脓毒症大鼠肺组织炎症反应相关基因表达变化

目的应用PCR Array技术检测脓毒症大鼠肺组织炎症相关基因的表达,从基因水平探讨其可能的作用机制。方法采用随机数字表法将30只清洁级健康雄性Wistar大鼠等分为对照组、脓毒症组。脓毒症组采用盲肠结扎穿孔术(CLP)进行建模,对照组仅行开腹、关腹,不予盲肠结扎穿孔,两组术毕均肌肉注射平衡液5ml/kg,并于术后24h取肺组织提取RNA后采用RT2 ProfilerTM PCR Array进行基因检测,应用计算机软件分析比较两组大鼠肺组织炎症相关基因表达的变化。结果大鼠建模后24h,与对照组比较,脓毒症组大鼠肺组织炎症反应相关基因表达12条基因出现表达上调,18条基因出现表达下调,主要涉及趋化因子、生长因子、白介素家族、抗炎相关细胞因子、TNF家族、干扰素。结论肺部炎症反应相关基因失衡的表达可能是脓毒症相关性肺损伤的主要原因。     

持续低氧条件下大鼠肠道微生物变化及其与心肌损伤的关联研究

【目的】研究持续性常压低氧对大鼠肠道微生物的影响,并分析其与低氧性心肌肥厚的关联性。【方法】雌性无特异性病原体(specific-pathogen-free, SPF)级SD (Sprague-Dawley)大鼠,按随机数字表法分为2组：常氧组和低氧组。实验开始后,低氧组大鼠置于低氧舱中,氧气浓度设定为10%,持续暴露30 d,常氧组大鼠正常条件饲养。每天记录大鼠体重,并于低氧前(0 d)和低氧后(30 d)分别收集粪便进行16S rRNA基因扩增子测序,测定肠道微生物组的变化。实验结束后进行血常规、血生化和器官指数分析;采用实时定量聚合酶链式反应(quantitativereal-time polymerasechainreaction,qRT-PCR)检测右心室组织中4种分子标志物心房利钠肽基因(atrial natriureticpeptide,ANP)、脑钠肽基因(brainnatriureticpeptide,BNP)、心肌肌球蛋白重链6基因(myosin heavy chain 6, Myh6)、心肌肌球蛋白重链7基因(myosin heavy chain 7, Myh7)...     

犀角地黄汤对脓毒症大鼠脾脏组织基因表达的影响

目的采用RNA-seq技术检测犀角地黄汤干预后脓毒症大鼠脾脏组织基因的表达变化,从基因水平探究其可能的作用机制。方法将45只清洁级健康雄性Wistar大鼠采用随机数字表法等分为对照组、脓毒症组、犀角地黄汤干预组。脓毒症组采用盲肠结扎穿孔术(CLP)进行建模。犀角地黄汤干预组大鼠除了CLP外,并于术前2天给予中药胃饲(胃饲量即浓煎剂总ml数/60×6.25×2),每天2次,于每天上、下午各给药1次;术后连续胃饲2天。对照组仅行开腹、关腹,不予盲肠结扎穿孔。3组术毕均肌肉注射平衡液5ml/kg;术后24h取脾组织提取RNA后采用RNA-seq进行基因检测,应用计算机软件分析比较3组大鼠脾组织基因表达的变化及涉及的相关通路。结果大鼠建模后24h,相对于对照组,脓毒症大鼠脾脏组织基因表达上调数为1030个,下调数为935个;犀角地黄汤干预组大鼠脾脏组织基因表达上调数为972个,下调数为1149个。犀角地黄汤干预组大鼠基因改变涉及的通路有Th17细胞分化相关通路、TNF信号传导相关通路、IL-17信号传导相关通路、细胞因子-细胞因子相互作用相关通路。结论犀角地黄汤可能主要通过调节Th17细胞分化相关通路、TNF信号传导相关通路、IL-17信号传导相关通路、细胞因子-细胞因子相互作用相关通路的信号表达,进而缓解脓毒症大鼠的炎症反应。     

