低硒大鼠心肌组织中微小RNA的差异表达研究

目的筛选低硒大鼠心肌组织中差异表达的微小RNA(miRNA),为克山病的发病机制研究提供参考。方法采用随机数字表法将30只清洁级SD大鼠分为对照组、低硒组、补硒组,每组10只。对照组大鼠给予AIN-93标准饲料喂养,低硒组及补硒组大鼠均给予AIN-93 M(低硒)饲料喂养;3组大鼠均喂养14周。之后补硒组大鼠给予亚硒酸钠溶液灌胃,对照组和低硒组大鼠则给予相同剂量蒸馏水灌胃;3组大鼠均连续灌胃3周。入组17周后留取3组大鼠外周血检测血清硒及血浆脑钠肽(BNP)水平;采用基因芯片测序筛选差异表达的miRNA,并行聚类分析及靶基因功能显著性分析;采用实时荧光定量聚合酶链反应(PCR)检测差异表达显著的miRNA表达情况。结果 (1)低硒组和补硒组大鼠血清硒水平低于对照组,血浆BNP水平高于对照组(P0.05);低硒组大鼠血清硒水平低于补硒组,血浆BNP水平高于补硒组(P0.05)。(2)基因芯片测序结果显示,有40个miRNA为硒敏感性miRNA。(3)基因本体(GO)功能富集分析结果显示,上述40个差异表达miRNA靶基因功能主要富集于脂代谢、心脏发育、细胞黏附、血管发育、轴突导向、细胞迁移调控、输尿管芽发育、跨膜受体蛋白酪氨酸激酶信号通路、细胞分化、有机氮化物反应、棕色脂肪分化、髓系祖细胞分化、细胞-基质黏附、细胞增殖调控、生物钟节律。靶基因投射的13条信号转导通路为肾细胞癌、转录调节、多巴胺能突触、焦点黏附、Rap信号通路、脂代谢、PPAR信号通路、Ras信号通路、Oxytocin信号通路、长期抑制、PI3K-Akt信号通路、MAPK信号通路、HTLV-1感染。(4)以表达最显著的8种miRNA作为验证基因,以对照组心肌组织miRNA为参照,补硒组大鼠心肌组织miRNA-374、miRNA-16、miRNA-199a-5p、miRNA-195、miRNA-30e*相对表达量低于低硒组,心肌组织miRNA-3571、miRNA-675、miRNA-450a*相对表达量高于低硒组(P0.05)。结论低硒会导致大鼠心功能损伤,低硒大鼠心肌组织中存在多个差异表达的miRNA,主要涉及15种生物学功能和13条信号转导通路,这些差异表达的miRNA可能参与克山病的发生发展。     

低硒与大鼠心肌组织脂质过氧化及心肌细胞损伤的关系

目的 :观察低硒喂养对大鼠心肌组织脂质过氧化物、心肌细胞超微结构及细胞凋亡的影响。方法 :将SD大鼠分为低硒饲料喂养及低硒饲料加硒喂养两组 ,至 12周时测血硒、谷胱甘肽过氧化物酶 （GPX）、心肌组织匀浆丙二醛 （MDA）水平 ,行心肌组织超微结构电镜观察及心肌细胞凋亡检测。结果 :低硒喂养组大鼠血硒及GPX水平较加硒喂养组显著为低 ,心肌组织匀浆MDA水平显著升高 ,心肌细胞线粒体肿胀增大 ,线粒体嵴排列紊乱 ,部分嵴溶解 ,部分线粒体空泡变 ,心肌细胞凋亡发生率显著升高。结论 :低硒可致心肌过氧化物损伤、心肌细胞线粒体损伤及细胞凋亡。     

