芹菜素对甲状腺癌BCPAP细胞生长及细胞周期的影响

研究芹菜素对人乳头状甲状腺癌BCPAP细胞生长的抑制作用及对细胞周期的影响。采用MTT法检测不同浓度芹菜素在24h对BCPAP细胞的抑制作用,以及12.5,25.0,50.0μmol/L芹菜素分别在24,48,72h对BCPAP细胞的抑制作用;通过明场细胞形态学照片分析,对比不同浓度芹菜素对BCPAP细胞形态的影响,评价其对细胞生长的抑制作用。利用流式细胞仪检测BCPAP细胞周期和凋亡。结果表明,不同浓度(12.5~100.0μmol/L)芹菜素对BCPAP细胞的毒性有明显剂量和时间依赖性,其24h的IC50值为40.65μmol/L。芹菜素对BCPAP细胞的形态学变化有显著影响,高浓度芹菜素强烈抑制BCPAP细胞数目的增长。芹菜素可使BCPAP细胞周期的构成发生明显的变化并诱导细胞凋亡。芹菜素对BCPAP细胞有较明显的细胞毒性和生长抑制作用,其抑制机制可能是使BCPAP细胞生长停滞在G2/M期并诱导细胞凋亡,使得细胞生存率下降,从而抑制细胞活性和数目增长。     

Fundamentals of cell proliferation: control of the cell cycle

Cell proliferation in higher eukaryotes is controlled by the extracellular environment and the state of differentiation. Many cells exist in a nondividing growth state termed quiescence. Some quiescent cells cannot proliferate and are said to be terminally differentiated. Others can be stimulated to divide in response to environmental signals or when cell replacement is needed. Finally, some cells undergo continual proliferation and differentiation. Growth regulatory factors generally act at specific stages of the cell cycle, most commonly during the first gap phase of the cell cycle. Once cells initiate DNA synthesis, they are generally committed to complete DNA replication. After DNA synthesis, additional signals determine whether cells in the last gap phase proceed through mitosis. In recent years, genes that appear to be critical for progression through the first two gap phases have been identified. Many are proto-oncogenes and therefore can neoplastically transform certain cells when mutated or inappropriately expressed. Growth factors that stimulate proliferation induce the expression of several proto-oncogenes; growth inhibitory factors often suppress proto-oncogene expression. As cells differentiate, the response to extracellular factors changes. In many cases, this may be due to intracellular controls that alter the response of certain proto-oncogenes to external signals.     

Composition of cell walls isolated from cell types of grain sorghum stems

Cell types were isolated from sorghum stems at two stages of development, anthesis and grain maturity, to study cell wall characteristics. Cell walls were isolated from epidermis (EPID), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) and analysed for total carbohydrate, acid insoluble lignin, total uronosyls, neutral sugars and hydroxycinnamic acids. In addition, walls from SCL, VBZ, IVB and PITH were subjected to chemical fractionation to separate wall carbohydrate into polysaccharide groups. Although wall characteristics were similar at both plant maturities, there were differences in lignin concentration, hydroxycinnamic acids, and carbohydrate composition among the cell wall types. Lignin was lowest in the PITH walls (169 g kg⁻¹) and highest in SCL and EPID (c 211 g kg⁻¹). Cellulose was most abundant in VBZ and SCL walls with greater secondary wall formation. Pectic materials were most abundant in PITH walls. Xylans were similar among wall types except for EPID that contained higher amounts of xylose. Releasable hydroxycinnamates were not as consistent among the cell wall types. Total ferulates, including ester linked and releasable ether linked, tended to increase from PITH to SCL (8 to 15 g kg⁻¹ CW) with an increase in the proportion etherified within the wall matrices (PITH 51%; SCL 66%). Total p‐coumarates showed opposite trends with PITH walls having significantly more (35 g kg⁻¹ CW) than VBZ or SCL (19 and 13 g kg⁻¹ CW). EPID walls contained the least pCA (6.5 g kg⁻¹ CW). Except for the hydroxycinnamates, compositional trends for the different wall types would reflect changes from primary walls to increased amounts of secondary wall. Neutral sugar analysis of indigestible residues indicated similar carbohydrate compositions among the cell wall types, with xylose being less degradable than all other wall sugars. © 1999 Society of Chemical Industry     

Practical cell labeling with magnetite cationic liposomes for cell manipulation

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10³–10⁵ cells for personalized cell processing, we determined that 10 μg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method.     

