斑马鱼胚胎发育过程中FGF3基因的表达

为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PcR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T／FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示：FGF3P基因在2hp胚胎就有表达,并持续至胚胎孵化,12hpf胚胎FGF3表达量达到高峰（P〈0．01）;胚胎发育过程中心表达部位以头、尾、咽弓为主。由此得出结论,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。     

鸡胚胎生殖细胞在鼠胚成纤维细胞饲养层上的生长

目的:探讨以鼠胚成纤维细胞为饲养层分离、培养鸡胚胎生殖细胞的方法和条件。方法:分离、培养12.5～13.5d鼠胚成纤维细胞。分离孵化5.5d鸡胚原始生殖细胞,原代培养时不使用饲养层,与性腺基质细胞共培养;继代培养时将其置于鼠胚成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞。结果:鼠胚成纤维细胞可连续传代18代以上(4个月),3～15代细胞可以用作饲养层细胞。分离的鸡胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过9代。集落未分化标志高碘酸希夫反应(PAS)呈强阳性,体外分化实验表明胚胎生殖细胞具有多能性。结论:用鼠胚成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。     

植物冷驯化相关信号机制

植物经过非致死温度的处理可以获得更强的抗冷能力叫做冷驯化,主要包括寒驯化和冻驯化 .在冷驯化过程中,质膜首先感受冷信号,调节胞质中IP3的含量,诱导胞质Ca2+浓度的升高,从而激活CBF基因的表达.至今已经克隆了大量的冷调控基因,组成了复杂的信号传导网络,其中ICE1-CBF-COR通路在植物的冷驯化过程中起到重要的作用.ICE1基因编码一个MYB类型的碱性螺旋 环-螺旋（bHLH）转录因子,在上游调节CBF和 其它转录因子的表达,提高抗冷性. HOS1蛋白通过泛素化介导的蛋白降解负调控ICE1,另外,CBF还通过转录的自我调控保持恰当的表达水平.基因的分析研究证明,RNA修饰和核质转运在植物的抗冷过程中也具有重要作用.在不依赖于CBF的途径中,转录因子HOS9和HOS10在调节抗冷有关基因的表达和提高抗冷能力方面具有至关重要的作用.     

植物冷驯化相关基因研究进展

冷驯化是与提高植物抗冷性有关的生物化学及生理学过程,主要包括寒驯化（cool acclimation）和冻驯化（freezing acdimation）。在冷驯化过程中,植物体内许多基因在转录水平上的表达受到影响,已经克隆了大量的相关基因,它们组成复杂的分子调控网络。目前研究表明不依赖ABA的低温信号转导途径是植物冷驯化机制的重要组成部分,其中CBF/DREB1是该调控过程的关键转录因子,与植物通过冷驯化而提高冰冻耐受能力密切相关。进一步利用转基因技术,可以有效地改善作物的耐冷性状。     

miR-223促进小鼠胚胎成纤维细胞复制性衰老

微小RNA（MicroRNAs（或miRNAs）是作为强大的基因表达调控子,广泛参与多种生命过程,在细胞衰老进程中的作用也日益受到关注。miR-223是一个典型的抑癌基因,可显著抑制细胞增殖能力。此外,miR-223与阿尔茨海默症、心血管疾病以及类风湿性关节炎等衰老相关疾病的发生发展密切相关。尽管如此,miR-223在细胞衰老进程中的作用及其分子机制尚未见报道。本研究通过连续传代建立了小鼠胚胎成纤维细胞（MEF细胞）的复制性衰老模型,并利用荧光定量qRT-PCR检测发现,miR-223在衰老MEF细胞中的表达水平显著上调。随后,通过转染miR-223模拟物Agomir-223在MEF细胞中过表达miR-223,结果显示过表达miR-223可显著促进MEF细胞的衰老表型并抑制其增殖能力,而抑制miR-223的表达可延缓MEF细胞的复制性衰老进程。进一步利用生物信息学方法预测获得多个miR-223的候选衰老相关靶基因,包括Rasa1、Ddit4和Smad1等。然而双萤光素酶报告系统结果显示,miR-223并不显著影响其萤光强度,表明它们很可能并不是miR-223的下游靶基因。综上所述,miR-223可显著促进MEF细胞复制性衰老,然而其调节细胞衰老进程的分子机制依然有待深入研究。     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ (PLDδ)基因敲除突变体与野生型植株进行冻害胁迫处理后, 比较2种基因型植株的抗冻性。结果发现, 经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型, 而未经低温驯化的PLDδ敲除突变体与野生型的抗冻性没有显著差异, 表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析, 发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节, 对脯氨酸和可溶性糖的积累起负调节作用, 但是参与低温信号转导物质ABA诱导抗冻性的过程。     

