The tat gene of human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic mice

Human T-lymphotropic virus type 1 (HTLV-1) is a suspected causative agent of adult T-cell leukemia. One of the viral genes encodes a protein (tat) that not only results in transactivation of viral gene expression but may also regulate the expression of certain cellular genes that are important for cell growth. Transgenic mice that expressed the authentic tat protein under the control of the HTLV-1 long terminal repeat were generated, and cell types that are permissive for the viral promoter and the effects of the tat gene on these cells were studied. Three of eight founder mice with high levels of expression of the transgene in muscle were bred and then analyzed. All developed soft tissue tumors at multiple sites between 13 to 17 weeks of age. This phenotype was transmitted to nine of nine offspring that inherited the tat gene and were available for analysis. The remaining five founders expressed the transgene in the thymus, as well as in muscle. This second group of mice all exhibited extensive thymic depletion and growth retardation; in all of these mice, death occurred between 3 to 6 weeks of age before tumors became macroscopically visible. The tat gene under the control of the HTLV-1 regulatory region showed tissue-specific expression and the tat protein efficiently induced mesenchymal tumors. The data establish tat as an oncogenic protein and HTLV-1 as a transforming virus.     

In vivo footprinting of MHC class II genes: bare promoters in the bare lymphocyte syndrome

Major histocompatibility complex (MHC) class II genes are coordinately regulated and show tissue-specific expression. With the use of in vivo footprinting, common promoter sites in these genes were found to be occupied only in cells that expressed the genes, in spite of the presence of the promoter binding proteins. In vivo analysis of mutant cell lines that exhibited coordinate loss of class II MHC expression, including several from individuals with bare lymphocyte syndrome, revealed two in vivo phenotypes. One suggests a defect in gene activation, whereas the other suggests a defect in promoter accessibility.     

A system for stable expression of short interfering RNAs in mammalian cells

Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.     

miR-486对绵羊骨骼肌卫星细胞增殖及PI3k-Akt信号通路相关基因表达的影响

【目的】明确miR-486对绵羊骨骼肌卫星细胞增殖及PI3K-Akt信号通路相关基因表达的影响,为揭示miR-486对绵羊骨骼肌发育的调控机制打下基础。【方法】以巴什拜羊1日龄羔羊后肢骨骼肌卫星细胞为研究对象,通过转染miR-486 mimics和miR-486 inhibitor致使绵羊骨骼肌卫星细胞中miR-486水平上调或下调,探究miR-486对骨骼肌卫星细胞增殖速度及PI3K-Akt信号通路中PTEN、GSK3b、PRKCα、Raf-1、MAPK1、PKN1、Casp9和SGK1等8个相关基因表达变化的影响。【结果】空白对照及转染miR-486 mimics、miR-486 mimics negative control和miR-486 inhibitor negative control的绵羊骨骼肌卫星细胞均表现出典型的S形增殖曲线,但各处理间的细胞增殖速度存在明显差异。当绵羊骨骼肌卫星细胞中miR-486水平上调可促使细胞进入快速增殖状态,而miR-486水平下调可使细胞保持静止状态。实时荧光定量PCR检测结果表明,当绵羊骨骼肌卫星细胞中miR-486水平上调时,可引起PTEN、Raf-1和MAPK1基因相对表达量极显著下调（P<0.01,下同）,PRKCα和PKN1基因相对表达量极显著上调,SGK1基因相对表达量显著上调（P<0.05）;而GSK3β和Casp9基因相对表达量在不同处理组绵羊骨骼肌卫星细胞间的差异均不显著（P>0.05）,可能在维持绵羊骨骼肌卫星细胞基本生命活动中发挥作用。【结论】miR-486通过调控PI3K-Akt信号通路相关基因的表达而参与绵羊骨骼肌卫星细胞生长发育及增殖,为深入研究miR-486对绵羊骨骼肌发育的调控机理及优质肉羊品种培育打下了理论基础。     

