应用基因表达谱芯片筛选丙型肝炎病毒核心蛋白反式调节基因

应用基因表达谱芯片技术对HCV核心蛋白表达质粒pcDNA3.1(－）-core转染的HepG2细胞和空载体处理的相同细胞差异表达的mRNA进行检测。基因表达谱芯片所检测的1152条目的基因均与GenBank中登录的基因，HCV核心表达质粒转染的细胞有95条差异表达基因，其中45条基因表达增强，50条基因表达降低。这些核心蛋白反式调节基因与细胞增殖、分化、凋亡、信号转导及免疫调节密切相关。本实验结果为进一步阐明HCV核心蛋白致病（癌）的分子生物学机制提供了理论依据。     

Aortic stiffness and aerobic exercise: mechanistic insight from microarray analyses

INTRODUCTION/PURPOSE: Regular aerobic exercise reduces aortic stiffness. However, the mechanisms by which chronic exercise lowers arterial stiffness are not known. To determine the molecular mechanisms of these changes, the alteration of gene expression in the aorta by aerobic exercise training was measured with the microarray technique. METHODS/RESULTS: The differences in expression levels of 3800 genes in the abdominal aorta of sedentary control rats (8 wk old) and exercise-trained rats (8 wk old, treadmill running for 4 wk) were compared by the microarray analysis. Aortic pulse wave velocity (PWV) was lower and systemic arterial compliance was higher (both P < 0.05) in the exercise-trained group than in the control group. Of the 323 genes that displayed differential expression (upregulation of 206 genes and downregulation of 117 genes), a total of 29 genes (24 upregulated and 5 downregulated genes) were identified as potential candidate genes that may be involved in vasodilation and arterial destiffening. Using real-time quantitative polymerase chain reaction, we confirmed the results of microarray analysis that prostaglandin EP2 receptor (PGE-EP2R), prostaglandin EP4 receptor (PGE-EP4R), C-type natriuretic peptide (CNP), and endothelial nitric oxide synthase (eNOS) genes were differentially expressed. Furthermore, there were modest correlations between arterial stiffness and levels of these factors. Differential expression of eNOS gene was further verified at protein level by using Western blot analysis. CONCLUSION: These results suggest that exercise training induces the altered expression in several genes including prostaglandin, CNP, and nitric oxide in the aorta and that these molecular changes (particularly eNOS as its protein expression was altered) may contribute, at least in part, to the beneficial effect of exercise training on aortic stiffness.     

Altered gene expression profiles by sodium/iodide symporter gene transfection in a human anaplastic thyroid carcinoma cell line using a radioactive complementary DNA microarray

BACKGROUND: The sodium/iodide symporter (NIS) is a membrane glycoprotein that mediates active 131I uptake during the treatment of cancer of the thyroid gland and extrathyroidal tissues. NIS gene transfection, a gene-therapy modality, has been introduced in many types of cancer, such as prostate cancer and breast cancer, and has demonstrated a high potential for the treatment of non-thyroidal cancers. AIM: To investigate the pattern of NIS gene expression and provide evidence of its beneficial effects in human anaplastic cancer ARO cells by using a radioactive complementary DNA (cDNA) microarray. METHODS: For cDNA microarray data analysis, superimposed images and clustergrams were prepared from basic radioactivity data obtained using a phosphoimager system. Gene expression profiles were constructed using the Z-transformed values of genes related to cancer biology. RESULTS: Radioactive cDNA microarray studies showed that 11 genes were upregulated (Z ratio > 1.5) and 31 genes were downregulated (Z ratio < -1.5) in response to NIS gene transfection. Of these differentially expressed genes, 33% were related to cell proliferation and apoptosis. Moreover, NIS gene transfection into an anaplastic thyroid cancer cell line affected the expression of the protein tyrosine phosphatase (PTP) family and Ras oncogene family, including Ras, Rac and Rab. CONCLUSION: The identification of changes in the patterns of gene expression may provide a better understanding of the response of molecular mechanisms to NIS gene transfection.     

