DNA甲基化对调控胰岛分化基因表达的影响

目的探讨DNA甲基化对调控胰岛分化基因表达的影响,为调控干细胞分化为胰岛细胞提供理论依据。方法采用甲基化DNA免疫共沉淀-实时定量PCR法(MeDIP-qPCR)检测129/J小鼠胚胎干细胞、NIT1细胞及NIH3T3细胞中Pdx-1、MafA、Nkx6.1和Oct4四种调控胰岛分化基因的DNA甲基化程度;同时采用实时定量RT-PCR检测上述3种细胞中4种基因mRNA表达水平,分析这些基因DNA甲基化水平差异与基因表达之间的关系。结果 Pdx-1、MafA和Nkx6.1基因在129/J小鼠胚胎干细胞和NIT1细胞中呈低甲基化,在NIH3T3细胞中则呈高甲基化,前两种细胞的甲基化程度明显低于后者(P<0.05);Oct4基因在129/J小鼠胚胎干细胞中未甲基化,在NIT1和NIH3T3细胞中呈低甲基化,NIT1细胞的甲基化程度明显低于NIH3T3细胞(P<0.05)。低甲基化的Pdx-1、MafA和Nkx6.1基因在NIT1细胞中可高效表达,而在NIH3T3和129/J小鼠胚胎干细胞中则未见表达(P<0.05);Oct4基因在129/J小鼠胚胎干细胞中可高效表达,在NIT1和NIH3T3细胞中则未见表达。结论转录起始区DNA甲基化程度可影响Pdx-1、MafA、Nkx6.1和Oct4基因的表达,参与β细胞的分化过程。     

多发性骨髓瘤p16基因甲基化与其预后的相关性

目的探讨多发性骨髓瘤p16基因甲基化与其预后之间的相关性。方法采用甲基化敏感的限制性内切酶联合聚合酶链反应方法(REP法),检测52名多发性骨髓瘤(MM)患者骨髓细胞p16基因第1外显子的甲基化状态,并检测存在p16基因第1外显子甲基化的MM患者p16基因的mRNA转录水平。对这52名患者的临床资料及预后因素进行分析。结果52名MM患者中,p16基因启动子区第1外显子甲基化率为53.84%(28/52)。存在p16基因第1外显子甲基化的MM患者中,p16基因mRNA阴性率为75%。p16基因甲基化患者的预后不良因素明显高于非甲基化患者。结论多发性骨髓瘤p16基因甲基化与其预后有关。     

烯效唑对干旱胁迫下薏苡抗氧化酶活性及DNA甲基化水平的影响

目的探讨烯效唑(S3307)对干旱胁迫下薏苡抗氧化酶活性及DNA甲基化水平的影响。方法将薏辽5号种子培养至幼苗的两叶一心期,实验分为3组:对照组(control):用Hoagland培养液处理;干旱胁迫组(PEG):用25%PEG的营养液处理;S3307缓解组(PEG+S3307组):经S3307预处理后,再用25%PEG处理。检测各组幼苗的形态指标(苗高、茎粗、根长、根数)及过氧化物酶(peroxidase,POD)、超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的活性,同时采用高效液相色谱(HPLC)法测定各组幼苗的DNA甲基化水平。结果经S3307预处理后,幼苗的株高和根长显著降低(P0.05),茎粗及根冠比显著增加(P0.05);SOD、CAT和POD抗氧化酶的活性显著升高(P0.05);幼苗叶片中5-甲基胞嘧啶(5-m C)比例比control组增加了34.29%(P0.001),增加幅度比PEG组降低了12.99%。结论 S3307可缓解干旱胁迫下薏苡的膜脂过氧化损伤及DNA甲基化水平的上升,具有良好的壮苗作用,为薏苡的抗旱性机理研究奠定了基础。     

