大鼠胰岛β细胞GPR40表达与内脏脂肪含量及胰岛素分泌的关系

目的:观察SD大鼠胰岛β细胞G蛋白偶联受体40(GPR40)的表达与内脏脂肪含量及胰岛素1相分泌之间的关系.方法:大鼠按体质量分为3组(100 g、200g及300 g组),行静脉糖耐量实验,胶原酶原位灌注法分离胰岛,免疫组织化学法结合激光扫描共聚焦显微镜技术对胰岛β细胞表达的GPR40进行定位,并行半定量分析.结果:随着大鼠体质量的增加,内脏脂肪含量明显增加,300g组大鼠胰岛β细胞GPR40表达较100g及200g组明显增加,3组大鼠胰岛素1相分泌无显著差异.结论:GPR40表达的变化可能与内脏脂肪含量及年龄因素有关,其表达水平对正常大鼠葡萄糖刺激的胰岛素分泌无明显影响.     

猪脂肪组织和原代脂肪细胞中GPR43基因的转录表达规律

根据GenBank已发表的人、小鼠及大鼠GPR43(G protein-coupled receptor 43)基因序列, 设计并合成一对引物, RT-PCR扩增获得猪GPR43基因cDNA, 并利用PCR技术检测该基因在不同猪种、不同发育阶段、不同部位脂肪组织及原代脂肪细胞中的转录表达规律。结果显示, 成功克隆猪GPR43 cDNA片段, 长度为486 bp (GenBank登陆号为EU122439); 同源性分析发现, 猪GPR43与人、小鼠和大鼠同源性达83%以上; GPR43 mRNA表达量在脂肪型猪种上显著高于瘦肉型猪种, 随月龄增长表达量逐渐上升, 且皮下脂肪表达量较内脏脂肪高; 在猪前体脂肪细胞诱导分化过程中, GPR43 mRNA表达量呈时间依赖性升高。揭示GPR43 mRNA表达与猪肥胖程度、年龄、脂肪沉积部位以及脂肪细胞分化程度密切相关。     

中链脂肪酸受体GPR84对小鼠糖脂代谢的影响

目前,全球肥胖及肥胖引发的并发症如2型糖尿病、心血管疾病等患病率呈上升趋势,并逐渐成为一个重大的公共卫生问题.与肥胖相关疾病的发病机制是由多种因素共同作用的结果,游离脂肪酸受体在其中就扮演着重要角色.G蛋白偶联受体84(GPR84)是中链脂肪酸(C9-C14)受体,其在糖脂代谢方面的功能尚不清楚.因此,探究中链脂肪酸受体GPR84在小鼠糖脂代谢方面的作用,具有重要意义.本文利用高脂饲料在GPR84野生型(WT)和基因敲除型(GPR84~(-/-))小鼠中诱导肥胖模型.研究发现,在常规饲料(normal chow,NC)喂养组和高脂饲料(high-fat diet,HFD)喂养组,与WT小鼠相比,GPR84~(-/-)小鼠在体重、摄食量、葡萄糖耐量、胰岛素敏感性、空腹血糖值、血清中胰岛素含量、组织器官重量、脂质合成与脂肪酸氧化、白色脂肪形成方面无显著性差异;在血脂水平,GPR84~(-/-)小鼠与WT小鼠相比,甘油三酯、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇无显著性差异;但是在高脂饲料喂养组,GPR84~(-/-)小鼠总胆固醇浓度显著性降低.同时,我们发现,在HFD组,与WT小鼠相比,GPR84~(-/-)小鼠肝脏中B类Ⅰ型清道夫受体表达显著上调.综上所述,中链脂肪酸受体GPR84在高脂诱导的肥胖模型中不影响小鼠糖脂代谢,但可能在由高胆固醇引起的高胆固醇血症发挥一定的功能.     

