脂肪肝超声定时量诊断与肝脏血流动力学指标的关系探讨

目的探讨脂肪肝超声定时量诊断与肝脏血流动力学指标的关系。方法将68例脂肪肝患者按AII值增高程度分为重度脂肪肝组20例、中度脂肪肝组27例、轻度脂肪肝组21例,另选择体检健康者20例纳入健康组,比较分析4组患者相关指标检查结果。结果脂肪肝患者病情严重程度与三酰甘油、总胆固醇水平有关;重度脂肪肝患者肝静脉和门静脉血流流速显著低于健康者(P<0.05)。结论声学密度定量技术对于脂肪肝患者的定量诊断具有重要临床意义,定量指标与患者肝脏各静脉血流流速存在相关性。

一种低轨卫星通信的定时提前计算方法

低轨(LEO)卫星网络作为地面网络的重要补充,是未来天地一体化网络的重要组成部分。由于LEO卫星的高移动速度以及星地通信的大传播距离造成了高传播时延,因此需要新的针对LEO卫星星地通信背景的上行链路的定时提前量(TA)的计算策略。本文基于LEO卫星的星地通信场景,介绍了TA及其在协议中的规定,并针对LEO卫星的特点,提出一种LEO卫星通信的定时提前计算方法。通过仿真分析验证了所提方案的有效性,为LEO卫星星地通信系统的设计提供了参考。

PIC16C54单片机爆光定时控制器的设计

普通相片扩印机配备的爆光定时器是用模拟电路制做的.本文作者用PIC16C54单片机设计了数字化的爆光定时器,其特点是定时准确,重复精度好,并可按10%比例加减剥光定时量.

鹿布鲁氏菌实时荧光定量PCR检测方法的建立

本研究依据布鲁氏菌的特异性基因Omp25c的部分片段作为靶基因,设计探针引物,且优化了反应体系,筛选出引物、探针的最优浓度配比。将扩增产物连接到PGEM-T载体上,制备标准品及标准曲线,建立鹿布鲁氏菌荧光定量PCR检测方法,并对其特异性、稳定性、敏感性进行评价。由标准曲线可知该方法的最低检测浓度可达到36拷贝/μL,比常规PCR灵敏度高出很多。

实时荧光定量PCR检测皱纹盘鲍Δ5脂肪酸去饱和酶基因表达方法的建立及应用

研究以皱纹盘鲍(Haliotis discus hannai Ino)体内存在的2条Δ5 Fad(Hdhfad1和Hdhfad2)为目的基因,β-肌动蛋白(β-actin)和核糖体蛋白S9(Ribosomal protein S9,RPS9)为内参基因,应用2–ΔΔCt方法建立了实时荧光定量PCR(Real-Time quantitative PCR,RT-qPCR)检测体系,并应用此体系分析了鲍鱼肌肉组织Δ5 Fad在不同饲料处理下的表达差异。实验饲料含有不同的脂肪源,分别是棕榈酸甘油酯(Tripalmitin,TP饲料)、富含二十碳四烯酸(Arachidonic acid,ARA)的油脂(AO饲料)和富含二十碳五烯酸(Eicosapentaenoic acid,EPA)的油脂(EO饲料)。结果表明:针对Hdhfad1、Hdhfad2、β-actin和RPS9所设计的引物特异性强。各引物对的PCR扩增效率(Efficiency,E)分别为1.05、0.99、0.97和0.98,满足2–ΔΔCt方法对E的要求。当退火温度为52℃,反应体积为25μL时,RT-qPCR的扩增效果最好。所建立的体系能够准确定量Δ5 Fad的基因表达。利用该方法分析Δ5 Fad在不同饲料处理下的表达结果显示,与TP对照组相比,EO和AO饲料显著降低了鲍鱼肌肉组织Δ5 Fad(Hdhfad1和Hdhfad2)的表达量。