冷藏对蓝莓果实细胞壁组分及其降解酶活性的影响

以"蓝丰"蓝莓果实为试材,研究了冷藏对蓝莓果实细胞壁组分及其降解酶活性的影响。结果表明:蓝莓果实的软化与离子结合型果胶(ISP)、共价结合型果胶(CSP)含量和多聚半乳糖醛酸酶(PG)、纤维素酶(Cx)、β-半乳糖苷酶(β-Gal)活性关系密切,其中,ISP含量和PG活性与果实硬度呈显著负相关,Cx和β-Gal活性与果实硬度呈极显著负相关,CSP含量与果实硬度呈极显著正相关;冷藏期间,随果实硬度的逐渐下降,CSP含量逐渐降低,ISP含量变化幅度小,PG和β-Gal活性呈上升趋势,Cx活性呈现先下降后上升趋势,3种酶均在贮藏的后期活性急剧上升;在冷藏的30 d,果实的细胞壁组分和相关酶活性变化较小,果实硬度下降缓慢;与采后自然后熟的果实相比,冷藏30 d的蓝莓果实常温货架期间Cx活性和β-Gal活性一直处于较低水平,PG活性高峰延晚出现,Cx活性高峰极显著降低,果胶甲酯酶(PE)活性未出现高峰。可见,冷藏30 d的蓝莓果实细胞壁组分含量及相关酶活性的变化一定程度受到抑制,果实状态保持良好。     

单细胞蛋白的利用

概括了单细胞的定义、种类、来源、生产工艺及其在饲料和食品工业中的应用;阐明了单细胞蛋白开发和生产的前景。     

单细胞蛋白的利用

概括了单细胞的定义、种类、来源、生产工艺及其在饲料和食品工业中的应用；阐明了单细胞蛋白开发和生产的前景。     

Single cell reporter assay using cell surface displayed Vargula luciferase

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.     

Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

The effects of bovine cumulus cell-conditioned medium on cloned bovine embryonic development and subsequent chromosome complement were examined using an air-dry procedure. Conditioned media were prepared using CR1aa supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). Nuclear-transferred embryos were reconstructed with nuclei from cumulus cells. Similar cleavage, morula, and blastocyst development was observed in conditioned media groups compared with the co-culture group. No differences (P > 0.05) were observed in the composition of blastocyst chromosomes after co-culture in different media, either with or without starvation of donor cells. The overall diploid blastocyst rate ranged from 75% to 84%. Chromosomal complement of blastocysts, however, was very different between conditioned medium and co-culture treatments. Overall incidence of chromosomal anomalies was 40% in conditioned medium, which was significantly higher (P < 0.001) than the co-culture group (20%). Moreover, a higher incidence (P < 0.05) of chromosomally abnormal blastocysts (41.5%) was observed after culture with FBS-containing conditioned medium than those cultured in BSA-containing conditioned medium (31.4%). No diploid improvement was observed after exchange of the culture system from conditioned medium to co-culture, or from co-culture to conditioned medium after the first 72 h of culture. The results of this study also indicated that the overall cell number was much lower (P < 0.01) in blastocysts with chromosomal abnormalities than those with a normal diploid state. We have concluded that medium conditioned with bovine cumulus cells increases the incidence of chromosomal anomalies in nuclear reconstructed embryos.     

Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line

Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 μM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G₁/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.     

植物甾醇对人神经母细胞瘤SH-SY5Y细胞增殖和凋亡的影响

初步探讨植物甾醇对人神经母细胞瘤SH-SY5Y细胞增殖和凋亡的影响。方法 采用体外细胞培养的方法,用不同浓度的植物甾醇与细胞共同培养,倒置显微镜观察细胞形态,MTT法测定细胞存活率,流式细胞术测定细胞凋亡率。结果 与对照组比较,从1μmol/L植物甾醇起即对细胞的存活率有明显影响,差异有统计学意义(P<0.01)；植物甾醇浓度由低到高,随着浓度的增加,对细胞的形态影响越明显,突触变短,胞体萎缩甚至死亡；不同浓度的植物甾醇可引起细胞不同程度的早期凋亡。结论 植物甾醇对体外培养的人神经母细胞瘤SH-SY5Y细胞有潜在的毒性作用,能够抑制细胞增殖和引起细胞凋亡。     

Isolates of cell types from sorghum stems: Digestion,cell wall and anatomical characteristics

Cell types were separated from internode 5 of sorghum stems to study the interrelationship between digestion characteristics and cell wall composition. Isolates of epidermis (EPI), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) were freeze-dried and ground for analysis. The cell fractions were digested in rumen fluid for times between 0 and 96 h, and wall composition measured using detergent extraction procedures. In-vitro dry matter digestibility (g kg^?1 after 48 h) of cell fractions was in the order of PITH (849-906) > IVB (794-816) > SCL (692-701) > VBZ (641-679) > EPI (608-628). Total cell wall content (CWC), indigestible CWC, and lignin content followed the inverse order. Lignin concentration on a dry matter or cell wall basis was highly correlated with indigestible wall residue after 96 h. The proportion of cell wall digested after 96 h was higher for SCL and VBZ cells (61·8-68·2%) than for PITH cells (48·4-56·1 %), despite the former having lignin content three to five times higher than that of PITH cells. Clearly, there were differences between the cell types in wall composition or chemical linkages between wall components that lead to the observed differences in wall digestion.     

