利用寡核苷酸芯片分析Smad3基因敲除小鼠关节软骨细胞基因表达谱的改变

此前发现 Smad3 基因敲除小鼠(Smad3ex8/ex8)的关节软骨细胞异常肥大分化,出现类似于人类骨关节炎的表型。为了进一步明确转化生长因子-β(TGF-β)/Smad3 信号通过调节哪些靶基因的表达来抑制关节软骨细胞的肥大分化,以及研究骨关节炎发病的分子机制,利用寡核苷酸芯片技术分析了 5 日龄 Smad3 基因敲除小鼠与野生型对照小鼠关节软骨细胞基因表达谱的改变。通过对差异表达基因的分析,发现在 Smad3 基因敲除小鼠软骨细胞中骨形态发生蛋白(BMP)与 TGF-β/细胞分裂周期基因 42(Cdc42)信号通路活性增强。此外,还发现其他信号通路,如生长激素(growth hormone)/胰岛素样生长因子 1(Igf1)以及成纤维细胞生长因子(Fgf)信号通路相关基因表达的改变。值得注意的是,还发现了 Smad3 基因敲除小鼠软骨细胞中蛋白合成与电子传递链相关基因的表达水平普遍上调,这意味着蛋白质合成速率的加快与细胞有氧呼吸的增强可能与关节软骨细胞的肥大分化和骨关节炎的发生相关。     

Notch3在血管平滑肌细胞中对Erk1/2的调控作用研究

Erk1/2活性在血管许多细胞功能中具有重要影响,而Notch3主要表达在动脉平滑肌细胞中,并且是发育过程中动脉成熟所必需的.为了探讨Notch3在血管平滑肌细胞中对Erk1/2信号通路的调控作用,采用siRNA基因敲除Notch3,γ-分泌酶抑制剂DAPT抑制Notch信号通路,质粒转染过表达Notch3活性区等方法,用Western印迹检测Notch3对血管平滑肌细胞中Erk1/2磷酸化水平,即Erk1/2活性的影响.同时,利用活性氧自由基（ROS）诱导激活Erk1/2;siRNA敲除Notch3表达致使血管平滑肌细胞中Erk1/2的磷酸化水平显著降低,并且抑制了ROS诱导的Erk1/2激活;同样,Notch通路抑制剂DAPT也抑制了ROS诱导的Erk1/2激活;而Notch3活性区NICD的过表达并没有改变血管平滑肌细胞中Erk1/2的磷酸化水平,但其延缓了ROS激活后Erk1/2活性的衰减.上述结果表明,Notch3可在血管平滑肌细胞中调控Erk1/2活性以及ROS诱导的Erk1/2信号激活.     

IL28B基因多态性检测的质粒标准品构建

人类白介素28b（IL28b）的SNP位点rs12980275的多态性（AA、AG和GG）与聚乙二醇干扰素、利巴韦林联合治疗的效果具有显著相关性。为了确保rs12980275预测丙型肝炎患者抗病毒治疗效果的价值,需要构建标准品作为rs12980275检测的标准对照。提取人类外周血基因组DNA,以IL28B SNP rs12980275为目的基因片段设计引物,进行PCR扩增;纯化目的片段与pGM-T Vector连接并转化到大肠杆菌中;提取重组质粒DNA,并进行PCR、测序鉴定。结果 IL28B SNP rs12980275目的片段制备成功,获得稳定的重组质粒,保证了目的片段的特异性与序列完整性。成功构建了IL28B基因SNP rs12980275突变检测的AA、AG和GG 3种质粒标准品,可作为预测丙型肝炎患者抗病毒治疗效果rs12980275突变检测的阳性质控物。     