稳定转染Dicer基因对HELF细胞功能的影响

目的观察正常人胚肺成纤维细胞（HELF）在稳定转染Dicer基因后增殖能力及迁移能力的变化。方法应用脂质体介导方法转染Dicer基因表达载体于HELF细胞,G418筛选,PCR检测整合情况,RT-PCR检测表达情况,Western-blot检测蛋白表达水平,确定稳定转染细胞株,MTT方法检测细胞增值能力,Transwell方法检测细胞迁移能力。结果Dicer基因稳定转染HELF细胞系建立,用RT-PCR和Western-blot方法检测到Dicer基因mRNA和蛋白表达水平都明显增强。与转染空载体的HELF细胞相比,转染Dicer基因的HELF细胞48、72、96h的MTT光吸收值明显升高（P〈0.05）,并且穿过人工重构基底膜的细胞数明显增多。结论Dicer基因稳定转染HELF细胞后,HELF细胞增殖能力和迁移能力都明显增强。     

低温诱导的黄瓜ccr18基因的cDNA克隆及其表达特性分析

采用mRNA差异显示银染技术克隆得到在黄瓜（Cucumis sativus L.)冷敏型品种“津研4号”低温锻炼中特异表达基因的cDNA克隆（ccr18),其大小为639bp。在基因组中以单拷贝或低拷贝形式存在。Northern blot分析显示ccr18基因在12、24、48和72h低温处理的黄瓜中表达,在6h低温处理及对照中没有表达。这表明ccr18基因与黄瓜低温锻炼相关,序列同源性比较表明,它与拟南芥(Arabidopsis thaliana)染色体ⅢBAC库中的F14P3基因组序列具有88％的同源性。     

人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

植物冷害机理及冷驯化的生理与分子学基础

介绍了近几年来植物冷害机理、冷驯化提高植物抗冷性的生理与分子生物学基础及冷信号转导的最新研究进展.     

低温锻炼对冰温贮藏牡丹切花抗冷性的影响

以牡丹品种‘玉面桃花’、‘清香白’和‘凤丹’切花为试验材料,测定室温对照(15~18℃保湿贮藏3d,RTS)、低温锻炼[(4±1)℃保湿贮藏3d,CAS]、冰温贮藏[(4±1)℃保湿贮藏3d,然后转入(-4±1)℃保湿贮藏3d,ITS]3种处理的牡丹切花花枝不同器官(花瓣、花药、子房、萼片、叶柄、叶片、茎杆)的过冷点(SCP)和冰点温度(FP),以及花瓣和叶片束缚水与渗透调节物质含量变化,明确低温锻炼对其切花抗冷能力的影响,为牡丹切花储运及销售过程中冰温贮藏温度控制提供参考依据。结果表明:经4℃低温锻炼3d的牡丹切花与室温对照相比,低温锻炼处理使3个牡丹切花不同组织的SCP和FP显著降低,但花瓣和叶片的束缚水、可溶性蛋白质、可溶性糖和脯氨酸含量均得到提高;冷锻炼后转入冰温贮藏处理进一步提高了牡丹切花花瓣和叶片的束缚水和可溶性蛋白质含量。研究发现,4℃低温锻炼显著提高了牡丹切花的抗冷性,使花枝能够更好地适应30~150d长期贮藏的-3~-4℃冰温环境。     

Pro-Angiogenetic Effects of Purified Extracts from Helix aspersa during Zebrafish Development

Helix aspersa is a species of land snail belonging to the Helicidae family, widespread in the Mediterranean and continental area up to Northern Europe. In some areas it is appreciated as a food, but is mostly considered a parasite of gardens and cultivated fields. The mucus of Helix aspersa has found multiple applications in the cosmetic and health fields. In the present study, we investigated for the first time the angiogenetic properties of purified extracts from Helix aspersa using a transgenic zebrafish line Tg (kdrl:EGFP). The angiogenesis induced by purified snail extracts was demonstrated by their capability to increase the three well-established parameters of angiogenesis: generation of intersegmental vessels, modeling of caudal venous plexus, and formation of sub-intestinal venous plexus. The effects appeared to be mediated by the vascular endothelial growth factor (VEGF) pathway, being prevented by pretreatment of embryos with the selective VEGF receptor antagonist SU5416, and supported by the increased VEGF mRNA levels found in snail-extract-treated embryos. Insufficient vascular supply is underlined by low VEGF signaling, primarily because of its indispensable role in preventing capillary loss. Our findings might have a pharmacological impact by counteracting VEGF hypofunction and promoting angiogenesis to maintain adequate microvascular and vascular density in normal and suffering tissues and organs.     

