恒河猴皮肤干细胞的体外培养纯化与鉴定

探索恒河猴皮肤干细胞的体外培养及纯化条件,为进一步的研究奠定基础. 通过组织块培养法和消化培养法 在体外培养恒河猴表皮细胞,然后用Ⅳ型胶原吸附法吸附20 min,获得快吸附细胞. 对快吸附细胞进行克隆培养,并进行免疫细胞化学双标染色、RT PCR鉴定 β1 整合素和角蛋白15的表达,用流式细胞仪鉴定纯化前后的细胞中 β1 整合素和角蛋白15的阳性细胞比例,并通过透射电镜观察细胞的超微结构. 组织块培养法和消化培养法均可获得表皮细胞,Ⅳ型胶原纯化后的细胞胞体较小,饱满,核/浆比例大,细胞镶嵌状排列. 细胞克隆分析显示,细胞全克隆生长率高. 细胞免疫荧光显示,分选后的细胞显示 β1 整合素和角蛋白15阳性. RT PCR检查呈现 β1 整合素和角蛋白15的特异性片段. 流式细胞仪检查显示,纯化前的细胞中角蛋白15阳性细胞占总细胞中的比例为8％, β1 整合素阳性细胞的比例为10.7％;纯化后,角蛋白15阳性细胞的比例为89.4％, β1 整合素阳性细胞的比例为88.5％. 通过组织块培养法和消化培养法均可培养获得活性良好的表皮细胞,Ⅳ型胶原吸附法是一种简便、有效的皮肤干细胞分离方法,可以为进一步的眼表上皮替代重建眼表提供足量的高纯度的干细胞建立可靠的物质基础.     

Biochemical, Immunochemical and Immunohistochemical Identification of Myosin Heavy Chains in Cultured Cells of Catharanthus roseus

In the pollen tubes of the lily Lilium longiflorum, myosin,composed of 170-kDa heavy chains is responsible for the intracellulartransport of organelles [Yokota and Shimmen (1994) Protoplasma177: 153]. Polypeptides of 170 kDa with similar antigenicityto this pollen-tube myosin have also been found in other angiospermcells [Yokota et al. (1995) Protoplasma 185: 178]. To clarifythe role of this type of myosin in cytoplasmic streaming, weprepared partially purified myosin fraction from cultured cellsof Catharanthus roseus by co-precipitation with F-actin. Ina motility assay in vitro with this fraction, rhodamine-phalloidin-labeledF-actin moved with an average velocity of 10.7 µm s^-1.This sliding velocity was similar to that of the cytoplasmicstreaming observed in intact cultured cells. Antibodies raisedagainst the 170-kDa heavy chain of pollen-tube myosin recognizedonly a single polypeptide of 170 kDa in this partially purifiedfraction. The same polypeptide was also identified by theseantibodies in a crude extract of proteins from cultured cells.The myosin-specific fluorescence was concentrated around thenuclei and was associated with particles of various sizes. Duallocalization using antibodies against myosin and against actinrevealed that these particles were preferentially co-localizedwith actin filaments. On the other hand, no component of thecrude extract or of the partially purified myosin fraction cross-reactedwith antibodies against heavy chains of myosin II from animalcells. These results suggest that the 170-kDa polypeptide is the myosinheavy chain and that this myosin generates the motive forcefor cytoplasmic streaming in cultured cells of Catharanthusroseus. (Received March 28, 1995; Accepted September 14, 1995)     

Histology of Shoot Bud Ontogeny from Seedling Root Segments of Brassica napus L.

Root explants of Brassica napus cultured in vitro form adventitiousshoots. The root buds originated at the base of the newly initiatedlateral root. Cells in association with the differentiatingphloem of the developing lateral roots were the sites for rootbud formation. A nodular mass of cytoplasmic cells developedby day 7 at the base of the lateral root. This group of cellscontinued to divide an enlarge. The cells in the peripheralregion of the nodular cell mass differentiated further intoa meristematic zone. The meristematic cells grew towards theperiphery of the cortex by crushing the outer layer of corticalcells. Further development of the meristematic layer resultedin the formation of shoot primordia with organized shoot apicalmeristems and leaf primordia.Copyright 1993, 1999 Academic Press Brassica napus, canola, cultured root segments, root buds     

Embryos and Plantlets from Cultured Anthers of Hybrid Grapevines

Embryos and plantlets were produced in large numbers from callusformed by cultured anthers of hybrid grapevines (Vitis viniferax Vitis rupestris). Anthers of Vitis vinifera produced smallamounts of callus or failed to grow in vitro. For embryo formationanthers containing uninucleate microspores were chilled (4 °C)for 72 h before culture with Nitsch medium containing 2, 4-D(5µM) and benzyladenine (1 µM). Highest yields ofembryos were with anthers cultured in darkness. For productionof normal plantlets embryos required chilling (4 °C) for2 weeks. Unchilled embryos produced mainly abnormal plantlets.Chilling was effective in promoting plantlet growth when appliedat any stage of embryogeny. In grapes ability to produce plantlets from cultured anthersis a genetically-determined trait and maleness, as distinctfrom hermaphroditism, may be a predisposing factor. Callus derivedfrom anthers contained both haploid and diploid cells but allplantlets produced so far are diploid. The genetic constitutionof plantlets, whether they are diploids of somatic origin ordiploids from spontaneously doubled haploid cells, is not yetknown and is being determined by standard genetic methods.     