oar-miR-133的表达对巴什拜羊骨骼肌细胞增殖和分化的影响

以巴什拜羊为对象,初步研究oar-miR-133的表达与巴什拜羊骨骼肌发育的关系。结果表明,oar-miR-133在巴什拜羊胎儿期第100天的骨骼肌和心肌中高表达;在巴什拜羊骨骼肌发育的不同阶段,oar-miR-133的表达量有明显的变化,在胎儿期的第100天和初生当天前后,骨骼肌中oar-miR-133的表达量均出现上调;当巴什拜羊骨骼肌卫星细胞处于增殖状态时,oar-miR-133表达量较低,而当骨骼肌卫星细胞处于诱导分化状态时,oar-miR-133表达量迅速升高,显著高于增殖状态(P0.05);当骨骼肌卫星细胞中的oar-miR-133表达水平升高时,骨骼肌卫星细胞几乎停止增殖,细胞有相互融合转化为肌管的趋势,同时细胞内可检测到细胞分化的标志基因MyHC基因的表达;生物信息学分析共预测到96个oar-miR-133的靶基因,GO分析及KEGG信号通路分析结果表明,这些基因在细胞内通过参与一些重要的信号通路而发挥其生物学功能。     

Targeting of nonexpressed genes in embryonic stem cells via homologous recombination

Gene targeting via homologous recombination-mediated disruption in murine embryonic stem (ES) cells has been described for a number of different genes expressed in these cells; it has not been reported for any nonexpressed genes. Pluripotent stem cell lines were isolated with homologously recombined insertions at three different loci: c-fos, which is expressed at a low level in ES cells, and two genes, adipsin and adipocyte P2 (aP2), which are transcribed specifically in adipose cells and are not expressed at detectable levels in ES cells. The frequencies at which homologous recombination events occurred did not correlate with levels of expression of the targeted genes, but did occur at rates comparable to those previously reported for genes that are actively expressed in ES cells. Injection of successfully targeted cells into mouse blastocysts resulted in the formation of chimeric mice. These studies demonstrate the feasibility of altering genes in ES cells that are expressed in a tissue-specific manner in the mouse, in order to study their function at later developmental stages.     

Trolox对猪肌肉干细胞增殖及分化的影响

【目的】探究抗氧化剂-水溶性维生素E的类似物（Trolox）通过调控活性氧对猪肌肉干细胞增殖及分化过程产生的影响,为进一步优化培养肉种子细胞在体外增殖及分化过程提供研究基础。【方法】首先在猪肌肉干细胞增殖过程中,添加不同浓度（0、50、100和200 μmol∙L^-1）的Trolox培养3 d,利用血细胞计数板进行细胞计数,统计细胞增殖倍数,同时利用CCK8技术检测不同浓度的Trolox对细胞增殖的影响;利用RT-qPCR技术检测不同浓度Trolox处理后表征干性的PAX7表达水平,Western Blotting技术检测PAX7蛋白的表达水平;通过CellROX荧光染料对细胞内活性氧进行染色并通过高通量高内涵活细胞共聚焦成像系统检测Trolox对活性氧的调控作用;进一步在猪肌肉干细胞体外成肌分化进程中添加Trolox处理,利用RT-qPCR技术检测分化早期标志基因MYOG、CAV-3及终末分化标志基因肌球蛋白重链（MyHC）的表达水平,并利用Western Blotting技术检测MyHC蛋白的表达,利用免疫荧光技术对MyHC进行染色并统计MyHC阳性细胞比例。【结果】细胞增殖倍数统计显示,50和100 μmol∙L^-1的Trolox处理组猪肌肉干细胞增殖倍数显著高于对照组（P<0.05）;CCK8测得50和100 μmol∙L^-1 Trolox处理组第3天的吸光值显著高于对照组（P<0.05）;100和200 μmol∙L^-1的Trolox显著上调了PAX7的表达（P<0.05）,对PAX7蛋白的表达有上调趋势,但无显著差异（P>0.05）;添加Trolox后,细胞内活性氧水平被极显著降低（P<0.001）;在分化进程中添加Trolox后,预分化阶段的MYOG和CAV-3及终末分化阶段的MyHC均显著上调（P<0.05）,而Western blotting结果显示MyHC蛋白的表达无显著变化（P>0.05）;免疫荧光结果显示MyHC阳性细胞比例有增多的趋势,但无显著差异（P>0.05）。【结论】Trolox通过降低细胞内的活性氧,促进猪肌肉干细胞的增殖以及分化进程。     