低硒对雄性大鼠性腺发育和分泌功能的影响

目的　研究雄性动物性腺的发育和功能与硒的关系。方法　分别以低硒饲料、补硒饲料和常规饲料喂养刚分窝的雄性Ｗ ｉｓｔａｒ 大鼠１４ 周和３９ 周后,分别采用２,３－二氨基萘（ＤＡＮ）荧光分光光度法和化学发光酶免疫法测定血和睾丸硒含量及血浆睾酮水平等指标。结果　饲养１４ 周后,低硒组大鼠不仅血和睾丸硒含量以及睾丸谷胱甘肽过氧化物酶（ＧＰｘ）活性显著低于对照组,而且其精囊腺重量、精囊腺指数和血浆睾酮水平等也显著低于对照组,但其睾丸中脂质过氧化物（ＬＰＯ）水平则显著高于对照组（ Ｐ ＜ ０．０５）。在低硒饲料中补充一定量的硒可在一定程度上纠正这些变化。饲养３９ 周后,低硒组精囊腺指数进一步降低,与对照组的差异达极显著程度（ Ｐ ＜ ０．０１）,甚至睾丸指数也降至显著低于对照组。结论　这些结果提示,硒不足对雄性动物性腺的发育和分泌功能有不良影响,适当补硒可作为男性不育症的辅助治疗手段。     

硒酵母和亚硒酸钠对克山病病区粮喂养大鼠血小板血栓素的影响

应用克山病病区低硒粮饲料喂养大鼠１４周，探讨补硒对血小板血栓素生成的影响。结果表明，补充两种硒制剂均可明显增高低硒大鼠血浆硒水平和血小板ＧＳＨ－Ｐｘ活性，降低ＡＤＰ诱导的血小板ＴＸＡ２水平，两种硒制剂的作用程度相似；补硒对血浆前列环素水平无显著影响。     

富含硒玉米对低硒大鼠血硒和GSH-Px活性的影响

为了研究富含硒玉米对低硒粮饲料喂养大鼠血硒状态的影响 ,观察了补充富含硒玉米或亚硒酸钠时和停止补硒后大鼠血硒水平和谷胱甘肽过氧化物酶 ( GSH- Px)活性的变化。结果显示 :1两种补硒形式均可有效地增高血浆、红细胞的硒水平和红细胞的 GSH- Px活性 ;2富含硒玉米增高和维持血浆和红细胞硒水平作用与亚硒酸钠相同 ;3富含硒玉米升高红细胞 GSH- Px活性的作用低于亚硒酸钠 ,但停止补硒后两者维持 GSH- Px活性作用相似。表明富含硒玉米能有效地改善低硒大鼠的血硒状态     

低硒环境下蛋氨酸对大鼠体内维生素E,硒及脂质过氧化物含量的影响

用低硒、低蛋氨酸饲料喂养大鼠8周后发现大鼠血清、肝脏及心肌中脂质过氧化物含量显著升高,血清维生素E显著下降,而心肌硒含量却显著升高。实验结果表明,在低硒环境下如降低饲料中蛋氨酸水平将导致大鼠体内脂质过氧化反应加剧。     

T-2毒素致低硒低蛋白大鼠心肌损伤实验观察

目的 探讨低硒低蛋白条件下T-2毒素对大鼠心肌的损伤作用以及补硒对该损伤的保护作用。方法 应用人工合成的低硒低蛋白饲料喂养Wistar大鼠，达到低硒状态后(6周)给予T-2毒素，按0．2mg／kg体重隔日灌胃24周，取大鼠心肌组织进行常规HE染色和电镜观察．并利用免疫组化技术检测心肌中的T-2毒素的含量。利用末端标记法检测心肌细胞凋亡发生情况。结果 T-2毒素灌胃组大鼠心肌T-2毒素表达明显，但灌胃各组之间差异不明显；低硒低蛋白组心肌损伤明显重于常硒常蛋白组．补硒保护作用明显。结论 T-2毒素能够在心肌组织中残留。低硒低蛋白能够加重T-2毒素对心肌的损伤作用．补硒能够起到一定的保护作用。     

