FOXO3a过表达对内皮祖细胞周期相关蛋白的调控

目的:观察FOXO3a(forkhead box O3a)的活性改变对内皮祖细胞(endothelial progenitor cells,EPCs)增殖和细胞周期相关蛋白表达的影响。方法:将携带突变激活FOXO3a基因的腺病毒载体Ad-TM(triple mutant)-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27kip1以及CDK2的表达水平。结果:构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM-FOXO3a组EPCs细胞生长缓慢,集落不明显;Western blot和CCK-8结果显示,Ad-TM-FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27kip1蛋白过表达,CDK2表达下调。结论:FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。     

FOX03a过表达对内皮祖细胞周期相关蛋白的调控

目的：观察FOXO3a（forkhead box O3a）的活性改变对内皮祖细胞（endothelial progenitor cells,EPCs）增殖和细胞周期相关蛋白表达的影响。方法：将携带突变激活FOXO3a基因的腺病毒载体Ad-TM（triple mutant）-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27^kip1以及CDK2的表达水平。结果：构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM—FOXO3a组EPCs细胞生长缓慢,集落不明显;Westem blot和CCK-8结果显示,Ad-TM—FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27^kip1蛋白过表达,CDK2表达下调。结论：FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。     

CEACAM1异常表达对白血病BALL-1细胞增殖的影响

通过si RNA技术抑制癌胚抗原相关粘附分子CEACAM1在人急性B淋巴细胞白血病细胞系BALL-1中的表达,体外实验研究异常表达于白血病B细胞的CEACAM1对细胞增殖的影响。应用CCK-8法测定细胞增殖发现CEACAM1表达下调后BALL-1细胞的增殖能力明显下降。细胞周期分析结果显示CEACAM1被抑制后细胞增殖状态表现为S期细胞百分比降低,G0/G1期细胞比例升高,提示CEACAM1表达下调是通过引起细胞周期停滞在G0/G1期来降低细胞增殖的,表明CEACAM1本身对白血病B细胞具有促进增殖的作用。     

小激活RNA上调抑癌基因p21表达及其应用前景

小激活RNA (small activating RNA, saRNA)是新近发现的能够靶向作用于基因启动子区域,并在转录水平诱导基因表达的小分子双链RNA。RNA激活通过重新开启内源性基因的表达、恢复蛋白质的天然功能来发挥治疗作用,在医学上具有巨大的应用前景。抑癌基因p21的主要作用是使细胞周期停滞于G1/S和G2/M期,从而抑制细胞增殖。该文就saRNA上调抑癌基因p21表达的生物学意义及saRNA药物应用的可行性作一综述。     

pLKO.1-shSIRT7重组质粒的构建及其对人肝癌PLC/PRF/5细胞系增殖的影响

基于近年来研究发现sirtuin 7 (SIRT7)与肝癌密切相关,为此,本研究获取SIRT7基因敲除小鼠的肝细胞基因表达谱数据,在m RNA整体水平上分析SIRT7在肝细胞中的作用;另外,构建特异性下调人SIRT7基因的短发夹RNA (shRNA)表达质粒,并初步研究SIRT7基因沉默对肝癌PLC/RPF/5细胞系的影响。应用DAVID、GSEA、Cytoscape等生物信息学相关工具分析基因表达谱数据。基于pLKO.1空载质粒构建p LKO.1-sh SIRT7重组质粒并通过酶切和测序鉴定。使用Lipofectamin 2000转染重组质粒进入PLC/RPF/5细胞,并通过PCR和Western blotting验证SIRT7基因的沉默效率。通过MTT实验检测细胞增殖能力的改变和流式细胞术检测细胞周期的变化,并使用PCR检测鉴定CDC4、TK1、CDC34、BCL2等基因表达水平的变化。基因表达谱分析结果显示,SIRT7基因敲除后,基因mRNA整体水平变化主要集中在与细胞周期、细胞凋亡等相关的通路中。重组质粒酶切与测序结果显示,成功构建了pLKO.1-shSIRT7重组质粒。转染后PLC/RPF/5细胞SIRT7基因的表达得到有效沉默,与对照组相比,沉默SIRT7基因的表达可导致细胞的增殖能力降低并引起G0/G1期的细胞比例增多,PCR实验显示CDC4与BCL2表达水平增高,TK1与CDC34表达水平降低。以上研究结果提示,在肝癌PLC/RPF/5细胞系中沉默SIRT7的表达可使更多的细胞阻滞在G0/G1期,从而降低其增殖能力,其机制可能与引起细胞周期相关基因的表达改变有关。     

P14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53,诱导细胞周期阻断或凋亡.利用高表达p14ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14ARF高表达能将细胞周期阻断在G1和G2期,p53,p2lcipl和p27kipl蛋白水平明显增强,而p-ERK1/2,CyclinDl和CyclinE蛋白水平下降,明显抑制细胞生长.提示p14ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