5-bromo-2'-deoxyuridine blocks myogenesis by extinguishing expression of MyoD1

The pyrimidine analog 5-bromodeoxyuridine (BUdR) competes with thymidine for incorporation into DNA. Substitution of BUdR for thymidine does not significantly affect cell viability but does block cell differentiation in many different lineages. BUdR substitution in a mouse myoblast line blocked myogenic differentiation and extinguished the expression of the myogenic determination gene MyoD1. Forced expression of MyoD1 from a transfected expression vector in a BUdR-substituted myoblast overcame the block to differentiation imposed by BUdR. Activation of BUdR-substituted muscle structural genes and apparently normal differentiation were observed in transfected myoblasts. This shows that BUdR blocks myogenesis at the level of a myogenic regulatory gene, possibly MyoD1, not by directly inhibiting the activation of muscle structural genes. It is consistent with the idea that BUdR selectively blocks a class of regulatory genes, each member of which is important for the development of a different cell lineage.     

猪骨骼肌卫星细胞分离培养、鉴定及其生物学特性

【目的】建立猪骨骼肌卫星细胞体外分离、纯化及鉴定的方法,并对其生物学特性进行探讨,为进一步研究猪肌肉生长发育提供良好的细胞模型。【方法】选取1日龄仔猪背最长肌为材料,无菌状态下将背最长肌剪碎为肉糜状。此后采用浓度为0.2%的Ⅰ型胶原酶消化90 min,再加入浓度为0.25%的胰蛋白酶37 ℃联合消化30 min。经终止消化、过滤、重悬后将分离得到的细胞置于37 ℃、5% CO₂细胞培养箱中培养。选用反复差速贴壁法对骨骼肌卫星细胞进行纯化,第一次纯化选择在细胞接种2 h后,将未贴壁细胞转移至新培养皿。上清液继续培养18 h后,对卫星细胞进行第二次差速贴壁纯化。当细胞密度达70%—80%时可对细胞进行传代或冻存处理。利用细胞免疫荧光技术检测P2代卫星细胞标志基因Pax7、MyoD的蛋白表达情况,并绘制卫星细胞生长曲线。分别添加不同诱导分化液使卫星细胞定向分化为肌细胞、脂肪细胞、成骨细胞,检测成肌分化标志基因MHC的免疫荧光,鉴定卫星细胞肌管形成情况;油红O染色及油红O定量鉴定卫星细胞诱导成脂分化效果;茜素红染色鉴定卫星细胞成骨分化能力,qRT-PCR检测成肌、成脂、成骨进程中关键基因的表达。【结果】通过两步酶消化和反复差速贴壁法分离纯化得到了纯度较高的卫星细胞,刚分离的细胞折光性强,贴壁后呈梭形或纺锤形,此后细胞延展并开始快速增殖。卫星细胞特异标志蛋白Pax7、MyoD细胞免疫荧光鉴定结果呈阳性,表明分离细胞为骨骼肌卫星细胞。骨骼肌卫星细胞增殖过程经潜伏期、生长期最终达到平台期,细胞生长曲线呈“S”型。当细胞生长至90%密度时,卫星细胞会出现自融合现象。对分离的骨骼肌卫星细胞成肌诱导分化后,可见邻近卫星细胞融合形成大量粗长肌管,多核肌管呈方向性排列,成肌标志蛋白MHC染色呈阳性。qRT-PCR结果显示标志基因MyoD、MyoG在成肌诱导分化过程中二者均呈先上升后下降趋势。经成脂诱导后细胞形态变为三角形,连续诱导发现脂滴出现并聚集成大脂滴,油红O染色可见大量红色葡萄样脂滴。油红O定量检测结果表明,成脂诱导过程中甘油三酯含量呈稳步上升趋势,各时间点均存在极显著差异（P<0.01）。qRT-PCR结果显示,PPARγ基因表达量在诱导中后期高表达;FABP4在诱导分化第6天达到最高,极显著高于其余时间点（P<0.01）;CEBP/β和HSL表达趋势一致,均呈先升高后降低趋势。诱导成骨分化后,发现细胞形态变为不规则状,诱导后期细胞复层生长形成骨钙结节,茜素红染色可见圆形不透明钙化结节,数量和密度较未诱导时期都明显增加,结果表明细胞出现成骨向分化。成骨标志基因BGLAP、RUNX2的表达量也随诱导进程呈稳步上升趋势,相比未诱导细胞差异极显著（P<0.01）。【结论】建立了基于联合酶消化和反复差速贴壁实现猪骨骼肌卫星细胞分离和纯化的方法,所得细胞增殖能力强且具有多向分化潜能,为猪骨骼肌卫星细胞作为种子细胞用于未来组织工程研究提供了技术平台。     