应用cDNA微矩阵基因芯片筛选运动性心肌肥大相关基因的初步研究

目的 :应用cDNAchip(cDNA芯片或微矩阵基因芯片 )技术筛选运动性心肌肥大相关基因。方法 :摘取运动组和对照组小鼠心脏 ,按一步法抽提总RNA并纯化mRNA ,将 2 30 4个点包括10 3个阴性及对照基因和 2 2 0 1条小鼠靶基因的PCR扩增产物 ,按微矩阵 (12× 12点× 16亚矩阵 ,点间距离 375 μm)点制于硅烷化玻片上 ,制备成cDNA芯片。将等量抽提纯化的运动组和对照组小鼠心肌组织mRNA ,分别与掺入的荧光分子Cy3和Cy5经逆转录合成cDNA荧光探针 ,混合后再与cD NA芯片杂交 ,通过芯片扫描仪对荧光信号图像进行扫描 ,经计算机分析比较运动组和安静对照组心肌组织中基因表达谱的变化。结果 :在 2 2 0 1条待研究基因中 ,两组小鼠心肌组织间存在差异表达的基因。具有显著表达差异的基因有 71条 ,其中上调基因有 37条 ,下调基因有 34条。结果显示 :cDNA芯片技术筛选运动性心肌肥大相关基因具有高通量、高敏度、高效率、大规模和并行性等优点。     

应用基因表达谱芯片技术研究膦甲酸钠处Jurkat细胞后的差异表达基因

目的 筛选膦甲酸钠处理人T淋巴细胞系Jurkat细胞后的差异表达基因。方法 应用基因表达谱芯片技术对膦甲酸钠处理的Jurkat细胞和以生理盐水处理的相同细胞的mRNA进行检测。结果 Jurkat细胞经膦甲酸钠处理后,所检测的1152条目的基因中,有94条产生差异表达,其中38条基因表达增强,56条基因表达降低。结论 应用基因表达谱芯片成功筛选了膦甲酸钠处理淋巴细胞后差异表达基因,为进一步阐明膦甲酸钠的免疫调节机制及深人了解膦甲酸钠用于治疗病毒性肝炎的药理作用机制提供依据。     

基于基因表达综合数据库筛选调控急性T淋巴细胞白血病超高剂量率放疗敏感性的枢纽基因

目的 通过生物信息学方法对基因表达综合(GEO)数据库中超高剂量率(FLASH)放疗的数据进行分析,寻找参与调控急性T淋巴细胞白血病FLASH放疗敏感性的枢纽(Hub)基因。方法 从GEO数据库中下载和提取接受FLASH放疗恶性肿瘤基因表达谱芯片数据,采用R软件进行差异基因的筛选,并对这些基因进行生物学功能、信号传导通路等分析。通过STRING在线软件分析差异基因的蛋白质相互作用(PPI) 网络,Cytoscape插件筛选Hub基因。最后,应用肿瘤基因组图谱(TCGA)和GTEx数据库验证Hub基因在急性T淋巴细胞白血病中的表达情况。结果 自GEO数据库中获得GSE100718芯片数据,共有12 800个基因与急性T淋巴细胞白血病放疗敏感性相关。选择表达量显著改变的61个基因进行进一步分析,这些基因参与代谢、应激反应、免疫应答等生物学过程。主要涉及氧化磷酸化、未折叠蛋白应答、脂质代谢等信号转导通路。通过PPI分析筛选出的Hub基因及后续验证表明HSPA5及SCD参与调控FLASH放疗敏感性,且在合并TRD/LMO2融合基因的急性T淋巴细胞白血病中显著高表达。结论 通过生物信息学分析可以有效筛选出调控FLASH放疗敏感性的Hub基因,基因表达谱可用于指导肿瘤患者分层以实现精准放疗。     

贝氏考克斯体毒力与毒力相关蛋白芯片的研制

目的:构建贝氏考克斯体毒力与毒力相关蛋白芯片.方法:根据贝氏考克斯体全基因组序列,筛选出约160个编码毒力及毒力相关蛋白的基因,采用PCR扩增基因片段,将获得的基因片段与pET32a表达载体连接,然后将该重组质粒转化大肠杆菌并使目的基因表达,采用Ni-NTA亲和层析柱纯化表达的目的重组蛋白.将纯化的重组蛋白点在醛基化玻片上制备蛋白芯片.结果:由贝氏考克斯体104个重组蛋白构建毒力与毒力相关蛋白芯片,通过荧光扫描仪分析蛋白芯片与贝氏考克斯体感染的小鼠血清反应,发现10个重组蛋白与该血清反应为强阳性.结论:所制备的毒力与毒力相关蛋白芯片具有贝氏考克斯体特异性,可用于贝氏考克斯体感染患者血清的检测.     