乙型肝炎病毒SYBR GreenⅠ实时荧光定量PCR方法的建立及临床应用

目的建立 SYBR Green I实时荧光定量 PCR检测乙型肝炎病毒 DNA的快速方法，并探讨其临床应用价值。方法根据GenBank公布的Hepatitis B virus gp1基因序列设计引物，建立SYBR Green I实时荧光定量PCR，并对反应体系和扩增程序进行优化。定量标准品通过基因克隆方法获得，同时以浙江省夸克公司 HBV DNA定量检测试剂盒作对照，应用于随机选取的100份乙型肝炎患者血清检测。结果 SYBR Green I 实时荧光定量 PCR 检出限范围为5＆#215;102 copies／ml～5＆#215;108 copies／ml，HBV DNA浓度与CT值有良好线性关系，无交叉反应，整个过程仅需2.5 h。在随机抽取的100份临床标本应用中，与浙江省夸克公司的 HBV荧光定量 PCR检测试剂相比，建立的 SYBR Green I实时荧光定量PCR体系灵敏度100％，特异度92.5％。结论 SYBR Green实时荧光定量PCR快速、简便、灵敏度高、特异度强，可用于乙型肝炎患者病情监测，有效指导临床用药，准确评价 HBV感染者的病情。

牛奶子实时定量PCR分析中内参基因的评价与验证(英文)

【目的】实时定量PCR结果的精确性在很大程度上取决于选择的内参基因的稳定性。通过评估候选管家基因的表达稳定性,筛选出用于牛奶子研究的最佳内参基因。【方法】设计简并引物从牛奶子中克隆12个潜在的内参基因片段,包括14-3-3、18S核糖体RNA(18SrRNA)、β-actin(Actin)、延长因子1-α(EF1-α)、真核起始因子4A(e IF4A)、3-磷酸甘油醛脱氢酶(GAPDH)、RNA聚合酶-Ⅱ(RPⅡ)、60S核糖体蛋白(RPL7)、翻译延长因子2(TEF2)、泛素连接酶E2(UBCE)、泛素(UBQ)和泛素延伸蛋白5(UBQ5)。采集牛奶子5个不同器官(根、茎、叶、花和红果)、4个成熟期的果实(绿果、黄果、深粉果和完全成熟的红果)、2种激素(脱落酸、赤霉素)处理4个时间点的绿果、热处理4个时间点的离体叶片、幼苗盐胁迫2个时间点的根茎叶,应用实时荧光定量PCR技术检测12个内参基因在各样品中的表达情况,采用基于CT值的ge Norm、Normfinder和Best Keeper及CT比较4种算法评价这些内参基因的稳定性,最终的排名则由Ref Finder算法产生。【结果】器官组稳定性好的前2位内参基因为UBCE和RPL7,果实成熟期组排名前2的为e IF4A和UBCE,激素处理组排名前2的为e IF4A和UBCE,非生物胁迫组排名前2的则为Actin和EF1-α,综合分析所有样品排名前3位的是e IF4A、RPL7和UBCE。分别用筛选出的稳定的e IF4A、RPL7、UBCE和不稳定的RPⅡ作为内参基因评价目的基因八氢番茄红素合成酶基因Eut Psy在果实成熟过程中的表达,结果显示分别以3个稳定的内参基因为单内参基因与以e IF4A同UBCE组合做内参基因所得到的结论一致,而RPⅡ给出的则不同。【结论】在应用荧光实时定量PCR技术研究牛奶子基因转录表达时,通常情况下e IF4A、RPL7和UBCE相对于其他9个候选内参基因更为可靠。研究结果为牛奶子及胡颓子属其他物种的基因表达分析奠定基础。

实时荧光定量PCR在食品致病菌及转基因成分检测中应用

与传统定性PCR技术相比,实时荧光定量PCR有更多的优点,其速度快,特异性更强、灵敏度更高、无污染性、自动化水平高等,且能对DNA模板进行定量。本文综述了实时荧光定量PCR技术原理、分类及其应用,并对其存在的问题和发展前景进行了展望。

From Phenotypes to Molecules: Revolutionizing Gut Microbiota Identification Methods