转染Netrin-1基因的真皮多能干细胞治疗大鼠脊髓损伤的研究

目的:观察转染Netrin-1基因的真皮多能干细胞(dMSCs)移植对大鼠脊髓损伤的修复作用。方法:取大鼠真皮组织,分离培养真皮多能干细胞,经转染Netrin-1基因和诱导,观察细胞形态变化,免疫细胞化学方法对分化细胞进行鉴定。Wistar大鼠在L4水平制成脊髓全横断损伤模型,伤处移植大鼠真皮多能干细胞或者转染Netrin-1基因的真皮多能干细胞。对大鼠进行动物行为学(BBB)评分和对损伤脊髓进行组织学检测。结果:转染Netrin-1基因的dMSCs诱导产生的神经元样细胞占总细胞数的比例为24.45±3.73%,而单独的真皮多能干细胞诱导产生的神经元样细胞占总细胞数的比例10.50±2.13%,二者差异显著(P<0.05)。BBB评分显示转染Netrin-1基因的dMSCs移植组明显高于单纯dMSCs移植组和空白对照组(P<0.05);转染Netrin-1基因的dMSCs移植组损伤脊髓结构的修复明显优于单纯dMSCs移植组和空白对照组。结论:转染Netrin-1基因的真皮多能干细胞移植较单纯dMSCs移植对大鼠脊髓损伤有更好的治疗作用。     

聚己内酯/柚皮素纳米纤维膜对白介素1β诱导的体外骨关节炎的作用研究

目的探讨聚己内酯(PCL)/柚皮素纳米纤维膜对白介素1β(IL-1β)诱导SD大鼠软骨细胞退行性变修复作用。方法使用静电纺丝技术分别制作PCL纳米纤维膜,PCL/柚皮素纳米纤维膜,使用扫描电子显微镜(SEM)观察纳米纤维膜的表征。提取3～5 d的SD大鼠软骨细胞,分为空白对照组、骨关节炎(OA)组、骨关节炎加PCL(OA+PCL)组、骨关节炎加PCL/柚皮素(OA+PCL/柚皮素)组,分别处理24 h后进行CCK-8实验检测各组SD大鼠软骨细胞的增殖情况,并进行活/死细胞染色,观察各组SD大鼠软骨细胞的毒性。提取各组SD大鼠软骨细胞的总RNA,使用qRT-PCR检测炎症及软骨标志基因的表达。结果与OA组相比,PCL/柚皮素纳米纤维膜组大鼠的细胞活性较高,细胞数量明显增多,炎症标志基因表达明显降低,软骨标志基因表达明显增高(均P0.05)。结论 PCL/柚皮素能降低IL-1β诱导的SD大鼠体外骨关节炎症标志基因的表达,对SD大鼠关节炎有一定治疗作用,且能上调关节炎细胞中软骨标志基因COL2a1的表达。     

下调PTEN基因对偏头痛大鼠三叉神经节CREB的调节作用

本研究利用RNAi重组腺病毒(AdR-siPTEN)下调偏头痛大鼠三叉神经节的PTEN(phosphatase and tensin homolog deleted on chromosome ten)基因,探讨其对偏头痛大鼠行为学的影响,以及经Akt(serine-threonine kinase)信号途径对CREB(cAMP responseelement-binding protein)的调控情况。实验采用健康雄性SD大鼠,随机分为假手术组(Sham)、硝酸甘油模型组(GTN)、Ad-RFP非特异siRNA处理空载体对照组(Vehicle+GTN)、AdR-siPTEN下调组(AdR-siPTEN+GTN)。用AdR-siPTEN重组腺病毒对大鼠进行预处理,然后通过硝酸甘油(glyceryl trinitrate,GTN)法建立大鼠偏头痛模型,进行大鼠挠头和爬笼次数的检测,并用RT-PCR和Western-blot法进行相关基因的mRNA和蛋白检测。结果表明,当PTEN基因表达下调时,有效缓解了偏头痛导致的挠头和爬笼行为,并激活Akt信号途径,增加其下游作用因子CREB的表达,进而可能经"PTEN/Akt/CREB"信号通路影响神经突触可塑性,参与了偏头痛的发病机制。     

替米沙坦改善糖尿病大鼠肾脏功能机制研究

目的研究替米沙坦对糖尿病大鼠24 h尿蛋白、血肌酐、肌酐清除率(Ccr)和血清尿素氮(BUN)等相关代谢指标的影响,且应用基因芯片探讨替米沙坦改善肾功能的机制。方法 30只SD大鼠,其中随机选取10只为正常对照组(给予等体积生理盐水)。选用20只大鼠,采用STZ法制备糖尿病模型,而后将16只造模成功的糖尿病大鼠随机分为替米沙坦治疗组(给予10 mg/kg/d的替米沙坦,n=8)和糖尿病模型组(给予等体积生理盐水,n=8)。三组大鼠均连续灌胃12周。每4周测定大鼠空腹血糖(FBG)和体重。12周末测定大鼠24h尿蛋白、尿肌酐、血肌酐和BUN水平。12周末处死大鼠,取肾脏组织进行基因芯片实验,并运用real time PCR进行验证。结果糖尿病模型组24h尿蛋白(P<0.01)、血肌酐(P<0.05)和BUN(P<0.01)比对照组显著升高,Ccr较对照组显著降低(P<0.05)。替米沙坦能改善糖尿病大鼠24h尿蛋白、血肌酐、Ccr和BUN水平。基因芯片结果显示替米沙坦组较糖尿病模型组有1541个基因发生显著改变,其中554个上调,987个下调。基因富集分析显示这些差异表达基因集中在氧化磷酸化通路和PPAR通路。Real time PCR证实替米沙坦组较糖尿病模型组ATP合成酶β亚基(Atp5b)、细胞色素c氧化酶亚基VIc(Cox6c)和NADH脱氢酶(辅酶Q)铁硫蛋白3(Ndufs3)基因显著下调。结论替米沙坦能有效改善糖尿病大鼠肾脏功能。替米沙坦的肾脏改善作用可能是通过线粒体氧化磷酸化通路和PPAR-γ通路调节。     