基因芯片技术在放射生物学研究领域的应用

机体对电离辐射反应非常复杂,受许多分子调节途径控制,p53就是这样的应激途径之一。已经知道它涉及100多个基因的活动,这些应激基因活动具有某种细胞特异性,DNA芯片技术可以完整地研究整个细胞或器官全部基因变化,可以通过基因分析发现对电离辐射的基因反应差异,从而建立一种新的分子放射生物学方法。应用该技术已经初步发现一些新的辐射反应基因活动,在不同基因背景这些基因活动变化很大,提示生物细胞对环境有害因素反应的复杂性及其在正常组织和肿瘤分化增殖过程中基因调控的重要意义,这种技术也可能成为肿瘤治疗和其他环境毒理研究的检测和预测方法。     

运动对心脏心钠素受体基因表达的影响

为探讨运动对心脏心钠素受体基因表达的影响,建立不同强度运动训练动物模型,采用免疫荧光组织化学法、原位杂交、激光共聚焦扫描和计算机图像分析技术,观察心脏心钠素受体的分布和运动对心脏心钠素受体基因表达的影响,结果表明,心钠素受体主要分布在心内膜、心肌纤维的肌膜、心肌纤维间的结缔组织和冠状动脉分支的血管壁.中等和大强度运动使心脏A型心钠素受体基因表达上调和C型心钠素受体基因表达下调,力竭运动使A型心钠素受体基因表达下调和C型心钠素受体基因表达上调.     

基因表达谱的生物信息学

DNA微阵列技术是继DNA重组技术、PCR扩增技术之后的又一重大生物技术。基于微阵列实验 ,可以同时观察在某一生命现象中成千上万个基因的动态表达水平。与过去的研究模式即单个基因的表达研究相比 ,分子生物学工作者的观念将由此发生巨大改变 ,使得人们能够在基因组水平上以系统的、全局的观念去研究生命现象及其本质。目前 ,微阵列技术已应用到肿瘤分型、肿瘤分类、基因功能研究、基因之间调控网络构建、药物靶位识别等许多方面 ,但是 ,从本质上讲 ,通过微阵列实验所直接获得的是一个基因表达谱 (即基因表达矩阵 ,其行表示基因 ,列表示实验样本 ) ,微阵列的实际应用就是通过对基因表达矩阵的生物信息学处理来实现的 ,因此 ,在由微阵列技术为基础的分子生物学研究中 ,生物信息学是其中及其重要的一环 ,本文就与基因表达谱相关的生物信息学方法作一综述     

Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells

Beta-irradiation used for systemic radioimmunotherapy (RIT) is a promising treatment approach for high-risk leukaemia and lymphoma. In bone marrow-selective radioimmunotherapy, beta-irradiation is applied using iodine-131, yttrium-90 or rhenium-188 labelled radioimmunoconjugates. However, the mechanisms by which beta-irradiation induces cell death are not understood at the molecular level. Here, we report that beta-irradiation induced apoptosis and activated apoptosis pathways in leukaemia cells depending on doses, time points and dose rates. After beta-irradiation, upregulation of CD95 ligand and CD95 receptor was detected and activation of caspases resulting in apoptosis was found. These effects were completely blocked by the broad-range caspase inhibitor zVAD-fmk. In addition, irradiation-mediated mitochondrial damage resulted in perturbation of mitochondrial membrane potential, caspase-9 activation and cytochrome c release. Bax, a death-promoting protein, was upregulated and Bcl-x_L, a death-inhibiting protein, was downregulated. We also found higher apoptosis rates and earlier activation of apoptosis pathways after gamma-irradiation in comparison to beta-irradiation at the same dose rate. Furthermore, irradiation-resistant cells were cross-resistant to CD95 and CD95-resistant cells were cross-resistant to irradiation, indicating that CD95 and irradiation used, at least in part, identical effector pathways. These findings demonstrate that beta-irradiation induces apoptosis and activates apoptosis pathways in leukaemia cells using both mitochondrial and death receptor pathways. Understanding the timing, sequence and molecular pathways of beta-irradiation-mediated apoptosis may allow rational adjustment of chemo- and radiotherapeutic strategies.     