The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many other aspects.Exploring the structure and function of the gut microbiota,as well as their key genes and metabolites,will enable the early diagnosis and auxiliary diagnosis of diseases,new treatment methods,better effects of drug treatments,and better guidance in the use of antibiotics.The identification of gut microbiota plays an important role in clinical diagnosis and treatment,as well as in drug research and development.Therefore,it is necessary to conduct a comprehensive review of this rapidly evolving topic.Traditional identification methods cannot comprehensively capture the diversity of gut microbiota.Currently,with the rapid development of molecular biology,the classification and identification methods for gut microbiota have evolved from the initial phenotypic and chemical identification to identification at the molecular level.This review integrates the main methods of gut microbiota identification and evaluates their application.We pay special attention to the research progress on molecular biological methods and focus on the application of high-throughput sequencing technology in the identification of gut microbiota.This revolutionary method for intestinal flora identification heralds a new chapter in our understanding of the microbial world.

Selection of Reference Genes in Transcription Analysis of Gene Expression of the Mandarin Fish, Siniperca chuasti

At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.

Developmental Changes of the FAS and HSL mRNA Expression and Their Effects on the Content of Intramuscular Fat in Kazak and Xinjiang Sheep

Twenty-four male Kazak sheep and 30 Xinjiang fine wool sheep at different ages were selected to investigate the development-dependent expression levels of fatty acid synthase （FAS） gene and hormone-sensitive lipase （HSL） gene in muscle and their effects on the contents of intramuscular fat （IMF）. Longissimus dorsal muscle was sampled to measure IMF and total RNA was extracted to determine FAS and HSL mRNA expression levels by real-time PCR. The results showed that： l） The IMF content increased continuously with growth and showed significant differences （P 〈 0.05） between different age groups in male Kazak sheep, but in Xinjiang fine wool sheep there was no such difference observed. Furthermore, the IMF contents in Kazak were much higher （P 〈 0.01） than that of the other breed from day 30 to 90. 2） FAS mRNA expression level was the highest （P 〈 0.05） on day 0 in Kazak sheep and then declined with growth, in the other breed the gene showed a d‘ecline-rise-decline-rise＇ expression manner as the animals grew. HSL mRNA expression level had a similar model in two breeds, in Kazak sheep it was the highest on day 0 （P 〈 0.05） and in Xinjiang fine wool sheep on day 30 （P 〈 0.01）, then both decreased after this term. 3） In male Kazak sheep, FAS and HSL mRNA expression level were both negatively related to IMF content （r= -0.485 （P = 0.02）, r= -0.423 （P = 0.05））, and the ratio of FAS/HSL expression exhibited significantly negatively related IMF contents. In male Xinjiang sheep, there were no obvious relationship between FAS and HSL expression and IMF content （P 〉 0.05）.

基于卷积神经网络的列车位置指纹定位算法研究

针对高速铁路隧道环境下采用位置指纹定位时定位精度低的问题,提出将深度卷积神经网络应用于列车位置指纹的定位中。首先采用2σ准则、模糊C均值聚类FCM及类数据加权,对采集到的下一代铁路通信系统LTE-R中的信号强度值进行预处理,降低异常值的影响,提高指纹数据的有效性;然后引入定时提前量,增强指纹特征值;接着将处理后的指纹数据量转换为灰度图片指纹条,基于图像样本建立FCM-CNN指纹定位模型;最后以现场实测数据为基础对定位模型进行测试验证。结果表明,相较于采用未经处理的数据作为样本的CNN模型及传统的位置指纹定位方法,基于FCM-CNN的列车位置指纹定位方法,提高了数据质量,在离线阶段具有较大的指纹采集间距,大幅减少了指纹采集工作量,模型训练时间较短,定位精度小于10 m的概率可达100%,满足列车在中密度线路对定位精度的要求。