RHCG基因在非小细胞肺癌中的表达及对细胞增殖的影响

为了探讨Rh type C glycoprotein (RHCG)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响及可能的作用机制,本研究使用荧光定量PCR法检测12对NSCLC及癌旁组织样本中RHCG mRNA的表达水平及pcDNA3.1-RHCG质粒对A549细胞RHCG m RNA的表达;采用CCK-8法检测细胞增殖能力;运用PI染色法检测细胞周期;使用免疫印迹法检p-PI3K、PI3K、p-AKT以及AKT蛋白表达水平。本研究发现,与癌旁组织比较,NSCLC中RHCG m RNA表达水平明显降低。RHCG过表达能抑制NSCLC细胞系A549细胞增殖能力。此外,RHCG过表达使A549细胞周期G1/S期转化发生阻滞。本研究还发现,RHCG过表达可下调A549细胞p-PI3K/PI3K和p-AKT/AKT水平。本研究表明,RHCG抑制NSCLC细胞增殖的作用与其抑制PI3K/AKT信号通路有关。     

沉默EZH2基因表达对宫颈癌细胞增殖能力影响的分析

本实验旨在探讨EZH2基因在宫颈癌中表达情况、对宫颈癌细胞增殖能力的影响及其机制。通过免疫组织化学法检测20例正常宫颈、20例CIN及60例宫颈鳞癌组织中EZH2蛋白表达;采用RT-PCR法检测4个宫颈癌细胞株中EZH2 m RNA的表达。通过Lipofectamin2000介导EZH2 si RNA瞬时转染C33A细胞株,Western blotting法检测si RNA的干扰效率。MTT、流式细胞仪检测EZH2沉默后细胞增殖及细胞周期变化;Western blotting检测p21表达变化。结果显示,正常宫颈组织、CIN、宫颈鳞癌中EZH2阳性表达率分别为15%、30%、76.7%,呈逐级升高趋势,后者与前两者见差异有统计学意义(p0.01);EZH2的表达同宫颈鳞癌细胞分化程度、间质浸润深度及淋巴结转移显著相关(p0.05)。EZH2 m RNA在宫颈癌细胞株He La、Si Ha、C33A及Caski中均表达,其中C33A细胞中表达最高。si RNA沉默EZH2基因明显抑制宫颈癌C33A细胞增殖,阻滞细胞周期于G1期;Western blotting检测p21表达上调。由此得出结论,EZH2基因在宫颈癌中高表达;沉默EZH2基因可通过上调p21蛋白的表达阻滞细胞周期于G1期、抑制宫颈癌细胞的增殖。     

过表达UHRF2通过上调p53及p21表达引起HEK293细胞G1期阻滞

UHRF2（ubiquitin like with PHD and ring finger domains 2）是新近发现的具有多个结构域的核蛋白,在细胞周期调控和表观遗传学中发挥重要作用．近期研究提示,UHRF2是肿瘤抑制蛋白p53的1个E3连接酶,在体内外能与p53相互结合并促进其泛素化,过表达UHRF2能使细胞周期停滞于G1期．然而,UHRF2介导的G1期阻滞及其与p53联系尚不清楚．通过共转染UHRF2质粒及p53特异性小干扰RNA(siRNAs)到HEK293细胞构建细胞模型,探索UHRF2引起细胞周期停滞与p53之间的关系．结果显示,UHRF2能促进HEK293细胞中p53的稳定,从而引起p21 (CIP1/WAF1)基因表达,并使细胞周期停滞于G1期;但在siRNA抑制p53的表达后p21(CIP1/WAF1)表达降低,UHRF2引起的细胞周期阻滞消除．研究结果提示,UHRF2可通过稳定p53,上调p21的表达,从而介导细胞周期阻滞于G1期;同时UHRF2可能参与细胞周期调控及DNA损伤反应（DNA damage response, DDR）．UHRF2稳定p53的具体分子机制及其在DDR中的作用有待进一步研究证明．     

贺谈老百岁华诞

谈家桢教授是国际著名遗传学家,我国现代遗传学的奠基人之一,他也是一位卓越的教育家和社会活动家。 1909年9月15日,谈家桢先生出生于浙江宁波。他就读于苏州东吴大学,1930年获理学学士学位。随后赴北京燕京大学攻读硕士学位,导师是我国现代遗传学奠基人之一的李汝祺教授,1932年获硕士学位。经导师推荐,谈家桢先生赴美国深造,师从当代遗传学宗师摩尔根,在遗传学家杜布赞斯基的指导下完成博士研究生学业,于1936年获美国加州理工学院哲学博士学位。     

Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.     

Expression and characterization of the C345C/NTR domains of complement components C3 and C5

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

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.     

Formation of Desacetylcephalosporin C in Cephalosporin C Fermentation

The origin of desacetylcephalosporin C in cephalosporin C fermentation broths was investigated. Esterase activity was detected in cell-free extracts of Cephalosporium acremonium, but these extracts failed to deesterify cephalosporin C. When cephalosporin C was added to sterile and inoculated fermentation media, the antibiotic decayed at nearly identical rates. The formation of desacetylcephalosporin C during the fermentation was measured by quantitative chromatography and by the incorporation of valine-1-(14)C into the molecule. The rate constants obtained from the results of these experiments were equivalent to those for the decay of cephalosporin C in sterile and inoculated media. The data demonstrate that desacetylcephalosporin C is produced by nonenzymatic hydrolysis of cephalosporin C.     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family

Summary A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F ^* 3 C4F ^*2.2 is composed of two C4F alleles and the other C4S ^* 5.1 C4S ^*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB ^*35, Bf ^* F, and HLAD/DR ^*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(a+) variant.This work was supported by grant No 12-1727 from the Danish Medical Research Council