cDNA RDA在类似普通2型糖尿病大鼠肾脏组织基因差异表达筛查中的应用

应用代表性差异分析 (cDNARDA)技术 ,对类似普通 2型糖尿病大鼠肾脏组织基因差异表达进行筛查 ,初步探讨类似普通 2型糖尿病大鼠肾脏损害发病的分子机制 .首先以类似普通 2型糖尿病大鼠肾脏组织作为实验组 (Tester) ,正常大鼠肾脏作为对照组或驱动组 (Driver)通过cDNARDA进行基因差异表达筛查 ;最终的差异产物亚克隆到Puc 18载体 ,测序及并进行生物信息学分析 ;半定量RT PCR对筛查到新的基因进行初步的鉴定 .结果发现 9个新ESTs ,2个新基因 .这 2个新基因分别与人及小鼠的丝氨酸蛋白酶抑制因子F ,及真核细胞转录启动因子 3亚单位 5 (EIF 3epsilon)基因有高度的相似性 (>90 % )并在类似普通 2型糖尿病大鼠肾脏组织表达上调 .推测 2个新基因分别是大鼠的丝氨酸蛋白酶抑制因子F及真核细胞转录启动因子 3亚单位 5 .两个新基因在类似普通 2型糖尿病大鼠肾脏组织表达上调 ,可能与类似普通 2型糖尿病大鼠肾脏损害相关 .同时 ,对新基因RS91进行了全长cDNA克隆     

红花注射液对气虚血瘀证模型大鼠基因调控作用的研究

目的:采用基因芯片技术,分别构建气虚血瘀证大鼠和红花注射液给药处理后气虚血瘀证大鼠的差异基因表达谱,比较并分析,筛选出红花能够治疗气虚血瘀证的关键基因群,并推测其起治疗作用的基因组调控机制。方法:15只SD大鼠随机分为模型组、给药组、空白对照组。模型组和给药组采用疲劳游泳和饥饿饲养处理。造模一周后,给药组尾静脉注射红花注射液(100mg/kg/d),模型组给予相同体积生理盐水;对照组不做任何处理。造模进行两周后处死大鼠,取血检验血流变指标并评价造模情况;另抽取足够的血分离mRNA并逆转录杂交基因芯片;扫描信号分析确定受红花注射液调控的基因;并通过基因数据库查询相关基因功能,结合相关文献分析初步探讨红花作用的机制。结果:两周后经过检验和观察发现模型组大鼠在不同切率下的全血粘度增加,并且其体征表现出虚弱和瘀血的状态、体重下降,确定造模成功;给药组大鼠则相对于模型组的各项检测指标和状态有所改善,确认药物有疗效。在差异基因的比较中,空白组相对于给药组上调基因252条,下调基因54条;给药组相对于模型组上调基因196条,下调基因32条;两次差异表达基因中有16条相同基因,这些差异基因涉及到炎症损伤、免疫调节反应等方面。结论:红花注射液对于气虚血瘀证有治疗作用,在基因层次上是通过抗炎症损伤机制实现的。     

紫苏干预COPD系统性炎症的大鼠模型研究

为揭示导入紫苏基因的新型烟草的生物学效应,将54只清洁级雄性SD大鼠随机分为健康对照组(CK)、普通烟草组(GT)和新型烟草组(NT),采用烟雾熏吸法构建COPD模型。对血清进行ELISA分析显示,香烟烟雾染毒56 d能诱发COPD大鼠的系统性炎症反应;但与GT组相比,NT组的CRP、IL-8以及TNF-α水平显著降低(P0.05)。取肝和脾组织进行IHC检测发现,COPD大鼠的NF-κB p65表达水平明显升高,且GT组与NT组脾组织间的差异显著(P0.05)。结果表明,导入紫苏基因的新型烟草能够抑制大鼠吸烟相关性COPD的全身性炎症反应,干预肺外组织NF-κB的过度活化,延缓疾病的进程。     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