东方蜜蜂微孢子虫的SNP与InDel位点鉴定及分析

【目的】东方蜜蜂微孢子虫Nosema ceranae是一种专性侵染成年蜜蜂中肠上皮细胞的单细胞真菌病原,广泛感染世界各地的蜂群。本研究拟利用已获得的东方蜜蜂微孢子虫纯净孢子的高质量转录组数据进行单核苷酸多态性（Single nucleotide polymorphism,SNP）和插入缺失（Insertion-Deletion,InDel）位点的鉴定和分析,旨在丰富东方蜜蜂微孢子虫的SNP和InDel信息,并为新型分子标记的开发提供基础。【方法】使用GATK软件识别东方蜜蜂微孢子虫的SNP和InDel位点。采用SnpEff软件预测变异位点发生的基因组区域及变异产生的影响。通过相关生物信息学软件分别将SNP和InDel位点所在基因分别比对GO和KEGG数据库,获得相应的功能和通路注释。【结果】共鉴定到28195个SNP位点,其中发生转换和颠换的SNP位点分别有21403和6792个;上述SNP位点的突变类型有12种,其中最丰富的突变类型为C/T;分布在CDS区的SNP位点最多,其次是基因间区、上游区、下游区和内含子区;最丰富的密码子突变类型是同义突变;SNP位点所在基因可注释到代谢进程、细胞组分和催化活性等43个GO条目以及代谢途径、核糖体和等次生代谢产物的生物合成等85条KEGG通路。共鉴定到2831个InDel位点,其中分布在基因间区InDel位点最多,分布在CDS区的InDel位点最少;最丰富的密码子突变类型是移码突变;InDel位点所在基因可注释到细胞进程、细胞和结合等38个GO条目以及代谢途径、次生代谢产物的生物合成及核糖体等73条KEGG通路。【结论】东方蜜蜂微孢子虫中存在大量的SNP和InDel位点,SNP位点的突变类型主要为转换,与其他物种类似;SNP与InDel位点的基因组功能元件分布规律和突变类型具有明显差异;SNP和InDel位点所在基因与东方蜜蜂微孢子虫适应宿主细胞内环境及病原增殖过程具有潜在关系。     

Lefty1对小鼠胚胎干细胞分化过程的影响

目的:研究Lefty1在小鼠胚胎干细胞分化过程中的作用。方法:根据染色质免疫沉淀测序结果,在临近Lefty1转录起始位点以及与之相距10 kb的上游区域有TGF-β信号通路Smad2/3蛋白的四个结合区域,通过CRISPR/Cas9方法获得四个区域敲除的单克隆细胞,利用荧光实时定量PCR(qRT-PCR)检测各细胞中Lefty1的转录水平,并用TGF-β信号通路的激活剂AC和抑制剂SB分别处理敲除的细胞,检测其对TGF-β信号的响应,最后通过胚状体形成实验,检测敲除细胞系在中内胚层分化过程中的标志分子Gsc和Mixl1的表达。结果:利用CRISPR/Cas9方法成功获得不同区域敲除的单克隆细胞,与野生型E14细胞相比,四种区域敲除的细胞中Lefty1 RNA含量明显降低,并且在干细胞状态和分化状态下,敲除细胞系对TGF-β信号的响应减弱。在胚状体形成的实验中,与野生型E14细胞相比,敲除细胞系在分化过程中Lefty1表达的基础水平明显降低,中内胚层分化的标志分子Gsc和Mixl1转录水平也明显下降。结论:在胚胎干细胞中,Lefty1转录起始位点附近以及上游10 kb的这四段区域通过TGF-β信号通路对Lefty1的转录发挥调控作用,从而影响中内胚层分化过程中的标志分子Gsc和Mixl1的表达。     