Cytoskeleton-Dependent Changes in the Structural Organization of Reticuloplasmins in Triticum aestivum Cells during Cold Acclimation and Treatment with Abscisic Acid

We studied the effects of the anti-microtubule drug, oryzalin, on the content and spatial organization of reticuloplasmins (Ca²⁺-binding marker proteins of the endoplasmic reticulum) in winter wheat seedlings after their cold acclimation (3°C, 7 days) and treatment with ABA (30 M). For identification and visualization of reticuloplasmins, we applied one-dimensional SDS-PAGE with subsequent Western blotting and indirect fluorescent microscopy. We used polyclonal HSP70 and CRH antibodies against BiP and calreticulin (Cal), respectively. On immunoblots, the brightest bands corresponded to polypeptides with mol wts of 58 kD (calreticulin) and 79 kD (BiP). The content of calreticulins in roots was shown to be higher than in leaves. Cold acclimation enhanced, and ABA treatment reduced, the concentration of calreticulins in root cells. Both treatments increased the BiP concentration in roots. Oryzalin (10 M, treatment for 3 h) did not affect the level of reticuloplasmins in roots of unhardened, cold acclimated, treated with ABA and with a combination of cold and ABA plants. However, both oryzalin and low-temperature treatments resulted in the accumulation of reticuloplasmins in the two spherical structures in the vicinity of the plasmalemma and nuclear envelope. After the combined action of oryzalin and low temperature, the cortical sphere of BiP proteins was shifted into the endoplasm and calreticulins appeared in the nuclear matrix. We believe that these changes in the reticuloplasmin localization are related to the rearrangement of the endoplasmic reticulum determined by the cytoskeleton modification. They result in the improved capacity of reticuloplasmins to control Ca²⁺ behavior and/or to the function as chaperones. The results obtained permit the conclusion that cytoskeletal proteins interact with reticuloplasmins, and this interaction might be involved in the transduction of the external and internal signals.     

大绒鼠冷驯化和脱冷驯化能量代谢特征的变化

通过测定冷驯化(5℃)到脱冷驯化(30℃)条件下,大绒鼠(Eothenomys miletus)的体重、摄入能、静止代谢率(RMR)、非颤抖性产热(NST)和血清瘦素含量等参数,探讨了血清瘦素浓度与能量收支的关系。结果表明,冷驯化可致大绒鼠体重下降,RMR、NST、摄入能升高,血清瘦素浓度降低;脱冷驯化后大绒鼠体重增加,RMR、NST、摄入能降低,血清瘦素浓度增加。血清瘦素含量与体重呈正相关,与RMR、NST、摄入能呈负相关。表明大绒鼠的体重、摄入能和产热能力具有较强的可塑性,且瘦素可能参与了大绒鼠适应冷驯化及恢复过程中的能量平衡和体重的调节。     

Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion

Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion‐related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3β during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell‐cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems‐oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell–substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering. J. Cell. Biochem. 109: 957–966, 2010. © 2010 Wiley‐Liss, Inc.     

烹调油烟挥发性有机物对人胚肺二倍体成纤维细胞活性氧水平的影响

目的：研究烹调油烟挥发性有机物（COFVOCs）对人胚肺二倍体成纤维细胞（HEIF）活性氧（ROS）水平的影响。方法：用活性炭采集烹调油烟挥发性有机物,对HELF进行染毒,采用MTT试验测得半数抑制浓度（IC50）,设计剂量和暴露时间,设立添加和不添加维生素C组,以2′,7′-二氯双氢荧光素二乙酸酯（DCFH-DA）和双氢罗丹明123（DHR123）进行标记,通过流式细胞术检测细胞ROS水平。结果：COFVOCs刺激HELF12、24、48h的IC50分别是104．9、111．9和127．2μg／mL;HEIF胞质内ROS平均荧光强度随COFVOCs剂量增加而增高;线粒体内ROS24、48h暴露于0．8、4μg／mL剂量与阴性对照组相比有统计学差异;维生素C预处理后,ROS平均荧光强度较未预处理时均有所降低,100μg／mL剂量组前后差异有统计学意义。结论：COFVOCs可引起HELF内ROS水平升高,维生素C对细胞的ROS升高有一定的抑制作用。