克山病区低硒粮饲养大鼠心脏收缩功能与舒张功能的变化

将Sprague-Dawley大鼠随机分为三组,分别用克山病区低硒粮饲料、病区粮加Na_2SeO_3饲料和本校常规饲料喂养,用以研究克山病区低硒粮对心脏收缩功能与舒张功能的影响。实验结果发现,动物饲养两月时,与补硒组及常规饲料组相比,低硒组大鼠的T值显著延长(P<0.05);动物饲养三月时,低硒组大鼠的dp/dt max、-dp/dt max显著减小(P<0.05),T值继续延长(P<0.05)。实行冠状动脉结扎术后,三组动物的各项心功能指标均发生严重损害,但常规饲料组的损害较轻,与低硒组相比,dp/dt max和T值有显著性差别(P<0.05)。实验结果说明,克山病区低硒粮能造成心脏收缩功能与舒张功能的损害,补硒则有保护作用。     

硒对心肌线粒体机能影响的研究Ⅲ.硒对大鼠心肌线粒体~(45)Ca摄取影响的动态观察

本文用克山病病区低硒和补硒饲料分别饲养大鼠,动态观察喂养1、2、3个月时硒对动物心肌线粒体~(45)Ca摄取的影响,结果表明低硒饲料可显著降低各月大鼠心肌线粒体的钙摄取,并且随喂养月份的增加降低得越来越显著。提示克山病病区低硒饲料使动物心肌线粒体钙含量升高的机理不在于钙摄取的增加,而可能与其钙释放的障碍有关。     

硒对克山病病区粮喂养大鼠血小板聚集性和5—HT释放反应的影响

用克山病病区低硒粮饲料喂养大鼠14周,探讨补硒(亚硒酸钠或硒酵母)对血小板聚集性和5—HT释放反应的影响。结果表明,补充两种硒制剂均能增加低硒大鼠血浆硒水平和血小板GSH—Px活性,降低ADP诱导的血小板聚集性和5—HT释放反应;两者的影响程度相似;补硒对血小板5—HT含量无显著影响。     

硒与维生素E的协同影响对心肌损伤保护的研究

通过在低硒富锰饲料中联合补充硒及ＶＥ喂养大鼠，并以亚硝酸钠作为诱发因素建立大鼠心肌损伤模型，观察硒与ＶＥ的协同作用对心肌损伤的保护效果。结果表明，在低硒环境下，富锰能显著提高心肌坏死检出率及降低机体抗氧化能力。单纯补充硒及ＶＥ均可对抗富锰的影响，但硒与ＶＥ的联合补充效果更佳。     

Dysregulated microRNA expression in rheumatoid arthritis families—a comparison between rheumatoid arthritis patients,their first-degree relatives,and healthy controls

Objective

Recent studies have demonstrated an altered expression of certain microRNAs in patients with rheumatoid arthritis (RA) as well as their first-degree relatives (FDRs) compared to healthy controls (HCs), suggesting a role of microRNA in the progression of the disease. To corroborate this, a set of well-characterized RA families originating from northern Sweden were analyzed for differential expression of a selected set of microRNAs.

Method

MicroRNA was isolated from frozen peripheral blood cells obtained from 21 different families and included 26 RA patients, 22 FDRs, and 21 HCs. Expression of the selected microRNAs miR-22-3p, miR-26b-5p, miR-34a-3p, miR-103a-3p, miR-142-3p, miR-146a-5p, miR-155, miR-346, and miR-451a was determined by a two-step quantitative real-time polymerase chain reaction (qRT-PCR). Statistical analysis including clinical variables was applied.

Results

Out of the nine selected microRNAs that previously have been linked to RA, we confirmed four after adjusting for age and gender, i.e., miR-22-3p (p?=?0.020), miR-26b-5p (p?=?0.018), miR-142-3p (p?=?0.005), and miR-155 (p?=?0.033). Moreover, a significant trend with an intermediate microRNA expression in FDR was observed for the same four microRNAs. In addition, analysis of the effect of corticosteroid use showed modulation of miR-103a-3p expression.

