人胚胎干细胞和分化细胞差别基因的筛选

人类胚胎干细胞(human embryonic stem cell,hESC)在增殖过程中如何保持未分化状态是干细胞生物学的核心问题之一,已有的LIF通路、Oct-4因子及Nanog基因的作用还不能对这一问题做出全面的解释。为进一步了解维持hESC未分化状态的机制,采用自行建立培养的hESC及分化的hESC细胞克隆(differentiated hESC,dhESC),应用抑制消减杂交(Suppression Subtractive Hybridization,SSH)结合反向cDNA斑点杂交技术,筛选出在hESC高表达、在dhESC中低表达或不表达的表达序列标签(Expressed Sequence Tag,EST)共105个。通过与GenBank比较,显示其中76个EST代表61个已知基因,18个EST代表15个假想基因,另有11个属于未知EST。选取其中8个克隆进行半定量RT-PCR分析,证实其中7个克隆在hESC中高表达,在dhESC中低表达或不表达。结果表明,hESC分化前后涉及多个基因的差异表达,对这些在hESC中高表达、在dhESC中低表达或不表达基因的进一步功能研究为阐明维持hESC未分化状态的机制提供了基础。     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.     

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

树干毕赤酵母酒精发酵碳元素代谢流向分析

实验对树干毕赤酵母（Pichia stipitis）进行了4个阶段共400 h连续恒化培养,在不同阶段以30.0 g/L葡萄糖作为基本碳源,添加30.0或15.0 g/L的木糖,通过控制温度（35±1）℃,进气量100～150mL/min,搅拌转速250～300 r/min。4个阶段共建立4个连续培养的"稳态"。对碳元素进行物料衡算发现,四个阶段碳元素回收率分别为118.0 %、105.6 %、113.5 %和94.7 %。对4个近似"稳态"的碳元素的代谢流向进行分析发现：将近50.0 %左右碳元素流向产物酒精,其次是CO2和酵母细胞;木糖醇浓度与流入底物中木糖浓度有直接关系,在相同发酵条件下流入的木糖浓度越大代谢生成木糖醇浓度也越高;实验所采用的通气条件更适合底物为30.0 g/L葡萄糖和30.0 g/L木糖混合液的连续发酵。     

胃印戒细胞癌与非印戒细胞癌临床病理分析

目的：通过比较胃印戒细胞癌与非印戒细胞癌的临床特征与病理资料,探讨胃印戒细胞癌的临床特征,病理特点及预后。方法：回顾性分析2005年1月至2007年1月在哈尔滨医科大学附属第三医院住院治疗的1138例胃癌患者临床资料,根据组织学类型分为胃印戒细胞癌组和非印戒细胞癌扣对两组的性别、年龄、家族史、肿瘤发生部位、浸润深度、淋巴结转移、远处转移及根治术后中位进展时间、1年生存率和3年生存率进行统计分析比较。结果：胃印戒细胞癌好发于女性（P〈0．05）;年龄≤55岁的患者发病率高于非印戒细胞癌（P〈0．05）,≥56岁的患者发病率低于非印戒细胞癌（P〈0．05）;肿块生长部位以胃中下部为主（P〈0．05）,侵犯胃上部者明显少于非印戒细胞癌（P〈0．05）;胃印戒细胞癌多数在胃壁组织内呈弥漫浸润性生长,垂直浸润能力较弱,突破浆膜层者明显少于非印戒细胞癌（P〈0．05）;根治术后中位进展时间胃印戒细胞癌（10个月）较非印戒细胞癌（12个月）提前2个月,进展风险增加（P〈0．05）;胃印戒细胞癌在家族史、淋巴结转移、远处转移及根治术后1年和3年生存率方面与非印戒细胞癌比较无差异。结论：胃印戒细胞癌多发于中青年女性;好发年龄小于56岁;发病部位以胃中下部为主;多呈弥漫性浸润性生长,且较少突破浆膜层;中位进展时间短,进展风险高。     

锡金海棠子叶再生体系的建立

锡金海[Malussikkimensis（Wenz．）Koehne]是我国稀有植物和优良苹果砧木种质资源。本研究探讨了影响锡金海棠离体再生的条件,建立了锡金海棠子叶高效离体再生体系。结果表明,子叶再生体系的最佳诱导培养基SC＋0．1mg·L-1 NAA＋2．0mg·L-1 TDZ,最佳分化培养基SC＋0．1mg·L-1 NAA＋1．5mg·L-1 TDZ,最佳继代增殖培养基SC＋2．0mg·L-1 6-BA＋0．2mg·L-1 NAA,最佳生根培养基为1／2MS＋I．0mg·L-1 IBA。炼苗后,移入珍珠岩、花生壳与草炭（1：1：1）的基质中,20d移栽成活率高达90％。     

Oligonucleotide biological activity: Relationship to the cell cycle and nuclear transport

Previous studies suggest that oligodeoxynucleotide (ODN) cellular uptake is cell cycle-dependent which may have important implications in cancer cell targeting. To further our understanding of ODN transport and activity, this study examines the relationships between the cell cycle, ODN cellular uptake, intracellular transport, and activity. An antisense c-myc ODN 21-mer was used to study ODN cellular uptake in Rauscher erythroleukemia cells synchronized by either chemical methods or flow cytometry. ODN uptake was examined using subcellular fractionation and confocal fluorescence microscopy. Western blot analysis was used to measure ODN-mediated decreases in c-myc protein levels. Intracellular ODN distribution and extent of uptake was influenced by the phase of the cell cycle, but the mechanism of uptake was not. The relative activity of the antisense ODN was positively correlated to ODN distribution to the cytosol, but negatively correlated to total cellular uptake. Although ODN total cellular uptake is positively influenced by the cell cycle, retention of the ODN in the cytosol (presumably extra-vesicularly) appeared to be relevant in determining the activity of an antisense ODN. Novel methods to target cytosol-acting drugs to the cytoplasm may therefore be warrented.     

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 10⁶–10⁸ peach mesophyll cells and protoplasts · gfw^-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml^-1. Purification of protoplasts on a sucrose gradient yielded about 10⁷ protoplasts · gfw^-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - FDA fluorescein diacetate - MES 2-M-morpholinoethane sulphonic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PVP polyvinylpyrrolidone     

siRNA沉默ERp57对细胞中CRT表达与定位的影响

探讨ERp57基因表达沉默对人小细胞肺癌A549细胞中CRT表达和定位的影响。利用siRNA技术获得ERp57基因表达沉默的人A549肺癌细胞株,分析该细胞株中ERp57基因以及CRT基因的蛋白表达水平,免疫荧光法检测细胞中CRT的表达和亚细胞定位,荧光法检测细胞凋亡。成功获得ERp57基因表达沉默的人A549肺癌细胞株。在该细胞中,CRT表达上调但仍定位于内质网中。用米托蒽醌处理对照细胞14 h后,可使CRT大量转移到细胞膜表面并发生簇集,但在ERp57表达沉默的细胞中,CRT的膜转移和簇集现象不明显。细胞凋亡分析显示,米托蒽醌处理细胞48 h后,所有细胞均出现凋亡细胞典型细胞核固缩、分裂现象。试验证明抑制ERp57蛋白表达会增加A549肺癌细胞中CRT的含量,但同时也阻断蒽环类药物诱导的CRT膜转移,提示ERp57也是介导肿瘤细胞免疫原性凋亡的重要因子。     

Pectate distribution and esterification in Dubautia leaves and soybean nodules,studied with a fluorescent hybridization probe

Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid) - NMR nuclear magnetic resonance spectroscopy - TTB 1,3,5-triazido-2,4,6-trinitrobenene