慢性腱病模型大鼠肌腱干细胞体外成脂和成肌腱的分化能力

背景:慢性腱病是一种常见的肌腱退行性病变,好发于运动员以及肌腱过度劳损的人群。由于慢性腱病的发病机制尚未阐明,临床上还缺乏有效的治疗手段。目的:体外研究比较慢性腱病大鼠和正常大鼠来源肌腱干细胞成脂、成肌腱分化能力。方法:从慢性腱病大鼠以及正常大鼠髌腱中分离培养原代肌腱干细胞,传代培养至第3代,体外观察细胞形态学变化。将两种来源肌腱干细胞(P3)单层培养至细胞融合,分为2组,成脂诱导组用成脂诱导培养基培养,对照组用基础培养基培养。成脂诱导分化21 d后,将两种来源肌腱干细胞的成脂诱导组和对照组分别行油红O染色定量分析。实时荧光定量PCR检测各组细胞成脂性基因C/EBPα和PPARγ2的m RNA的表达。将两种来源的肌腱干细胞体外单层培养至70%-80%融合时,行实时荧光定量PCR检测慢性腱病来源肌腱干细胞以及正常肌腱干细胞成肌腱相关基因Col1a1,Scx,Tnmd和Dcn的m RNA的表达。结果与结论:肌腱干细胞体外培养至第3代时,正常肌腱来源的肌腱干细胞保持细长纺锤形的典型的干细胞形态,而慢性腱病来源的肌腱干细胞虽然形态发生改变,但仍保持纺锤形形态。肌腱干细胞(P3)体外成脂诱导分化21 d后,慢性腱病来源的肌腱干细胞胞体变大、变圆,可见大量油红O染色阳性的细胞,油红O染色阳性率显著高于正常大鼠(P=0.004)。实时荧光定量PCR结果显示,慢性腱病大鼠来源肌腱干细胞的成脂性基因(C/EBPα和PPARγ2)m RNA的表达均显著高于正常大鼠(P=0.004),肌腱特异性基因Col1a1,Scx,Tnmd以及Dcn m RNA表达量均明显低于正常大鼠(P=0.009)。说明与正常大鼠来源的肌腱干细胞相比,慢性腱病来源的肌腱干细胞体外成肌腱分化的能力减弱,而成脂分化的能力增强,此结果为进一步揭示慢性腱病发病机制提供了细胞生物学依据。

长链非编码RNA HOTAIR在胃癌中的表达及临床意义

目的:探讨长链非编码RNA HOTAIR在胃癌及癌旁组织中的表达及其与临床病理特征之间的关系。方法:利用实时定量PCR法检测55例胃癌组织及其配对癌旁正常组织中长链非编码RNA HOTAIR的表达情况,并分析其与胃癌临床病理学特征之间的联系。结果:实时定量PCR法检测HOTAIR在胃癌组织中的相对表达量为0.0232±0.0073,其配对癌旁正常组织相对表达量为0.0071±0.0017,差异具有统计学意义(P<0.05)。长链非编码RNA HOTAIR的表达与胃癌患者肿瘤淋巴结转移、远处转移、TNM分期显著相关(P<0.05),与年龄、性别、分化程度等无关(P>0.05)。结论:长链非编码RNA HOTAIR在胃癌组织中表达显著高于癌旁组织,与胃癌转移、TNM分期等病理因素相关,可能在胃癌的侵袭、转移过程中起重要作用。

嗜热四膜虫两类不同金属硫蛋白的功能补偿分析

为了分析嗜热四膜虫两类金属硫蛋白之间的关系,研究分别构建了MTT1-MTT3和MTT2-MTT4的基因敲除载体,通过同源重组获得敲除大核MTT1-MTT3和MTT2-MTT4的两种嗜热四膜虫突变体细胞株△MTT1-MTT3和△MTT2-MTT4。两种突变体细胞株暴露在Cd2+、Cu2+和H2O2的生长表现出显著不同,△MTT1-MTT3突变体细胞对Cd2+的耐受性显著下降,而△MTT2-MTT4突变体细胞对Cu2+和H2O2的耐受性均显著下降。实时荧光定量PCR分析不同突变体中其他MTT基因的表达变化,在△MTT2-MTT4突变体细胞株中,MTT5的表达水平下调,在500μmol/L Cu2+处理后,△MTT2-MTT4突变体细胞中MTT1、MTT3和MTT5表达相对野生型分别上调6.1、9.5和8.5倍。在△MTT1-MTT3突变体细胞中,MTT2、MTT4和MTT5的表达水平下调,当5μmol/L Cd2+处理后,△MTT1-MTT3突变体细胞株MTT5表达水平相对野生型上调2.9倍,而MTT2和MTT4表达水平相对野生型分别下降了4.9倍和2.5倍。结果表明嗜热四膜虫中的金属硫蛋白MTT1、MTT3和MTT5主要参与细胞的重金属解毒功能;而MTT2和MTT4主要参与细胞内正常的新陈代谢功能,不同的金属硫蛋白基因之间的表达存在相互调控和功能补偿。

Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.