Conclusions

We confirm that microRNAs seem to be involved in the development of RA, and that the expression pattern in FDR is partly overlapping with RA patients. The contribution of single microRNAs in relation to the complex network including all microRNAs and other molecules is still to be revealed.

无淋巴结转移结肠癌微小核糖核酸表达谱的初步研究

目的:探讨无淋巴结转移结肠癌微小核糖核酸(microRNA)表达谱,筛选与结肠癌早期发生、发展相关的特异性microRNA.方法:选取手术、病理证实未发生淋巴结转移的结肠癌及大于癌旁5 cm结肠组织标本各3例,抽提分离miRNA,与Agilent microRNA基因芯片杂交,并进行图像和数据分析.运用microRNA特异引物对差异表达的microRNA进行荧光定量实时聚合酶链反应(RT-RCR)验证.结果:筛选出在结肠癌中差异表达的microRNA有14个,其中12个microRNA表达水平增高,分别为miR-106b、miR-135b、miR-18a、miR-18b、miR-196b、miR-19a,miR-224、miR-335、miR-424、miR-20a*、miR-301b和miR-374a;2个microRNA表达水平降低,分别为miR-378和miR-378*.miR-106b和miR-19a下游存住符合要求的靶基凶数目众多.检测了芯片中显示表达上凋的miR-18a和miR-135b在3例结肠癌组织和癌旁组织中的表达,结果显示miR-18a和miR-135b在3例痛组织和癌旁组织中表达均上调;荧光定量RT-PCR结果与microRNA芯片的结果符合.结论:miR-106b、miR-135b、miR-18a、miR-18b、miR-196b等microRNA在结肠癌与癌旁组织中存在差异表达,预测其下游靶基因中大部分与肿瘤相关,提示它们可能在结肠癌早期发生、发展讨程审起着重要的调控作用.     

硒和维生素E对大鼠心肌单胺类物质和单胺氧化酶的影响

本文观察了硒（Ｓｅ）和维生素Ｅ（ＶＥ）对大鼠心肌去甲肾上腺素（ＮＥ）、多巴胺（ＤＡ）、５羟色胺（５－ＨＴ）含量和单胺氧化酶Ａ（ＭＡＯ－Ａ）、单胺氧化酶Ｂ（ＭＡＯ－Ｂ）活性的影响，发现经低Ｓｅ、低ＶＥ饲料喂养１０周，并且处死前经２次冰浴刺激的大鼠心肌ＮＥ含量明显增高，ＭＡＯ－Ａ活性降低，ＭＡＯ－Ｂ活性增加，通过补充饲料中Ｓｅ和ＶＥ，对上述指标有不同程度的改善，并以联合补充Ｓｅ和ＶＥ效果最佳。本研究结     