利用基因芯片技术筛选秦川牛公牛与阉牛肌肉组织差异表达基因

为了筛选秦川牛公牛和阉牛肌肉组织差异表达的基因,探讨二者肉质差异的分子生物学机理,文章利用Affymetrix公司生产的牛基因组芯片技术,分别检测了3头36月龄秦川牛公牛与阉牛背最长肌肌肉组织的mRNA表达水平变化;运用Significance Analysis of Microarrays(SAM)法对秦川牛公牛和阉牛基因表达谱进行了差异分析;并通过分子注释系统平台(MAS2.0)对差异表达基因进行了功能富集类分析和调控通路分析;最后应用实时定量PCR技术对部分差异表达基因进行了验证。基因表达谱分析结果显示,在36月龄秦川牛的肌肉组织中,共检测到约11000个探针,代表大约10000个基因。共筛选出差异表达基因143条,主要涉及胶原纤维的组织和合成、细胞粘附、细胞生长调控、泛素介导的蛋白分解代谢和横纹肌收缩等生物学过程;在分子注释系统数据库中注释到的显著调控通路为细胞外基质受体反应、细胞通讯、焦点粘连和紧密连接等;所验证的差异表达基因的荧光定量PCR结果与芯片结果基本一致。结合已有的文献报道,文章初步认为ECM受体反应、细胞通讯、焦点粘连、紧密接头等调控通路及COL3A1、COL1A1、COL1A2、SPP1、FBN1、MMP2、ECM1、MYH3、MYH8、S100A4、ASPN、CFD等基因可能是参与调控秦川牛阉割前后肉质性状差异的重要调控通路和基因。此外,还筛选出一些尚未在GenBank上登陆的序列,推测可能是未知的新基因,它们在牛肉质代谢过程中的作用还需进一步的研究证明。     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury, the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS. The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorporation of different fluorescence (Cy3, Cy5) labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray chips. The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0. Among the 14112 target genes, 293 genes were found to be significantly differentially expressed, in which 188 genes were up-regulated and 105 genes were down-regulated. Based on the analysis of biological functions of those differentially expressed genes, it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reactions, cell synthesis, metabolism, apoptosis and transportation in liver cell, which might be quite important for elucidating the regulatory network of gene expression associated with the liver damage, also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury,the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS.The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorpora-tion of different fluorescence(Cy3,Cy5) labeled dUTP as the hybridization probes.The mixed probes were hybridized to the cDNA microarray chips.The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0.Among the 14112 target genes,293 genes were found to be significantly differentially expressed,in which 188 genes were up-regulated and 105 genes were down-regulated.Based on the analysis of biological functions of those differentially expressed genes,it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reac-tions,cell synthesis,metabolism,apoptosis and transportation in liver cell,which might be quite im-portant for elucidating the regulatory network of gene expression associated with the liver damage,also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.     

Adenylate deaminase. A multigene family in humans and rats

Multiple AMP deaminase (AMP-D) isoforms have been found in vertebrates, and tissue-specific inherited deficiencies of AMP-D have been described in two unrelated clinical syndromes suggesting there may be more than one AMP-D gene in higher eukaryotes. Using a newly isolated cDNA cloned from an adult rat brain library and a previously reported cDNA cloned from adult rat skeletal muscle, two linked AMP-D genes have been identified in rat and man. ampd1 is expressed at high levels in skeletal muscle of the adult rat. ampd2 is the predominant gene expressed in non-muscle tissues and smooth muscle of the adult rat, and it is also the predominant gene expressed in embryonic muscle and undifferentiated myoblasts. Both genes are expressed in cardiac muscle of the adult rat. The peptides encoded by these two genes have distinct immunological properties. The conservation of nucleotide sequence and exon/intron boundaries in these two genes suggests they arose by duplication of a common primordial gene around 150 million years ago.     

利用GenMAPP筛查鼻咽癌差异表达基因

利用GenMAPP软件对鼻咽癌和正常鼻咽上皮基因微阵列表达谱结果进行分析,筛查鼻咽癌差异表达基因. 结果显示：在17 000个基因中,与正常鼻咽上皮相比,在鼻咽癌中发生2倍以上差异表达的基因共有339个,其中有160个基因在鼻咽癌中表达上调,179个表达下调. 这些基因分别与细胞增殖、基因转录、凋亡、信号转导、DNA损伤修复、肿瘤分化和浸润转移及细胞周期调节等相关. 鼻咽癌的发生发展存在多基因表达调控的改变,对其差异表达基因的研究有助于阐明鼻咽癌发生发展机制.