基于差异蛋白质组学技术解析xCT缺陷诱导的细胞死亡机制

目的：对Sut黑色素细胞和野生型黑色素细胞进行高分辨率差异蛋白质组学分析,探讨xCT缺陷引起细胞生长抑制的机制。方法分离Sut黑色素细胞和野生型黑色素细胞总蛋白,进行双向电泳和串联质谱分析,获得数值经Mascot软件处理后通过NCBI及Swiss-Prot蛋白质数据库检索,结合双向凝胶电泳相应点的表观等电点、相对分子质量、匹配肽段的多少和序列覆盖率等综合分析筛选差异蛋白质;逆转录-PCR分析Sut细胞差异蛋白mRNA表达水平,用蛋白免疫印迹法对xCT缺陷的Sut黑色素细胞进行自体吞噬分析。结果与结论得到20个明显上调或下调的蛋白,经MALDI-TOF质谱分析鉴定差异蛋白质,发现与自体吞噬和囊泡运输相关的调控因子（ Anxa3、Hist 1h2bk、NDRG1、CaM）以及与细胞转移相关的调控因子（S100A-4、S100A-6、波形蛋白）表达发生了改变;对自噬标志蛋白分析发现,在xCT缺陷的Sut细胞中发生了自体吞噬。结果表明,xCT缺陷可能通过与细胞外基质的黏附异常以及激活Sut黑色素细胞中自噬相关的信号通路,从而导致细胞的生长抑制和自噬性死亡。     

认知相关基因的生化通路与蛋白质互作网络分析

目的用生物信息学的方法揭示认知相关基因所调控的生物过程及通路,构建认知基因的特异蛋白网络,并挖掘其主要功能。方法在GO、phenopedia、GLAD4U3个数据库中,收集认知相关的基因,进行基因本体分析、通路分析及通路串话分析;从人类蛋白互作网络中提取认知相关特异蛋白网络,并通过聚类分析找出重要功能模块。结果明确了126个认知相关基因,这些基因主要参与学习或记忆、神经发育等生物过程;多条与神经突触发育、信号转导、成瘾相关的通路参与认知过程,且各个通路之间存在紧密的串话作用。认知相关基因之间存在显著的互相作用关系(P<10^-16),相比背景基因具有更高节点度(P=1.39×10^-4)以及介数中心性(P=2.07×10^-5);聚类分析挖掘出胺转运调节、胆碱能突触传递和学习等重要功能模块。结论认知是一个非常复杂的过程,涉及多个具有关键功能的基因,如HTR3A、CHRNA6、BDNF等,基因间通过紧密互相作用,调控多个生物过程及通路,包括胆碱能突触,多巴胺能突触、血清素能突触等,这些通路彼此协同发挥功能。     

辐射相关基因芯片的制备及其在肺癌细胞中的应用

目的 寻找参与肺癌细胞辐射抗性的基因。方法构建辐射相关的基因芯片，初步筛选出在不同辐射敏感性肺癌细胞系中差异表达的基因。为了评价芯片结果的可靠性，我们对一些差异表达基因进行了RT-PCR验证。结果和辐射敏感的NCI-H446细胞相比，辐射抗性的A549中有18个基因有明显的改变，8个基因是上调，10个基因是下调。在照射后6h和24h，A549细胞中分别有22个基因(19个基因上调，3个下调)和26个基因(8个上调，18个下调)的差异表达；NCI-H446细胞中分别有17个基因(9个基因上调，8个下调)和18个基因(6个上调，12个下调)差异表达。从这些基因中，我们发现一些参与细胞增殖和抗凋亡的基因，在照射后的A549细胞中是增高的，而在NCI-H446细胞中是降低的。另外一些参与DNA修复的基因，在A549中比在NCI-H446细胞中有更高的表达。结论一些参与细胞DNA修复、细胞周期调控、细胞增殖和抗凋亡作用的基因可能有助于A549细胞辐射抗性的形成。     