MicroRNA expression pattern in intraductal papillary mucinous neoplasm

Background/Aims: Intraductal papillary mucinous neoplasms (IPMN) are precursor lesions of fatal pancreatic cancer. Physiological function of microRNA is to regulate the stability and translation of mRNA. The aberrant microRNA expression is commonly observed in many cancers. The aim of this study was to analyze the expression pattern of microRNA in IPMN and evaluate the role of the microRNA.Methods: Using two paraffin-embedded IPMN tissues, microRNA expression of normal tissue, IPMN adenoma and carcinoma were compared by cDNA-mediated annealing, selection, extension and ligation microarray assay. Using real time PCR, expression levels of aberrantly up-regulated microRNAs were assessed in another 20 IPMNs, four pancreatic cancer cell lines (Panc1, MiaPaCa-2, XPA-3, BxPC-3) and immortalized pancreatic ductal cell line (HPNE). Effect of suppressing highly over-expressed two microRNAs in pancreatic cancer cell lines with anti-microRNA inhibitors were evaluated using CCK-8 assay.Results: Among aberrantly expressed 122 microRNAs in IPMN, miR-552, miR-25*, miR-183, miR-1300, miR-196a, miR-182*, and miR-30c-1* were consistently increased more than 3-fold. On average, miR-196a and miR-183 increased 10,824 folds and 26,519 folds in four pancreatic cancer cell lines compared with HPNE. These two microRNAs were also over-expressed in 20 IPMNs compared with HPNE. After applying anti-miRNA inhibitors, cell survival of four pancreatic cancer cell lines decreased by 24.5% with anti-miR-196a and by 14.2% with anti-miR-183 on average.Conclusions: Aberrant expression of 122 microRNAs was observed in IPMN. Two microRNAs, miR-196a and miR-183-increased in IPMN and pancreatic cancer cell lines compared with immortalized dancreatic ductal cell line. The inhibitions of these microRNAs repressed cell proliferation of pancreatic cancer cell lines. (Korean J Gastroenterol 2011;58:190-200).     

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes

Aims/hypothesis

MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease.

Methods

MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells.

Results

MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation.

Conclusions/interpretation

We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.     

Identification and study of differentially expressed miRNAs in aged NAFLD rats based on high-throughput sequencing

Introduction and objectivesHepatic microRNA (miR) expression profiles were explored in aged rats with NAFLD, in order to clarify the molecular mechanisms underlying the pathophysiological processes of aging-related NAFLD.Patients or materials and methods24 aged rats (18-month-old) and 24 young rats (2-month-old) were randomly divided into two subgroups according to diet, control group and NAFLD group. After 8 weeks of administering 45% high-fat diet or normal diet, total hepatic RNA was extracted from liver tissues of the aged rats. Differentially expressed microRNAs (DE-miRs) in aged NAFLD group were detected and screened out using high-throughput sequencing technology. The data were subjected to Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses using a bioinformatics approach. The sequencing results were further verified by RT-qPCR.ResultsCompared with the aged control liver tissues, 6 significantly upregulated miRs (miR-881-3p, miR-871-3p, miR-335, miR-223-3p, miR-155-5p, miR-146b-5p) and 4 significantly downregulated miRs (miR-182, miR-193-3p, miR-31a-5p and miR-96-5p) were identified in the aged NAFLD liver tissues. These DE-miRs were found to be involved in the regulation of cell signaling transduction and metabolism processes, probably affecting signaling pathways relevant to insulin secretion and some senile diseases. RT-qPCR results corroborated the sequencing results and demonstrated that 6 significantly upregulated miRs were not identified in the young group.ConclusionsA total of 10 DE-miRs identified in the aged NAFLD rats were involved in some certain insulin secretion and age-related functional pathways, which may serve as novel candidate targets for the diagnosis and treatment of aging-associated NAFLD.     

硒和维生素E对大鼠心肌腺苷酸及氧化损伤的影响

目的：研究低硒（Ｓｅ）低维生素Ｅ（ＶＥ）饮食大鼠心肌腺苷酸含量和氧化损伤的关系。方法：使用高效液相测定心肌腺苷酸含量和使用生化方法测定心肌ＭＤＡ含量和ＳＯＤ、ＧＳＨ－Ｐｘ活性。结果：与补充Ｓｅ和／或ＶＥ饮食大鼠相比，低Ｓｅ低ＶＥ饮食大鼠心肌ＡＭＰ，ＡＤＰ含量无明显差异，而ＡＴＰ含量明显降低，ＭＤＡ含量增加，ＳＯＤ，ＧＳＨ－Ｐｘ活性降低，表明Ｓｅ和ＶＥ影响ＡＴＰ含量与其抗氧化作用有关，而其中以联合补充Ｓｅ和ＶＥ效果最佳