究表明七氟醚麻醉和静脉注射丙泊酚麻醉都能在冠状动脉旁路移植术手术后对心肌缺血再灌注损伤起到保护作用,但其相关的机制不是很清楚。本研究首次分析了吸入性七氟醚处理组特异性差异表达基因、静脉注射丙泊酚处理组特异性差异表达基因以及两组共有的差异表达基因,共三组差异表达基因,通过比较七氟醚和丙泊酚使用后引起的基因表达上的差异来分析两种麻醉方式的作用特点,以期找到影响二者作用效果的关键性基因。随后本研究又对三组差异表达基因进一步进行了功能注释和分析,并探索了三组差异表达基因所在的互作功能模块,然后对互作功能模块进行功能富集分析。以期从更加细致的分子互作角度阐释吸入性七氟醚麻醉和静脉注射丙泊酚麻醉的心肌保护机制。本研究的意义在于从基因层面阐述吸入性麻醉剂七氟醚和静脉麻醉剂丙泊酚在冠状动脉旁路移植手术中心肌细胞保护机制的作用特征和关键性的作用基因,为探索静吸复合麻醉方式在该手术中可能发挥的作用提供理论依据。
[Abstract]:Coronary artery bypass grafting ( CABG ) is an effective method for coronary artery bypass grafting ( CABG ) and coronary artery bypass grafting ( OPCABG ) for coronary artery bypass grafting ( CCABG ) and off - pump coronary artery bypass grafting ( OPCABG ) . OPCABG has been widely used in the treatment of coronary heart disease . However , OPCABG is widely used in the treatment of coronary heart disease .

The results showed that the levels of IL - 10 and IL - 1 receptor in patients with coronary artery bypass grafting could be reduced by lowering the level of myocardial ischemia induced by myocardial ischemia .

Although a large number of researches have been conducted to prove that the heptafluoroether and propofol have an effective cardioprotective effect , the potential mechanism of myocardial protection is still unclear . The research uses bioinformatics technology to analyze the specific differential expression gene of the inhalation heptafluoroether treatment group , the specific differential expression gene of intravenous propofol treatment group and the two groups of common differential expression genes , and the three groups of differentially expressed genes are analyzed .
Then we performed functional annotation and analysis on three groups of differentially expressed genes .
In order to find out the mechanism of myocardial protection in anesthesia and intravenous injection of propofol from more detailed molecular interaction , this paper lays a theoretical foundation for the protection of myocardium in the perioperative period of CABG . The results are as follows :

1 . The expression profiles of myocardial tissue microarray in coronary artery bypass grafting ( GSE4386 ) were downloaded from GEO ( Gene Expression Omnibus ) . The expression profiles of differentially expressed genes induced by propofol group and propofol group were screened by Affy packet in R language .

2 . Compared with the differentially expressed genes screened in the group of propofol anesthesia and intravenous anesthetic propofol , the specific differential expression genes were obtained under the induction of the inhalation of heptafluoroether , 290 of which were specific differential expression genes and 275 differentially expressed genes which were induced by intravenous anesthetics .
There was significant difference in the expression level of propofol in propofol group ( P = 0.02206 ) .
There was a difference between the two groups in the two groups ( P = 2.98e - 14 ) , that is , the results of the analysis were consistent with the screening results of the differentially expressed genes .

3 . GO analysis was carried out on the specific differential expression gene of the group of heptafluoroether and propofol by the DAVID online tool to enrich the key biological processes involved in these differences .
The specific differential expression genes in propofol treatment group were most significantly involved in response to organic matter ( n = 45 ) . The comparison between the two groups of specific differential expression genes and the genes annotated by the common differential expression genes was compared with the COG direct homologous cluster database . The COG functional classification of three types of differential expression genes from cellular _ component , molecular _ function and biological process was very similar .

4 . The functional modules of the specific differential expression gene were identified as : CISH , PTPN1 , SOCS1 , IL15RA , RCAN1 , IL6R , FOSB , JUNB and TNFAIP3 .

In this study , the cardioprotective effects of heptafluoroether and propofol in CABG were systematically reviewed from the molecular biology perspective . The results showed that the combination of the inhalation anesthetic and propofol had a synergistic effect in the protection of cardiac muscle .

Subject innovation and significance

In this study , we analyzed the specific differential expression gene , intravenous propofol treatment group specific differential expression gene and two groups of different expression genes , and then analyzed the functional characteristics of the three groups of differentially expressed genes , and then analyzed the functional enrichment of the three groups of differentially expressed genes .

The significance of this study is to elucidate the role and key role of propofol and propofol propofol in coronary artery bypass grafting ( CABG ) in coronary artery bypass grafting ( CABG ) , and provide theoretical basis for exploring the possible role of propofol in the operation .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R614.1

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