敲减DTX4表达抑制C2C12细胞成肌分化

DTX4（Deltex 4 homolog）蛋白属于Deltex家族成员|Deltex家族是Notch信号通路的调节因子. 已知Notch信号通路在成肌分化中发挥重要作用. 然而,DTX4是否参与调控肌肉发育尚未有报道. 本研究探索DTX4对成肌分化的影响及作用机制. 实时定量PCR和蛋白质印迹分析揭示,伴随小鼠C2C12成肌细胞（myoblast）分化为肌管（myotube）过程,成肌分化标志蛋白肌球蛋白重链（myosin heavy-chain,MyHC）、肌细胞生成素(myogenin)表达逐渐升高,DTX4 mRNA及蛋白质表达水平也逐渐升高. 通过顺序专一的siRNA敲减DTX4表达后,C2C12成肌细胞肌管面积和肌管融合指数明显减少|MyHC、肌细胞生成素蛋白表达水平明显降低|但ERK信号通路未见明显变化.上述结果表明,敲减DTX4表达抑制C2C12细胞成肌分化.我们的结果提示,DTX4可能参与C2C12细胞成肌分化.     

小鼠肌肉生长抑制素基因短发夹RNA促进MyoD在C2C12细胞中的表达

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子-β(transforming growth factor-β,TGF-β)超家族,主要功能为负向调节骨骼肌的生长.肌肉生长抑制素基因敲除小鼠肌肉出现显著增加,而将干涉该基因的短发夹RNA注射并电击转化入大鼠胫前肌则引起肌肉重量、肌纤维以及MHCⅡ表达的增加.通过与小鼠肌肉生长抑制素基因表达载体共转染HEK293细胞,筛选到两条能够高效抑制小鼠肌肉生长抑制素基因表达的小干涉RNA.构建了这两条小RNA的表达载体Mst-shRNA1和Mst-shRNA2,用其分别转染小鼠C2C12成肌细胞,并通过G418药物筛选和流式细胞仪富集整合了短发夹RNA表达载体的阳性细胞.通过采用Real-time PCR和Western blot分析,检测到在分别整合了Mst-shRNA1和Mst-shRNA2的C2C12细胞中,内源性肌肉生长抑制素基因的mRNA水平分别下降了10.2%和35.5%,蛋白质表达则分别下降了29.3%和64.7%.同时,在这两组中MyoD的表达上升了24.4%和40.4%,证明通过RNA干涉实现的肌肉生长抑制素基因的抑制导致了下游MyoD基...     

Arg缓解MSTN对猪肌肉间充质干细胞成脂-分化的抑制

间充质干细胞可分化为脂肪细胞并沉积脂质,从而增加动物脂肪沉积.因此,猪肌肉间充质干细胞被认为是肌内脂肪的重要来源之一.本研究主要在体外探讨了肌肉生长抑制素（MSTN）对猪肌肉来源间充质干细胞成脂分化的影响及Arg的调控作用.结果表明,外源添加MSTN活性蛋白极显著地降低了胞内甘油三酯水平,而添加Arg或MSTN抗体则表现相反的作用（P〈0.01）.同时添加Arg可缓解MSTN对间充质干细胞脂质沉积的抑制作用（P〈0.01）.生脂转录因子表达模式分析表明,外源添加MSTN抑制了细胞PPARγ2和aP2的表达,添加Arg和MSTN抗体增强了细胞ADD1的表达（P〈0.01）.此外,同时添加MSTN蛋白和Arg较仅添加MSTN蛋白极显著增强了细胞ADD1和PPARδ的表达（P〈0.01）,同时添加MSTN和Arg抗体较仅添加MSTN抗体显著增强了细胞ADD1,PPARδ,C/EBPα,PPARγ2和LPL的表达（P〈0.05）.由此可见,MSTN抑制了猪肌肉来源间充质干细胞成脂分化,添加Arg至少部分地通过上调ADD1和PPARδ的表达来缓解MSTN对成脂分化的抑制作用.     

共表达HIV-1中国流行株gp120与IL-18重组鸡痘病毒的构建及其免疫原性观察

为筛选我国的HIV-1疫苗候选株,以鸡痘病毒282E4中国疫苗株为载体,构建了共表达中国流行株HIV-1外膜蛋白gp120和IL-18的重组鸡痘病毒,并将该重组鸡痘病毒免疫BALB/c小鼠,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体水平。结果显示,HIV_1外膜蛋白gp120和IL-18不但可在重组鸡痘病毒感染的鸡胚成纤维细胞中表达,而且可在重组鸡痘病毒感染的哺乳动物细胞中表达。重组病毒具有良好的免疫原性,可诱导小鼠产生特异性抗体和脾特异性CTL反应,且IL_18发挥了免疫佐剂的作用。本研究结果为制备安全、有效的HIV-1基因工程活载体疫苗奠定了基础。     

转基因高表达卵泡抑素1对斑马鱼肌肉生长促进作用研究

抑肌素是转化生长因子β超家族成员,其主要功能是对肌肉的生长发育起负调控作用.在哺乳动物中,抑肌素信号是通过其特异性的与激活素受体ⅡB亚基结合而发挥作用.因此,其信号传递作用可以被其结合蛋白以及ActR ⅡB的结合蛋白卵泡抑素所抑制.在小鼠肌肉组织中,高表达卵泡抑素可以导致肌肉组织肌纤维的增生和肥大.为了研究鱼类卵泡抑素...     