Relationship of cell wall composition to in vitro cell wall digestibility of maize inbred line stems

The phenolic equipment of maize stem tissues was investigated in relation to the feeding value of the detergent fibre components. Sixteen maize inbred lines, including three brown‐midrib 3 mutants and their normal counterparts, were selected for highly divergent in vitro cell wall digestibility. These lines were grown during two years. Maize stems were analysed for detergent fibre concentration, esterified and etherified p‐hydroxycinnamic acids, lignin content and structure and in vitro digestibility. A large genotypic variation was found for neutral detergent fibre, cell wall phenolic composition and cell wall digestibility. Within the normal maize lines the in vitro neutral detergent fibre digestibility (IVNDFD) of stem fractions was negatively correlated with their Klason lignin content. A multiple regression model based on esterified p‐coumaric acid and lignin composition as two explanatory variates accounted for 58% of the IVNDFD variation. In this study, three normal maize inbred lines displaying a lignin content and a cell wall digestibility level close to those observed in the three bm3 lines could be detected, which opens up new breeding avenues. © 2000 Society of Chemical Industry     

Phenylpropanoid metabolite supports cell aggregate formation in strawberry cell suspension culture

Plant cells in suspension culture tend to aggregate and form large clumps. In suspension culture, large cell aggregates are frequently subjected to hydrodynamic shear stress; however, a certain degree of cell aggregation is often required for cell growth and metabolite production. Thus, controlling cell-aggregate size is desired to establish high productivity of useful products using plant cell suspension culture. In this study, we focused on the relationship between cell-aggregate formation and secondary metabolism. We found that anthocyanin concentration showed a good correlation with cell-aggregate size in the cultured strawberry cell line FAR (Fragaria ananassa R), which produces anthocyanin and other phenylpropanoid metabolites constitutively without illumination. This result suggests that there is a relationship between cell-aggregate formation and the accumulation of phenylpropanoid metabolites. To investigate the direct effect of phenylpropanoid metabolism on cell-aggregate formation, the time course of cell-aggregate size was monitored when phenylpropanoid metabolism was suppressed by a metabolic inhibitor, L-alpha-aminooxy-beta-phenylpropionic acid (AOPP), a specific inhibitor of phenylalanine ammonia lyase which is the starting and key enzyme of the phenylpropanoid pathway. In the absence of AOPP, the average diameter of cell aggregates increased on day 8 of culture. This increase in cell-aggregate size was completely suppressed by the addition of 0.1 mM AOPP, without any reduction in cell growth rate or soluble protein content. These results indicate that cell-aggregate formation is directly supported by a secondary metabolite produced from the phenylpropanoid pathway, suggesting that cell-aggregate size can be controlled by AOPP without inhibition of primary metabolism.     

Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.     

Mechanical characteristics of artificial cell walls

Artificial plant cell walls were produced from bacterial cellulose and cell wall constituents. The artificial cell walls were stored at low, medium and high relative humidity, and then subjected to micro-mechanical tests. From chemical composition and microstructure analysis it was found that, among all artificial cell wall materials produced, the most representative analogue of natural apple cell wall was based on bacterial cellulose supplemented with xyloglucan and pectin. Uniaxial tensile tests revealed that the different cell wall materials differed in their mechanical properties; increasing the humidity during storage resulted in a decrease in the value of the secant modulus. The cell wall model material obtained may be used for the simulation of the effect of external factors on the physical and chemical properties of cell walls.     

3种肽对细胞氧化损伤的保护作用及促细胞增殖能力比较

目的 探究小麦肽、大豆肽和海参肽对H2O2诱导的L929细胞氧化应激损伤的保护作用及促细胞增殖能力比较。方法 首先通过测定L929细胞的增殖率方法来确定L929细胞的浓度及H2O2的浓度, 建立L929细胞H2O2损伤模型, 测定3种肽对L929细胞的氧化损伤保护能力, 再通过测定细胞周期及细胞凋亡率的方法来比较3种肽的促细胞增殖能力。结果 海参肽的氧化损伤保护能力优于大豆肽和小麦肽; 200 μg/mL大豆肽和海参肽处理后的成纤维细胞增殖指数相比于空白对照都有显著增加, 即大豆肽和海参肽可以通过影响细胞周期提高增殖指数而促成纤维细胞增殖, 且同浓度海参肽的促成纤维细胞增殖能力优于同浓度的大豆肽, 而小麦肽对细胞周期的影响无显著性差异。海参肽的抑制成纤维细胞凋亡作用最强, 经200 μg/mL海参肽作用后, 细胞的凋亡率由空白对照的的(3.59±0.11)%降至(1.89±0.09)%。结论 海参肽对细胞氧化损伤保护及促细胞增殖能力最强, 大豆肽次之。