Kiss1和GPR54基因多态性与多囊卵巢综合征相关性分析

本研究旨在探讨Kiss1和GPR54基因多态性与多囊卵巢综合征的相关性。利用超声检查卵巢体积、血清睾酮、游离雄激素指数情况;临床评估患者身高(cm)和体重(kg)、BMI、静息血压、痤疮和黑棘皮病的分布;ELISA酶联免疫法检测血清中的kisspeptin和睾酮水平,使用Next generation sequencing方法(LGC group, Germany)对基因(Kiss1, GPR54)进行测序。结果显示,PCOS患者比对照组女性具有更高的BMI和mFG评分,PCOS患者血清Kisspeptin和睾酮浓度显著提高,且LH浓度也显著高于对照组(p<0.05)。GPR54和Kiss1 2个基因在患者体内存在多态性;测序分析结果显示GPR54基因存在的2个新的SNP位点(chr19:918686, A→G和chr19:918735, A→G),这2个新的多态性位于内含子区域(内含子2),Kiss1基因也存在两个SNP,位于非翻译变体5的末端(rs5780218)和外显子3 (rs4889),即GPR54基因存在A→G多态性,Kiss1基因为CTT→CT/G→C多态性,且相关性关联分析结果表明,GPR54基因型多态性(Chr19:918735)与PCOS风险增加相关(p<0.05);而Kiss1 SNP的基因型与PCOS风险之间没有关联。此外,PCOS与GPR54和Kiss1基因的单倍型没有显著关联。本研究推论对PCOS发生风险的遗传影响可能不仅是通过直接改变Kiss1/GPR54相互作用,而且还可能通过改变个体与环境因素的相互作用。     

Kisspeptins/GPR54神经内分泌系统对哺乳动物生殖活动的调控

哺乳动物的生殖活动受到神经内分泌系统的精确调控。近年来的研究表明,KISS-1基因的表达产物Kisspeptins及其G蛋白偶联受体（G protein coupled receptor 54,GPR54）在这一调控网络中发挥着重要作用,与哺乳动物青春期启动、发情排卵及季节性繁殖等过程密切相关。该文就近年来Kisspeptins/GPR54系统对哺乳动物生殖活动调控的研究进展进行简述。     

KISS-1/GPR54基因及其在生殖中的作用

KISS-1及其受体GPR54基因对青春期的正常启动具有重要作用。青春期开始前后, 动物下丘脑中KISS-1和GPR54 mRNA水平很高, Kisspeptins(KISS-1基因产物)通过激活GPR54增加促性腺激素的释放, KISS-1基因的表达受性腺类固醇激素的调控。GPR54基因突变可以导致人和鼠的特发性促性腺激素分泌不足性腺机能减退症和促性腺激素依赖性性早熟。文章还介绍了KISS-1、GPR54基因的结构、表达、多态性以及和其它生殖调控因子之间的相互关系。     

特异性干扰ICP27基因的siRNA对HSV-1病毒复制的影响

针对单纯疱疹病毒1型(herpes simplex virus 1,HSV-1)的ICP27基因和其他疱疹病毒相关基因的高度保守区设计小干扰RNA(small interfering RNA,siRNA),研究其抑制病毒复制的效果。首先构建相应的小发夹RNA(small hairpin RNA,shRNA),然后通过病毒滴度测定、real-time PCR和细胞致病变效应(cytopathic effect,CPE)检测所设计的siRNA抑制病毒复制的能力。结果显示,所设计的shRNA-2(靶序列起始位置815)和shRNA-3(靶序列起始位置1 367)具有明显地抑制病毒复制的效果。尤其是shRNA-3,抑制病毒复制的效果更明显,在病毒滴度实验中,与阴性对照相比,其抑制倍数为81,同时可以下调ICP27基因的mR NA表达水平。实验结果表明shRNA-3能够显著抑制HSV-1病毒复制的能力,可以作为HSV-1感染性疾病的补充治疗手段,其对应的靶序列可以作为抗HSV-1新的靶标。     