干扰Sema7A抑制C2C12成肌细胞的增殖和分化

为研究脑信号蛋白家族（Semaphorins）成员Sema7A对成肌细胞增殖和分化的影响,本文设计并合成了Sema7A基因的小干扰RNA（small interfering RNA,siRNA）,用此siRNA转染C2C12成肌细胞．通过Hoechst核染和流式细胞术检测细胞增殖情况,免疫荧光检测肌管的形成情况,real-time qPCR和Western印迹技术检测成肌标记基因的变化.结果显示,干扰Sema7A后,C2C12成肌细胞增殖减慢,处在G2和S期的细胞所占的比例明显下降,而G1期细胞的比例升高．免疫荧光检测结果显示,干扰Sema7A后,肌管的直径及MyHC+细胞所占比例均显著降低．Real-time qPCR和Western印迹结果也显示,肌肉分化标志基因MyoD、MyoG、MyHC的mRNA及蛋白质表达均下降．进一步检测Sema7A受体下游信号通路发现,干扰Sema7A后,其下游信号分子PI3K和AKT的磷酸化水平被下调．以上结果表明,Sema7A可以调节C2C12成肌细胞的增殖和分化,可能是通过其受体作用于PI3K/AKT信号通路实现的,这为进一步研究Sema7A在骨骼肌发育中的作用提供实验基础.     

外源性重组人睫状神经营养因子抑制成人成肌细胞的体外分化

为探讨外源性重组人睫状神经营养因子(rhCNTF)在成肌细胞分化中的作用,实验观察了0-10 ng/mlrhCNTF对成人成肌细胞体外分化的影响。结果表明,与对照组相比,2.5-10 ng/ml rhCNTF能显著抑制成肌细胞的体外分化(P<0.01),并呈量-效依赖关系,且这种抑制作用是可逆的。Western Blot分析提示,这种抑制作用伴有成肌细胞分化期特异标志myogenin和p21表达量的显著降低(P<0.01),以及成肌细胞增殖期特异标志myf5和desmin表达量的显著增加(P<0.01)。因此可以认为,外源性rhCNTF能可逆地抑制成人成肌细胞的体外分化并保持增殖。     

人Sema4C重组腺病毒载体的构建及其在小鼠成肌细胞系C2C12中的表达

利用Ad5腺病毒载体系统构建人Sema4C基因重组腺病毒表达载体并在成肌细胞系C2C12中表达,并初步探讨Sema4C基因在成肌发育过程中的可能作用。利用脂质体介导重组腺病毒载体转染HEK293细胞,包装出完整的腺病毒;将重组腺病毒载体感染C2C12成肌细胞后,利用激光共聚焦显微镜观察发现12h即有绿色荧光表达,24h后绿色荧光蛋白表达最强;流式细胞仪检测病毒的感染效率几乎达100%。WB检测结果表明感染重组腺病毒载体组C2C12细胞Sema4C蛋白的表达量明显高于空载体对照组（P<0.01）。为了进一步观察Sema4C基因对C2C12细胞增殖分化的影响,流式细胞仪检测了病毒感染48h后C2C12细胞的增殖指数,并对感染后诱导分化的C2C12细胞的分化情况进行了观察。我们的结果首次表明,过表达外源性人Sema4C基因不仅能使C2C12细胞的G0/G1期比例增加,细胞的增殖指数下降,同时在分化培养条件下还能促进C2C12细胞肌管的形成。     

编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长

肌生长抑制素(myostatin,MSTN)是转化生长因子 β(transforming growth factor-β,TGF-β)家族成员之一,是一种肌肉生长抑制因子.解除MSTN的生长抑制功能是提高畜禽肌肉产量的一种有效途径.TGF-β 的半胱氨酸节结构基元(cystine knot motif)能够稳定MSTN...     

低氧抑制C2C12细胞的体外分化

目的:研究低氧环境对C2C12细胞分化的影响,为探讨肌肉的发生和骨骼肌的损伤修复机理提供理论依据.方法:培养C2C12细胞,分别在常氧和低氧(3%O2)条件下诱导分化.免疫细胞化学方法检测成肌细胞终末分化的标志蛋白MI-IC(肌球蛋白重链)的表达;Western blot检测MHC以及MRFs(成肌调控因子)的表达.结果:在常氧条件下诱导分化的C2C12细胞融合形成肌管并表达MHC蛋白,而在低氧条件下培养的C2C12细胞几乎很少融合形成肌管并表达MHC蛋白;同时低氧下调了C2C12细胞中MRFs的表达.结论:低氧抑制了C2C12细胞的体外分化.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.