SOX6 基因3'UTR 区野生型和突变型重组质粒的构建与鉴定

目的:构建含单核苷酸多态性(SNP)位点rs1065024的SOX6基因3'UTR双荧光素酶报告基因载体,并用生物信息学软件预测与rs1065024位点区域相结合的mi RNA,为进一步研究此SNP位点的功能及mi RNA与SOX6基因3'UTR区之间的关系奠定基础。方法:提取人全血基因组DNA,以基因组DNA为模板,通过PCR扩增含SNP位点在内的SOX6基因3'UTR片段,经过胶回收纯化后,将回收的目的片段插入双荧光素酶报告基因载体p MIR-REPORT中,再经DH5a转化扩增,挑单克隆进行菌落PCR并进行质粒提取,对质粒进行双酶切鉴定,最后进行DNA测序鉴定。针对SNP进行定点突变,构建出野生型和突变型重组质粒,并用生物信息学软件预测出与SNP位点相结合的mi RNA。结果:经单菌落质粒测序验证显示带有T碱基的SOX6基因3'UTR重组质粒p MIR-REPORT-3'UTR-T构建成功;经定点突变,成功将p MIR-REPORT-3'UTR-T质粒转变为p MIR-REPORT-3'UTR-C,经比对未引入任何其他突变;生物信息学预测显示,rs1065024位点位于mi R-190b、mi R-190a-5p、mi R-451b、mi R-4791与SOX6基因3'UTR的结合区域,其多态的改变可以影响mi RNA与m RNA的结合效率。结论:本研究成功构建了含SNP位点rs1065024的p MIR-REPORT-SOX6-3'UTR野生型和突变型重组质粒,为今后SOX6基因3'UTR的SNP位点的功能及mi RNA与SOX6基因3'UTR区之间的关系研究奠定基础。     

黔北人群TGFα基因多态性与非综合征性唇腭裂的相关性研究

本研究旨在探讨黔北地区人群TGFα基因3个SNP位点的多态性及其与非综合征性唇腭裂的相关性。采用PCR和测序方法对86个对照儿童(其中核心家系41例)和116个NSCL/P儿童(其中核心家系52例)的TGFα基因rs11466297、rs473698和rs115055578 3个SNP位点进行扩增和测序;对样本群体进行Hardy-weinberg平衡检测,对2组人群进行基因型频率、等位基因频率比较及OR分析;对病例组核心家系进行HHRR和TDT检验。对照组与病例组人群rs11466297基因型均为AA野生型,rs473698位点包含GG型、GC型和CC型,rs115055578位点基因型均为GG野生型。对于rs473698位点,对照组和病例组均符合Hardy-Weinberg平衡法则(p>0.05),2组人群的基因型和等位基因频率分布差异均无统计学意义(p<0.05);rs473698位点未发现传递不平衡现象(p>0.05)。黔北地区人群TGFα基因rs11466297和rs115055578位点以野生纯合型为主,rs473698位点具有多态性,但其多态性与黔北地区人群区非综合征性唇腭裂的发生可能没有相关性。     

腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响

主要嗅觉表皮(main olfactory epithelium, MOE)是哺乳动物感知气味分子的主要嗅觉器官。在MOE组织内,大多数嗅觉神经元通过cAMP信号传导通路感知气味信息。作为嗅觉cAMP信号通路的主要成员之一,腺苷酸环化酶3（adenylyl cyclase 3, ac3）基因敲除小鼠嗅觉探测功能丧失。除cAMP信号传导通路外,MOE内AC3相关因子AC2和AC4,以及肌醇1,4,5-三磷酸（inositol 1,4,5-trisphosphate,IP3）信号通路和Sonic Hedgehog（Shh）信号通路均有表达。然而,敲除ac3是否会对ac2和ac4以及IP3和Shh信号通路成员产生影响,尚不清楚。本文以AC3缺失（AC3-/-）及其野生型小鼠（AC3+/+）MOE为材料,采用实时荧光定量PCR（qRT-PCR）和免疫荧光组织化学方法,发现AC3缺失后,MOE内的ac2和ac4,以及IP3信号通路中的IP3受体ip3r1及钙调蛋白calm1和calm2表达水平均明显降低。Shh信号通路中的受体patched(ptch)与smoothened(smo)、以及核转录因子gli1与gli2的表达也受到了影响。总之,AC3基因缺失不但导致小鼠MOE组织中cAMP信号通路受损,同时AC3相关因子,IP3信号通路和Shh信号通路的传导也受到抑制。本文对于阐明AC3基因敲除小鼠嗅觉丧失的原因及其嗅觉探测机制具有重要启示作用。     