下调Fsp27基因表达联合杨梅素干预对3T3-L1细胞脂解的影响

目的:下调脂肪特异性蛋白27(Fsp27)基因表达联合杨梅素干预,观察对3T3-L1细胞中脂质代谢的影响,并探究脂滴发生、发展变化的调控机制。方法:常规培养3T3-L1前脂肪细胞,采用\"鸡尾酒\"法诱导其分化为成熟脂肪细胞。脂质体法转染sh-Fsp27干扰载体,以杨梅素浓度为100μmol/L的完全培养基干预成熟脂肪细胞72h。油红O染色,观察脂滴形态及大小的变化;酶法测定细胞内甘油及甘油三酯的含量,观察细胞脂质代谢的变化。Western blot检测Fsp27、激素敏感性甘油三酯脂肪酶(HSL)、甘油三酯脂肪酶(ATGL)以及丝裂原活化蛋白激酶(MAPK)信号通路蛋白的表达。结果:1. 3T3-L1细胞诱导分化后,形态由纤维样变成圆形,并伴随有细胞体积的增大。2.与对照组相比,杨梅素组和转染组细胞中甘油三酯含量下降,甘油含量升高(P 0. 05)。与其他三组相比,联合干预组细胞中甘油三酯含量减少,甘油含量增加(P 0. 05)。3.与对照组相比,其余三组细胞内Fsp27蛋白的表达量均降低,ATGL和PPARγ的表达量升高(P 0. 05)。另外,联合干预组和杨梅素组细胞内HSL的表达量和p-p38MAPK/p38MAPK的比值均大于sh-Fsp27组和对照组(P 0. 05)。结论:1. Fsp27基因沉默与杨梅素联合干预可以更大程度地促进脂肪分解代谢。2.杨梅素可通过激活MAPK信号通路,上调HSL和ATGL的蛋白表达来发挥其促脂解的作用; sh-Fsp27干扰载体通过调节PPARγ和Fsp27蛋白的表达,增加ATGL含量来加速脂肪分解。     

舒林酸调节IKK通路对3T3-L1细胞IRS-1酪氨酸磷酸化的影响

目的探讨舒林酸通过调节IKK通路对分化成熟3T3-L1细胞胰岛素受体后信号转导蛋白胰岛素受体底物1（IRS-1）蛋白酪氨酸/丝氨酸（Tyr/Ser）残基磷酸化表达的影响。 方法用地塞米松、IBMX和胰岛素三联培养诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色观察脂肪细胞形态。诱导分化成熟的脂肪细胞如下分组干预,实时荧光定量PCR检测不同浓度炎症因子IL-1 β（0,1,10,100 ng/ml）和（或）不同浓度IKK特异阻断剂舒林酸（0,0.1,1,10 mmol/L）对诱导分化成熟的脂肪细胞IKK通路激活状态的影响。Western Blot检测IL-1β和（或）舒林酸对诱导分化成熟的脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化状态的影响。采用单因素方差分析进行统计学分析。 结果实时荧光定量PCR和Western Blot结果显示,IL-1β 10 ng/ml组诱导成熟脂肪细胞IKKβ mRNA较对照组相对表达水平增加,分别为[（2.85±0.16）﹪,（1.00±0.12）﹪,P < 0.01];而IRS-1酪氨酸的磷酸化相对表达量较对照组下降,分别为[（0.72±0.26）﹪,（1.00±0.24）﹪,P < 0.01]。进一步予舒林酸（1?mmol/?L、10?mmol/L）干预后较对照组显著逆转IL-1β诱导脂肪细胞IRS-1酪氨酸磷酸化的表达水平,分别为[（1.72±0.16）﹪,（1.90±0.08）﹪,（1.00±0.13）﹪,P < 0.01],同时下调IRS-1丝氨酸磷酸化的表达水平[（0.79±0.16）﹪,（0.66±0.08）﹪,（1.00±0.10）﹪,P < 0.05]。 结?论IL-1β通过促进诱导分化成熟脂肪细胞IKKβ的表达,激活脂肪细胞IKK炎症通路,抑制脂肪细胞IRS-1酪氨酸残基磷酸化的表达,舒林酸通过调节脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化的表达,改善脂肪细胞胰岛素受体后信号转导。     

沉默IQGAP1基因对小鼠胰腺祖细胞增殖的影响

为探讨沉默IQ结构域三磷酸鸟苷合酶-激活蛋白1(IQ motif containing GTPase-activating protein 1,IQGAP1)基因的表达对胰腺祖细胞增殖的影响,本研究将靶向IQGAP1基因的小干扰RNA(siRNA)转染胰腺祖细胞,qPCR,Western blotting验证IQGAP...     

Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes

To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function.     

Amelioration of β^654-thalassemia in mouse model with the knockdown of aberrantly spliced β-globin mRNA

Large amounts of aberrantly spliced mRNA from the β654 allele was present in erythroid cells, which might impair the erythropoiesis.A therapeutic strategy for β-thalassemia was explored by knocking down the aberrantly spliced mRNA of β-globin. Lentiviral vector with siRNA fragment targets on the specific portion of β654-globin aberrantly spliced pre-mRNA was constructed. In HeLa β654 cells, the siRNA vector could reduce approximately 60% of aberrantly spliced mRNA, which was assessed by RT-PCR and qRT-PCR. Furthermore, a disease model of β654 thalassemia mice with lentiviral-mediated siRNA was produced by subzonal injection (named Hβi-Hbbth-4/Hbb+transgenic mice). Our results showed that the hemotological parameters were improved in Hβi-Hbbth-4/Hbb+ transgenic mice. This study provides a potential way for β654-thalassemia therapy by knocking down the aberrantly spliced β-globin mRNA, whilst supporting that the aberrantly spliced β-globin mRNA may aggravate the disease.     

RNA干扰与技术

RNA干扰(RNA interference,RNAi)是由双链RNA诱导的、序列特异的基因沉默机制。它是自然存在于植物、线虫和果蝇中抵抗外来基因(包括病毒、转座子)入侵的方式。在哺乳动物细胞中,能够人工诱导RNA干扰,沉默有同源序列基因表达。这一新技术具有特异性、高效性。因此,正被用来研究人类基因组的功能、肿瘤和抗病毒感染等。     

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.     

Identification and characterization of small RNAs involved in RNA silencing

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.     

Genome-wide application of RNAi to the discovery of potential drug targets

Progress is being made in the development of RNA interference-based (RNAi-based) strategies for the control of gene expression. It has been demonstrated that small interfering RNAs (siRNAs) can silence the expression of target genes in a sequence-specific manner in mammalian cells. Various groups, including our own, have developed systems for vector-mediated specific RNAi. Vector-based siRNA- (or shRNA) expression libraries directed against the entire human genome and siRNA libraries based on chemically synthesized oligonucleotides now allow the rapid identification of functional genes and potential drug targets. Use of such libraries will enhance our understanding of numerous biological phenomena and contribute to the rational design of drugs against heritable, infectious and malignant diseases.     

Altered function and expression of the orphan GPR135 at the cardiovascular level in diabetic Wistar rats

Abstract

Cardiovascular complications are the main cause of mortality in patients with diabetes, these have been associated with changes in function and expression of receptors coupled to G proteins (GPCR), which include orphan receptors which some of them tend to modify in diabetes, although others are not known, such as GPR135. For this reason, the objective of this work was to study the expression of the orphan receptor GPR135 in brain, heart, kidney, aorta, lung, spleen and liver of diabetic rats, as well as its function by the administration of siRNA (small interfering RNA) and curves to isoproterenol. Our results showed that GPR135 is expressed in all tissues analyzed and its expression is modified due to diabetes, we also observed that the responses to isoproterenol increase in diabetic rats administered with siRNA. Therefore, we conclude that the orphan receptor GPR135 is expressed in different tissues and its expression tends to be modified due to diabetes, besides that it is functional and that it seems to be coupled to Gi/o protein which has negative chronotropic and inotropic effects, therefore, we do not rule out that it participates in the cardiovascular complications associated with diabetes.     

Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.