慢性髓细胞白血病相关蛋白CMLAP的基因克隆化研究

目的了解慢性髓细胞白血病(CML)基因表达谱的规律,并对在CML中特异性高表达的一个新基因进行克隆、分析与鉴定。方法应用基因表达谱芯片技术,比较CML患者和正常人外周血单个核细胞(PBMC)基因的表达差异。检索核苷酸序列数据库(GenBank)和蛋白质一级结构序列数据库(SwissProt),对差异表达的基因进行生物信息学分析,与已知功能基因序列进行同源性比较。根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列,确定新基因序列,据此设计并合成该基因序列的特异性引物,提取CMLPBMC的总RNA,以RT-PCR技术扩增获得该新基因的全长序列,并对克隆的基因及其编码产物的序列进行分析。结果在CML患者PBMC中克隆一个新的基因,经测序证实,其编码序列全长为1872个核苷酸(nt),编码产物由624个氨基酸残基(aa)组成,命名为CMLAP。在GenBank中注册,注册号为AY762229。结论基因表达谱芯片技术与生物信息学技术相结合,发现并鉴定、克隆了在CML中高表达的新基因CMLAP,为进一步研究CML发生发展的分子生物学机制奠定基础。     

Gene expression analysis in human malignant melanoma cell lines exposed to carbon beams

PURPOSE: To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. MATERIALS AND METHODS: We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. RESULTS: Twenty-two genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle - related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including the tumor protein p53 (p53) target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). CONCLUSION: Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle - related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.     

Gene expression changes: five years after creation of elastase-induced aneurysms

Purpose

Intracranial saccular aneurysms are associated with chronic remodeling of the arterial wall. The pathobiology of aneurysm growth and rupture is poorly understood. The present study was performed to study the gene expression patterns in elastase-induced saccular aneurysms in rabbits 5 years after aneurysm creation, compared with unoperated control arteries.

Materials and Methods

Elastase-induced saccular aneurysms were created in 25 rabbits and followed up for 5 years. Thirteen rabbits died during follow-up for reasons unrelated to the aneurysms. RNA was isolated from aneurysm tissue and the control contralateral common carotid artery in five of the 12 surviving animals, and analyzed for gene expression by using human gene microarrays. Genes with statistical differences between groups (P < .05 and fold change ≥ 1.5 and ≤ 0.75) were considered differentially expressed. Real-time polymerase chain reaction (RT-PCR) was used for confirmation of gene microarray findings for selected genes.

Results

Fifty-three of 13,353 genes (0.4%) were differentially expressed in the aneurysms compared with the unoperated control arteries. Molecular and functional pathway analysis revealed that immunoregulatory molecules, growth factors, cell adhesion molecules, and structural molecules were differentially expressed in the aneurysms compared with controls. RT-PCR results of selected genes confirmed the differential expression identified by using the gene chip microarray.

Conclusions

Significant modulation in a variety of biochemical and cellular functions in chronic aneurysms provides molecular insights into the pathophysiology of saccular aneurysms.     

rno-miR-203a-3p and Mex3B contribute to cell survival of iliopsoas muscle via the Socs3-Casp3 axis under severe hypothermia in rats

Forensic diagnosis of fatal hypothermia is considered difficult because no specific findings, such as molecular markers, have been identified. Therefore, determining the molecular mechanism in hypothermia and identifying novel molecular markers to assist in diagnosing fatal hypothermia are important. This study aimed to investigate microRNA (miRNA) and mRNA expression in iliopsoas muscle, which plays a role in homeostasis in mammals, to resolve the molecular mechanism in hypothermia. We generated rat models of mild, moderate, and severe hypothermia, then performed body temperature-dependent miRNA and mRNA expression analysis of the iliopsoas muscle using microarray and next-generation sequencing. Analysis showed that rno-miR-203a-3p expression was lower with decreasing body temperature, while Socs3 expression was significantly increased only by severe hypothermia. Luciferase reporter assays suggested that Socs3 expression is regulated by rno-miR-203a-3p. Socs3 and Mex3B small interfering RNA-mediated knockdown showed that suppressing Mex3B could induce the activation of Socs3, followed by a change in caspase 3/7 activity and adenosine triphosphate levels in iliopsoas muscle cells. These findings indicate that rno-miR-203a-3p and Mex3B are deactivated by a decrease in body temperature, whereby it contributes to suppressing apoptosis by accelerating Socs3. Accordingly, the rno-miR-203a-3p-Socs3-Casp3 or Mex3B-Socs3-Casp3 axis may be the part of the biological defense response to maintain homeostasis under extreme hypothermia.