无规甲基化β-环糊精与苯乙烯包结物的制备和表征

在水溶液中制备了无规甲基化β-环糊精(RAMEB)/苯乙烯包结物。利用热重-差热分析、紫外-可见光谱及核磁共振波谱等检测手段研究了无规甲基化β-环糊精与苯乙烯的包合作用,对包结物的结构、性能进行了表征和分析。运用紫外-可见光谱测试溶液吸光度的变化,摩尔比法表明无规甲基化β-环糊精与苯乙烯包结物的摩尔比为1∶1。Benesi-Hildebrand方程进一步确定了包结反应的包结比以及计算出该温度下的平衡常数,表明无规甲基化β-环糊精与苯乙烯形成了摩尔比为1∶1的包结物。     

人T-bet和HBV-L共表达质粒的构建及体外表达

目的构建人转录因子T-bet基因与乙肝病毒表面抗原大蛋白全长基因(hepatitis B virus surface antigen large protein,HBV-L)的共表达载体,并检测其在体外的表达。方法利用PCR及基因重组技术,以人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的cDNA为模板,扩增获得人T-bet全长基因,以含有HBV-L的质粒pcDNA3.1-HBV-L为模板,扩增获得HBV-L基因,将上述2个基因克隆至真核表达质粒p IRES中,获得共表达质粒p IRES-T-bet-HBV-L。将该质粒转染293T细胞,经RT-PCR、ELISA和细胞免疫荧光试验检测其在真核细胞中的表达。结果限制性酶谱分析及测序证实重组质粒p IRES-T-bet-HBV-L构建成功;该质粒在体外转染293T细胞后,可表达T-bet与HBV-L两者的mRNA;细胞免疫荧光试验和ELISA检测结果证实,该质粒转染293T细胞后可表达T-bet和HBV-L蛋白。结论成功构建了共表达质粒pIRES-T-bet-HBV-L,该质粒能在293T细胞中表达。     

热力学基本方程及相关关系式的新记忆情景构建和运用

针对热力学基本方程及其衍生关系式和麦克斯韦关系式易混淆、难记忆的难题，文章构建了一种基于4个缩写词组和2个规则的新记忆情景，并结合具体例题展示了该记忆情景的实践运用。与已报道的记忆方法相比，文章所创建的记忆情景简单、高效，便于学生快速记忆和应用这些关系式，能够显著提高学生的学习效率和解题能力。     

血管紧张素转换酶基因rs4340和rs4343多态性与心房颤动的相关性

目的探讨血管紧张素转换酶(Angiotensin converting enzyme,ACE)基因rs4340和rs4343多态性与心房颤动(简称房颤)的相关性。方法选择重庆地区4家三甲医院就诊的102例房颤患者及同期住院的无房颤病史患者100例,抽取患者静脉血,分别提取基因组DNA,采用单核苷酸多态性-限制性片段长度多态性(Single nucleotide polymorphism,restriction fragmentlength polymorphism,SNP-RFLP)法及基因测序检测ACE基因rs4340和rs4343的基因型。结果房颤组ACE基因rs4340多态性的基因型及等位基因分布与对照组相比,差异无统计学意义(P>0.05),房颤组rs4343的基因型和等位基因分布与对照组间差异有统计学意义(P<0.001或P=0.001)。与对照组相比,房颤组GG+AG基因型频率明显高于AA基因型频率(P<0.001)。II/AA基因型在房颤组中出现的频率明显少于对照组(P=0.001),而II/AG基因型在房颤组中出现的频率明显高于对照组(P=0.002)。房颤组中rs4340和rs4343各基因型左房前后径与右房横径差异均无统计学意义(P>0.05);而将两位点联合分析发现,携带II/AA基因型房颤患者的左房前后径和右房横径均明显小于其他基因型(P<0.001),同时,携带II/AG基因型房颤患者的左房前后径和右房横径均明显大于其他基因型(P<0.001)。结论 ACE基因rs4343多态性与房颤显著相关,II/AA基因型是房颤发生发展的保护因子,而II/AG基因型是预测房颤发生发展的危险因子。     