红鳍笛鲷视杆蛋白和长波敏感视蛋白的基因克隆及表达规律分析

【目的】克隆红鳍笛鲷(Lutjanus erythropterus)视杆蛋白(RH1)基因和长波敏感视蛋白(LWS)基因,并分析其在早期发育不同阶段和成鱼不同组织的表达规律,为探究笛鲷属鱼类适应从表层浮游逐步转为下层底栖光环境生活的过程提供理论基础。【方法】利用RACE克隆红鳍笛鲷RH1基因和LWS基因cDNA全长,利用生物信息学软件对2个基因序列及编码的氨基酸序列进行分析,并利用实时荧光定量PCR检测其在红鳍笛鲷6个胚胎发育时期(原肠下包1/2期、胚孔封闭期、视囊期、晶体出现期、心脏跳动期、孵化出膜期)和5个仔鱼发育时期(1、3、10、15和20 d),以及成鱼不同组织(脑脏、心脏、肝脏、肌肉、黑色皮肤、红色皮肤、胃和视网膜)的表达规律。【结果】红鳍笛鲷RH1基因和LWS基因cDNA全长分别为1723 bp和1302 bp,其中RH1基因具有465 bp的3'-UTR和196 bp的5'-UTR,开放阅读框(ORF)长度为1062 bp,编码353个氨基酸残基;LWS基因具有213 bp的3'-UTR和15 bp的5'-UTR,ORF长度为1074 bp,编码357个氨基酸残基。系统发育分析结果显示,2个视蛋白基因均先与鲈形目等鱼类聚为一支,再与鳉形目、鲽形目和鲤形目等聚为一支,最后再与陆生脊椎动物聚为一支。实时荧光定量PCR检测结果显示,RH1基因在孵化出膜后15和20 d的相对表达量显著高于其他时期(P<0.05,下同),LWS基因在孵化出膜后20 d的相对表达量显著高于其他时期;2个视蛋白基因在成鱼不同组织的表达模式相似,在视网膜上的相对表达量均显著高于其他组织。【结论】红鳍笛鲷RH1和LWS基因的表达具有组织特异性,且在早期发育阶段的表达水平与红鳍笛鲷的生活习性变化相关,表明红鳍笛鲷RH1和LWS基因表达与其生活光环境的变化密切呼应,在早期发育变态阶段的光线调节,以及适应黑暗环境等方面发挥重要作用。     

转录组分析组蛋白去乙酰化酶抑制剂TSA对毛白杨悬浮细胞表达谱的影响

组蛋白去乙酰化酶HDAC具有调节细胞增殖、诱导细胞分化、凋亡的作用, 是近年生物学研究热点之一, 而在木本植物方面的作用却鲜有报道。目的本实验用组蛋白去乙酰化酶抑制剂曲古抑菌素A(Trichostatin A, TSA)对杨树悬浮细胞进行处理, 通过改变细胞的组蛋白乙酰化水平, 来探讨其对基因表达谱变化的影响。方法用0.5 μmol/L的TSA处理悬浮细胞, 10 d后收集材料用于RNA-Seq分析, 比较经过TSA处理与对照组的悬浮细胞之间转录组表达差异情况。结果通过显微镜观察发现, 对照组细胞呈均匀椭圆形, 而经过TSA处理后的细胞出现圆形及长条形细胞。通过转录组分析得到4 465个差异表达基因(DEGs), 其中2 363个基因上调, 2 102个基因下调。差异表达基因主要富集在细胞周期、苯丙素生物合成、细胞壁结构成分和乙酰化相关等途径。结论通过分析TSA处理和对照组的悬浮细胞基因表达差异, 发现TSA通过影响组蛋白乙酰化水平直接或间接影响细胞生长、细胞壁组分和细胞周期的相关基因, 为认识组蛋白乙酰化对植物生长发育的影响提供依据。      