Overexpression of IGF2R and IGF1R mRNA in SCNT-produced Goats Survived to Adulthood

The procedure of somatic cell nuclear transfer （SCNT） is likely to affect the expression level of growth-related genes especially imprinting genes. In this study, expressions of growth-related genes including three imprinting genes （H19, IGF2, and IGF2R） and four non-imprinting genes （IGF1, IGFIR, GHR, and GHSR） in adult nuclear transferred （NT） goats were investigated by real-time PCR. The expressions of these genes in adult clones were found largely normal, but IGF2R and IGFIR were more highly expressed in cloned goats than in non-NT goats （P 〈 0.01）. Analysis on mono-allelic expression pattern of imprinting genes indicated that mono-allelic expression patterns of H19 and IGF2 in cloned goats were similar to that in non-NT goats. In addition, the sequence of goat IGF2 gene and the putative amino acid sequence were obtained. The 986 nucleotide cDNA of goat IGF2 gene contained an open-reading frame of 540 nucleotides coding for 179 amino acids. Both cDNA sequence and amino acid sequence of IGF2 in goat showed their higher homology with that in sheep than in cattle; the partial cDNA fragments of H19, IGF2R, GHSR, IGFIR, and GHR in goat were also cloned and sequenced, which shared higher sequence identities with those in sheep than in cattle.

贵州地方猪骨骼肌中3种microRNAs的表达差异

为探明miR-1、miR-133、miR-206基因在不同品种猪肌肉组织中的表达及其与屠宰性能间的关系,以6月龄大白猪、糯谷猪、关岭猪及黔南黑猪的背最长肌为材料,采用实时荧光定量PCR方法,研究了3种microRNAs(miRNAs)在贵州地方猪种和大白猪背最长肌中的表达差异。结果表明:成年期miR-133的表达量高于另外2种miRNA分子。miR-133在糯谷猪中的表达量是大白猪的1.58倍(P<0.05),与其眼肌面积、花油重高度负相关,与屠宰率高度正相关。miR-206在糯谷猪中的表达量是大白猪的2.23倍(P<0.05),与其瘦肉率高度负相关,与板油重中度正相关。在关岭猪背最长肌中miR-133的表达量最高,是大白猪的2.67倍(P<0.05),miR-133和miR-206的表达量与眼肌面积、胴体重高度正相关,与花油重、板油重和脂肪比负相关;但miR-206的表达量明显低于其他猪种。不同品种猪中miR-133和miR-206存在差异表达,对地方猪品种的骨骼肌生长和脂肪沉积有一定的调节作用。

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Quantitative Study on Expression of MSTN Gene in Different Tissues of Tibetan Sheep at Different Ages

[Objective] The aim was to investigate the difference in MSTN gene expression in different tissues of Tibetan sheep at different ages.[Method] According to the sequence（NM_001009428.1）published in GenBank,a pair of specific primers was designed to amplify part of cDNA sequence of MSTN by using QRT-PCR technique.The relative expression level of MSTN gene in rennet stomach,rumen,leg muscle and cardiac muscle of Tibetan sheep at different ages were analyzed.[Result] After normalization with β-actin gene,the relative expression level of MSTN gene in the 6-month-old Tibetan sheep was the highest and it was 2.52 times than that in 12-month-old Tibetan sheep（P0.05）,the relative expression level of MSTN gene in leg muscle was the highest among all tissues and it was 3 984.78 times than that in rumen（P0.01）.[Conclusion] The results established theoretical foundation for the correct use of MSTN antibody.