白细胞介素-1B和肿瘤坏死因子-α基因多态性与胃溃疡、胃癌易感性的相关性

目的探讨人群中白细胞介素-1B(IL-1B)和肿瘤坏死因子-α(TNF-α)基因多态性与胃溃疡、胃癌易感性的相关性。方法选取幽门螺杆菌阳性的39例胃溃疡患者、35例胃癌患者和70名健康对照者,采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分析法检测该人群中IL-1B-511、TNF-A-308和TNF-A-857基因多态性。结果疾病组与对照组中IL-1B-511各基因型频率的分布差异无统计学意义。与对照组比较,胃溃疡组TNF-A-308各基因型的频率分布、TNF-A-857各基因型的频率分布以及胃癌组TNF-A-308各基因型的频率分布差异均有统计学意义。经Logistic回归分析,与携带TNF-A-308G/G者比较,携带TNF-A-308A/A者发生胃溃疡的危险性为OR=21.62(95%CI:2.07～226.13);与携带TNF-A-857C/C者比较,携带TNF-A-857T/T者发生胃溃疡的危险性为OR=5.37(95%CI:1.28～22.50);与携带TNF-A-308G/G者比较,携带TNF-A-308A/A者发生胃癌的危险性为OR=16.41(95%CI:1.62～116.55)。结论TNF-α基因多态性与胃溃疡、胃癌的易感性相关。     

IL-1β-31、IL-10-819和TNF-α-1031基因多态性与幽门螺杆菌感染相关性胃溃疡及胃癌易感性的关系

目的分析人群中IL-1β-31、IL-10-819和TNF-α-1031基因多态性与幽门螺杆菌(H.pylori)感染相关性胃溃疡及胃癌易感性之间的关系,为临床诊断及预防该病提供新的思路和方法。方法选取H.pylori阳性的51例胃溃疡患者、43例胃癌患者和100例健康对照者,采用PCR-限制性长度片段多态法和多重引物特异PCR法,检测其IL-1β-31、IL-10-819和TNF-α-1031位点,分析其多态性。结果在胃溃疡组中TNF-α-1031各基因型的频率分布与健康对照组比较,差异有统计学意义。在胃癌组中TNF-α-1031各基因型的频率分布与健康对照组比较,差异有统计学意义;Logistic回归分析表明,与携带TNF-α-1031T/T者相比,携带TNF-α-1031C/C者发生胃溃疡的危险性为OR=5.84(95%CI:1.00～33.84),发生胃癌的危险性为OR=6.95(95%CI:1.19～40.63)。在疾病组和健康对照组中,IL-10-819和IL-1β-31各基因型频率的分布差异无统计学意义。结论 TNF-α-1031基因多态性与胃溃疡、胃癌的易感性相关。     

CAD技术与GPS和卫星影像图技术相结合在通信线路及资料管理维护上的应用

对通信线路通信管道、人井、电杆和附属设施、光电缆的维护是基层通信分公司日常工作中的重要内容,把建立在网络技术之上的CAD制图技术与网络卫星影像图、GPS定位技术相结合,绘制完成CAD辖区平面图能够快速确定通信线路设施的地理位置、名称、区段长度、容量等信息,为通信企业生产施工,管道人井、线路资料的管理和使用,提供了一个很好的维护平台.     

Maize DNA Methylation in Response to Drought Stress Is Involved in Target Gene Expression and Alternative Splicing

DNA methylation is important for plant growth, development, and stress response. To understand DNA methylation dynamics in maize roots under water stress (WS), we reanalyzed DNA methylation sequencing data to profile DNA methylation and the gene expression landscape of two inbred lines with different drought sensitivities, as well as two of their derived recombination inbred lines (RILs). Combined with genotyping-by-sequencing, we found that the inheritance pattern of DNA methylation between RILs and parental lines was sequence-dependent. Increased DNA methylation levels were observed under WS and the methylome of drought-tolerant inbred lines were much more stable than that of the drought-sensitive inbred lines. Distinctive differentially methylated genes were found among diverse genetic backgrounds, suggesting that inbred lines with different drought sensitivities may have responded to stress in varying ways. Gene body DNA methylation showed a negative correlation with gene expression but a positive correlation with exon splicing events. Furthermore, a positive correlation of a varying extent was observed between small interfering RNA (siRNA) and DNA methylation, which at different genic regions. The response of siRNAs under WS was consistent with the differential DNA methylation. Taken together, our data can be useful in deciphering the roles of DNA methylation in plant drought-tolerance variations and in emphasizing its function in alternative splicing.     