Differentiation-linked leukemogenesis in lymphocytes

Most human lymphoid malignancies preserve a pattern of gene expression reflecting their proliferative activity and the development level of clonal expansion and maturation arrest. Characteristics of leukemia and other cancer cells frequently considered to reflect aberrant differentiation may more often reflect clonal selection of cell types that are normally infrequent and transitory. The differentiation status of progenitor or mature lymphoid cells influences which genetic elements are at risk of being exploited, via mutation, recombination, or deletion, for clonal advantage. These alterations may frequently arise spontaneously as a consequence of the unique developmental and functional programs of lymphoid cells and have as a major phenotypic consequence the stabilization of transitory cellular phenotypes.     

鸭胚骨骼肌成肌细胞中MyoD1和Myf5基因的表达与分析

以高邮鸭和金定鸭为研究对象,采用实时荧光定量PCR方法测定生肌调节因子MyoD1和Myf5基因在不同发育阶段鸭胚（13、15、17、19、21和23胚龄）骨骼肌成肌细胞中的表达水平,分析MyoD1和尬庐基因在鸭胚胎期骨骼肌成肌细胞中的表达模式和差异．结果表明,Myf5和MyoD1基因在胸腿肌成肌细胞中呈现不同的表达规律：胸肌成肌细胞中,Myf5基因的表达呈“低→高→低→较高”的趋势,类似反“-”形;MyoD1基因的表达呈“低-高-低”的趋势,在金定鸭17胚龄时达到高峰后迅速下降（P〈0．01）,并维持在低水平,类似“∧”形,而在高邮鸭17胚龄时达到高峰后维持高水平至19胚龄（P〉0．05）,再下降并维持在低水平（P〈0．05）,类似“∩”形．腿肌成肌细胞中,岣筘基因的表达呈“低-高-低”的趋势,类似“∧”形;MyoD1基因的表达呈“较高→低→高→低”的趋势,类似“－”形．结果提示：Myf5和MyoD1基因在骨骼肌成肌细胞中的表达具有时间和品种依赖性;两基因在两品种鸭胸腿肌中均在17胚龄时达到高峰,表明17胚龄可能是成肌细胞分化和增殖的一个重要时期．     

Regulatory Expression of Peroxisome Proliferator Activated Receptors Genes During Fatty Liver Formation in Geese

In order to investigate the expression pattern of peroxisome proliferator activated receptor （PPAR） genes before and after overfeeding, and estimate the effect of expressed PPAR levels on weights of fatty liver and abdominal fat in geese, the RT-PCR products of PPAR genes in heart, liver, spleen, lung, kidney, stomach, small intestine, brain, breast muscle, leg muscle, and abdominal fat were determined before and after overfeeding. RT-PCR was used to determine the expression levels of PPAR genes. Quantity one software was used to analyze absorbency, and the expression level of GAPDH gene was used as contrast. Expression levels of PPAR-α were relatively high in most of detected tissues but undetectable in abdominal fat tissue before overfeeding, and the level was evidently increased in lung, appeared in abdominal fat tissue, and reduced in the other tissues after overfeeding. Expressed PPAR-γ levels were relatively high in liver, spleen, lung, small intestine, and abdominal fat, and relatively low in the other tissues before overfeeding. Expressed PPAR-γ levels were enhanced in liver, spleen, lung, stomach, and kidney but decreased in abdominal fat and without obvious changes in the other tissues. Expression patterns of PPAR genes show tissue-specific manner. In addition, expression patterns of PPAR-α are different from PPAR-γ after overfeeding. It might suggest that different functions of PPAR subtypes are responsive to overfeeding.     

广西麻鸡m6A甲基转移酶基因表达与肌纤维类型及成肌分化的关系

[目的]肌纤维类型及其转化的调节是改善肉质的重要途径,最新研究表明,m6A RNA甲基化修饰在肌肉的分化中发挥着重要作用.以优良的地方品种——广西麻鸡作为研究对象,研究其性成熟日龄不同部位肌纤维类型的组成差异,探究m6A甲基化转移酶相关基因甲基化转移酶样蛋白3/14(methyltransferase like 3/1...