中华蜜蜂的单核苷酸多态性和插入缺失突变位点鉴定

【目的】本研究拟利用已获得的中华蜜蜂Apis cerana cerana幼虫肠道的转录组数据对单核苷酸多态性（Single nucleotide polymorphism,SNP）和插入缺失（Insertion-Deletion,InDel）突变位点进行挖掘和分析,旨在丰富中华蜜蜂的SNP和InDel信息,并为新型分子标记的开发提供基础。【方法】根据有效读段与东方蜜蜂Apis cerana参考基因组的比对情况,采用GATK软件识别单碱基错配和碱基的插入缺失情况,再利用ANNOVAR软件对SNP位点和InDel位点进行分析。通过相关生物信息学软件将SNP和InDel位点所在基因分别比对GO和KEGG数据库,以获得相应的功能和通路注释。【结果】共鉴定到中华蜜蜂的58 919个SNP位点,包括24 548个纯合位点和34 371个杂合位点;发生转换和颠换的SNP位点分别有49102和9817个;数量最多和最少的突变类型分别是C/T和T/G;分布在外显子区的SNP位点数量最多,达到22 649个;此外,发生同义突变的SNP位点数量最多,其次是非同义突变;SNP位点所在基因可注释到46个GO条目和121条KEGG通路。共鉴定到6 551个InDel位点,包括3 270个插入突变和3 281个缺失突变;分布在内含子区InDel位点最多,共计2 793个;发生移码插入的InDel位点最多;进一步分析结果显示InDel位点所在基因可注释到27个GO条目和28条KEGG通路。【结论】本研究鉴定到中华蜜蜂的大量SNP位点和InDel位点,解析了SNP和InDel位点的突变类型、基因组功能元件分布和密码子突变类型,并揭示SNP和InDel位点对中华蜜蜂的重要生物学过程具有潜在影响。     

gp130介导的信号转导通路在哺乳动物着床中的作用

IL—6相关细胞因子家族成员包括LIF、IL-6、IL-11以及它们的共同受体gp130,在哺乳动物的着床过程中起着重要的作用。LIF敲除的小鼠不能着床。IL—11Rα敲除的小鼠不能完全发生蜕膜化,从而导致妊娠的失败。IL—6敲除的小鼠着床数和着床胚胎的存活率均降低。这些细胞因子通过与受体结合,激活下游信号分子STAT,从而形成了gp130/Jak／STAT信号转导通路,并且STAT3基因敲除的小鼠也不能着床。这些细胞因子通过gp130/Jak／STAT信号转导通路在着床过程中起着重要的作用。了解此信号通路在看床中的作用对解决一些不明原因的不孕症,以及开发着床相关的避孕药物等具有重要意义。     

Ubiquitin-specific proteases in inflammatory bowel disease-related signalling pathway regulation

The exact pathogenesis of inflammatory bowel disease (IBD), a chronic gastrointestinal inflammatory disease comprising Crohn’s disease and ulcerative colitis, remains unclear. Studies on ubiquitination, which regulates the degradation of inflammation signalling pathway molecules, and deubiquitination have provided novel insights. Targeting the ubiquitin-specific protease (USP) family of deubiquitinases elucidates IBD signalling pathway mechanisms and possibly, IBD therapeutic solutions. Here, we characterised USPs as chief regulators of pro-inflammatory signalling pathways, including nuclear factor-κB and transforming growth factor-β; analysed the relationship between USPs and IBD pathogenesis in terms of genetic susceptibility, intestinal epithelial barrier, immunity, and gut microbiota; and discussed future research prospects.Subject terms: Inflammatory bowel disease, Ubiquitylation     

GPR48-Induced keratinocyte proliferation occurs through HB-EGF mediated EGFR transactivation

GPR48 can mediate keratinocyte proliferation and migration. Our investigations showed that AG1478, an inhibitor of EGFR tyrosine kinase, could block GPR48-mediated cellular processes. AG1478 treatment of Gpr48^+/+ cells also decreased phosphorylation of EGFR, ERK and STAT3. Subsequent screening using conditioned media immunodepleted of EGFR ligands identified HB-EGF as the ligand responsible for phosphorylation of EGFR, ERK and STAT3. HB-EGF was reduced in Gpr48^−/− cell culture medium, but its addition restored the phosphorylation of EGFR, ERK, STAT3, as well as cell proliferation. Confirmation that GPR48 mediates EGFR signaling pathway through HB-EGF was subsequently performed using an inhibitor of HB-EGF.     

Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production

IL-23R gene variants have been identified as risk factors for two major inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, but how they contribute to disease is poorly understood. In this study, we show that the rs10889677 variant in the 3'-untranslated region of the IL-23R gene displays enhanced levels of both mRNA and protein production of IL-23R. This can be attributed to a loss of binding capacity for the microRNAs (miRNAs) Let-7e and Let-7f by the variant allele. Indeed, inhibition and overexpression of these miRNAs influenced the expression of the wild type but not the variant allele. Our data clearly demonstrate a role for miRNA-mediated dysregulation of IL-23R signaling, correlated with a single nucleotide polymorphism in the IL-23R strongly associated with IBD susceptibility. This implies that this mutation, in combination with other genetic risk factors, can lead to disease through sustained IL-23R signaling, contributing to the chronicity of IBD.     

p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease

Inflammatory bowel diseases (IBD)--Crohn's disease and ulcerative colitis--are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-alpha, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38alpha-delta), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38alpha, JNKs, and ERK1/2 were significantly activated in IBD, with p38alpha showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38alpha to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn's disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-alpha. In vivo inhibition of TNF-alpha by a single infusion of anti-TNF-alpha Ab (infliximab) resulted in a highly significant transient increase of p38alpha activity during the first 48 h after infusion. A significant infliximab-dependent p38alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-alpha gene expression, which could be inhibited by SB 203580. In conclusion, p38alpha signaling is involved in the pathophysiology of IBD.     

Association of the myosin heavy chain 9 gene single nucleotide polymorphism with inflammatory bowel disease

BackgroundTo date, the cause of inflammatory bowel disease (IBD) remains a mystery. A balance between cell proliferation and apoptosis maintains intestinal tissue homeostasis. Dissociation-induced myosin-actin contraction results in stem cell apoptosis. This study aiming to evaluate the influence of the myosin heavy chain 9 (MYH9) gene single nucleotide polymorphisms (SNPs) on inflammatory bowel disease.Subjectsand methods: The study carried on eighty patients with IBD and seventy controls. All participants subjected to history taking, thorough physical examination, colonoscopy and laboratory investigations. Genotyping performed for rs4821480 and rs3752462 by SNP assay real-time PCR methods.ResultsOn analyzing rs3752462 CT and TT genotypes were significantly more frequent in IBD patients as compared to controls with 4.6 fold increase in the risk of IBD. While on analyzing rs4821480, The TG and GG genotypes have significant increased distribution among the IBD patients as compared to the controls with 5.3 fold increase in the risk of IBD and higher prevalence of GG genotype in patients with low hemoglobin level and higher BMI.ConclusionThe rs3752462 T allele and rs4821480 G allele of MYH9 are associated with more susceptibility to IBD.     

Interleukin-6 trans-signaling in inflammatory bowel disease

The pathogenesis of inflammatory bowel disease (IBD) is complex, involving a wide range of molecules including cytokines. Recent investigations support the important role of an interleukin-6 (IL-6) signaling pathway in the development of IBD. However, the molecular mechanisms of this pathway in the intestine remain incompletely understood. The circulating and intestinal levels of IL-6 as well as soluble IL-6 receptor (sIL-6R) are increased in patients with IBD. It is remarkable that the mucosal T cells of IBD patients are extremely resistant to apoptosis and that a large fraction of these cells express membrane-bound gp130 but not IL-6R. The accumulated evidence strongly supports the hypothesis that the development and perpetuation of IBD relies on the increased formation of IL-6/sIL-6R complexes interacting with membrane-bound gp130 on T cells via trans-signaling. These studies suggest that IL-6 trans-signaling may play a role in the development of IBD; they therefore imply the possibility of a selective therapeutic strategy to target this signaling.     

Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells: role of BRAF mutation and fibroblast growth factor signaling

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.