Methylation: An Ineluctable Biochemical and Physiological Process Essential to the Transmission of Life

Methylation is a universal biochemical process which covalently adds methyl groups to a variety of molecular targets. It plays a critical role in two major global regulatory mechanisms, epigenetic modifications and imprinting, via methyl tagging on histones and DNA. During reproduction, the two genomes that unite to create a new individual are complementary but not equivalent. Methylation determines the complementary regulatory characteristics of male and female genomes. DNA methylation is executed by methyltransferases that transfer a methyl group from S-adenosylmethionine, the universal methyl donor, to cytosine residues of CG (also designated CpG). Histones are methylated mainly on lysine and arginine residues. The methylation processes regulate the main steps in reproductive physiology: gametogenesis, and early and late embryo development. A focus will be made on the impact of assisted reproductive technology and on the impact of endocrine disruptors (EDCs) via generation of oxidative stress.     

Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase

Dnmt3a‐C, the catalytic domain of the Dnmt3a DNA‐(cytosine‐C5)‐methyltransferase, is active in an isolated form but, like the full‐length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a‐C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation‐sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a‐C by optimization of the sequence of the DNA substrate, we analyzed its flanking‐sequence preference in detail by bisulfite DNA‐methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence‐preference profile for Dnmt3a‐C from the ?6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position ?2, A over G at ?1, a pyrimidine at +1, and A and T over G at +3. We designed one “good” substrate optimized for methylation and one “bad” substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a‐C substrate can be applied in enzymatic high‐throughput assays with Dnmt3a‐C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.     

Conversion of allylic hydroxy oleate to conjugated linoleic acid and methoxy oleate by acid-catalyzed methylation procedures

Conjugated linoleic acid (CLA), a term describing a group of conjugated octadecadienoic acids that are both naturally occurring and formed during food processing, is the subject of considerable current research because of the recently reported antioxidant and anticarcinogenic properties of these compounds. Allylic hydroxy oleates (AHOs), secondary products of lipid autoxidation, have also been found in foods. By means of high-performance liquid chromatography with ultraviolet detection, gas chromatography/mass spectrometry and gas chromatography/matrix isolation/Fourier transform infrared spectroscopy, we determined that currently used acid-catalyzed methylation procedures convert AHOs to CLA and other products that potentially yield high values in determination of CLA in foods. A mixture of AHOs, containing mainly (8- and 11-)hydroxy-9-octadecadecenoates, was synthesized and tested by methylation procedures with the following catalysts: BF₃, HCl, NaOMe and tetramethylguanidine. Both the BF₃ and the HCl procedures converted AHOs to CLA. The base-catalyzed procedures did not convert AHOs to CLA.     

Apolipoprotein B mRNA editing and apolipoprotein gene expression in the liver of hyperinsulinemic fatty zucker rats: Relationship to very low density lipoprotein composition

We previously demonstrated increased apolipoprotein B (apoB) mRNA editing, elevated levels of mRNA for the catalytic component of the apoB mRNA editing complex, apobec-1, and increased secretion of the product of the edited mRNA, apoB48, in very low density lipoproteins (VLDL) in primary cultures of Sprague-Dawley rat hepatocytes following insulin treatment. In order to determine the effect of in vivo hyperinsulinemia on these processes, we determined apoB mRNA editing, apobec-1 expression, hepatic expression of mRNA for apoB and other VLDL apoproteins, and the quantity and composition of plasma VLDL in the hyperinsulinemic fatty Zucker rat. Total apoB mRNA content of the livers of the fatty rats and lean littermates did not differ, however, edited apoB message coding for hepatic apo B48, and abundance of mRNA for the catalytic subunit of the apoB mRNA editing complex, apobec-1, was increased by 1.7-and 3.3-fold, respectively, in fatty rats. ApoCIII mRNA abundance was increased in livers of fatty rats as well, but the abundance of hepatic apoE mRNA in the fatty animal was not different from that of the lean rat. Hepatic apoAI mRNA abundance was also increased in the fatty rats. Associated with increased apoB mRNA editing, was the 1.7-fold increase in the fraction of apoB in plasma as apoB48 in fatty rats. VLDL-triglyceride and-apoB in plasma were 15-and 3-fold higher, respectively, in fatty Zucker rats compared to lean littermates, indicating both enrichment of VLDL with triglycerides and increased accumulation of VLDL particles. Increased hepatic expression of mRNA for apoCIII and apoAI was associated with increased content of apoC (and relative depletion of apoE) in VLDL of fatty rats, and plasma apoAI was increased in fatty Zucker rats, primarily in the HDL fraction. The current study provides further evidence that chronic exposure to high levels of insulin influences both the quantity of and lipid/apoprotein composition of VLDL in plasma. The increased apoC and decreased apoE (as well as increased triglyceride) content of VLDL in the fatty Zucker rat observed in the current study may affect VLDL clearance and therefore may be a factor in the observed accumulation of VLDL in the plasma of the fatty hyperinsulinemic Zucker rats.     

羊布氏菌vjbR基因的克隆及原核表达

目的克隆并原核表达羊布氏菌强毒株16M vjbR基因。方法 PCR扩增羊布氏杆菌16M株vjbR基因,亚克隆至原核表达载体pET-28a中,构建重组原核表达质粒pET-28a-vjbR,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经His Trap FF纯化后,进行Western blot分析。结果扩增的vjbR基因大小为708 bp,与预期一致;重组原核表达质粒pET-28a-vjbR经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为29 000,诱导4 h表达量可达6.84 mg/ml;纯化的重组蛋白纯度为65.7%,能被羊布氏杆菌阳性血清所识别。结论成功在大肠杆菌中表达了羊布氏菌VJBR蛋白,为其功能的研究、羊布病诊断试剂盒的研制及亚单位疫苗的研发奠定了基础。     

Aberrant Methylation of 20 miRNA Genes Specifically Involved in Various Steps of Ovarian Carcinoma Spread: From Primary Tumors to Peritoneal Macroscopic Metastases

Our work aimed to differentiate 20 aberrantly methylated miRNA genes that participate at different stages of development and metastasis of ovarian carcinoma (OvCa) using methylation-specific qPCR in a representative set of clinical samples: 102 primary tumors without and with metastases (to lymph nodes, peritoneum, or distant organs) and 30 peritoneal macroscopic metastases (PMM). Thirteen miRNA genes (MIR107, MIR124-2, MIR124-3, MIR125B-1, MIR127, MIR129-2, MIR130B, MIR132, MIR193A, MIR339, MIR34B/C, MIR9-1, and MIR9-3) were hypermethylated already at the early stages of OvCa, while hypermethylation of MIR1258, MIR137, MIR203A, and MIR375 was pronounced in metastatic tumors, and MIR148A showed high methylation levels specifically in PMM. We confirmed the significant relationship between methylation and expression levels for 11 out of 12 miRNAs analyzed by qRT-PCR. Moreover, expression levels of six miRNAs were significantly decreased in metastatic tumors in comparison with nonmetastatic ones, and downregulation of miR-203a-3p was the most significant. We revealed an inverse relationship between expression levels of miR-203a-3p and those of ZEB1 and ZEB2 genes, which are EMT drivers. We also identified three miRNA genes (MIR148A, MIR9-1, and MIR193A) that likely regulate EMT–MET reversion in the colonization of PMM. According to the Kaplan–Meier analysis, hypermethylation of several examined miRNA genes was associated with poorer overall survival of OvCa patients, and high methylation levels of MIR130B and MIR9-1 were related to the greatest relative risk of death.