Cellular Growth in Roots of a Gibberellin-Deficient Mutant of Tomato (Lycopersicon esculentum Mill.) and its Wild-type

The role of gibberellins in regulating the growth of tomatoroots was investigated by comparing various cellular parametersin cultured roots of the gibberellin-deficient mutant gib-l/gib-lwith those in roots of the near-isogenic wild-type. In addition,wild-type roots treated with 0?1 µM 2S,3S paclobutrazol,an inhibitor of gibberellin biosynthesis, and mutant roots treatedwith 0?1 µM GA₃ were also compared: the former roots constitutea phenocopy of the mutant, whereas the latter roots appear tobe ‘normalized’ and similar to wild-type. The elongationof mutant and phenocopied roots were similar, their maximumelongation rates being about half or two-thirds that of wild-typeor GA₃-treated mutant roots, respectively. These rates wereinterpreted in terms of the numbers and lengths of cells withinthe meristematic and non-meristematic portions of the elongationzone. Mean meristem length tended to be shorter in both themutant and the 2S,3S paclobutrazol-treated wild-type roots thanin the other two types of root. A major difference between thetwo pairs of mutant and normal roots was their mean final celllengths: mean lengths of cortical cells of the mutant and 2S,3Spaclobutrazol-treated roots were, respectively, 39% and 25%shorter than the mean length of wild-type roots. Final celllength in the GA³-treated mutant roots were similar to wild-type.By contrast, the diameters of mature cortical cells of the mutantand phenocopy were about 20% greater than the diameters of equivalentwild-type or ‘normalized’ mutant cells. The meanvolumes of cortical cells in all four types of roots showedno significant differences. Knowledge of the distribution ofcortical cell lengths, widths and volumes along the root axis,together with information about the rate of root elongation,permitted comparisons of the relative elemental growth ratesof each of these three cellular parameters. The available evidence suggests that the level of endogenousgibberellins in mutant roots is lower than in wild-type roots.The present results, therefore, suggest that endogenous gibberellinsare necessary for normal growth of cultured tomato roots andthat they regulate the relative amounts of growth at the longitudinaland transverse walls of the cells which, in turn, affects theshape of the elongating cells. Key words: Cell growth, cultured roots, gibberellin, gib-l mutant, Lycopersicon esculentum, 2S,3S paclobutrazol, relative elemental growth rate, root meristem     

Regulation of Tubulin Degradation in Isolated Zinnia Mesophyll Cells in Culture

Tubulin degradation in isolated Zinnia mesophyll cells in culturewas investigated by pulse-chase labeling with [³⁵S]-methionineand two-dimensional electrophoresis. Tubulin degradation changesdynamically during culture. Almost no tubulin degradation occursin the cells on the first day in culture. Treatment of thesecells with colchicine activates the degradation of tubulin,but not of proteins other than tubulin. In the presence of colchicine,the and ß-subunits of tubulin are degraded togetherand the half life of each subunit is approximately 6 h. After2 d in culture, there is active degradation of tubulin evenin the absence of colchicine. Colchicine did not inhibit new synthesis of tubulin in Zinniacells. This is very different from the results reported in culturedmammalian cells, whereby unpolymerized tubulin elevated by colchicine-treatmentdepresses its own synthesis. These and previous results dealing with changes in the leveland synthesis of tubulin in cultured Zinnia cells (Fukuda 1987),are discussed in relation to the regulation of tubulin metabolismin cultured Zinnia cells. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Aoba-yama, Sendai, 980 Japan. (Received September 5, 1988; Accepted December 20, 1988)     

Development of Photosynthetic Structure and Function during Light-Induced Greening in Photomixotrophic Cells of Arachis hypogaea L.

The synthesis of chlorophyll and development of photochemicalactivities were complete within 70–80 h in greening leaves,whereas these processes continued for 8 days with an initiallag of 8 h for pigment synthesis in greening Arachis hypogaeaL cells. The activity of photosystem I in cultured Arachis cellswas detected earlier (24–36 h after illumination) thanthat of photosystem II (42–54 h after illumination) andthe development of the latter coincided with the synthesis ofa 46,000 dalton polypeptide of the thylakoid membranes. Experimentalstudies with cultured cells have the advantage in that the temporalsequence of the assembly of membrane components and associatedfunctions are determined easily because of longer developmentalperiod of chloroplast. (Received January 29, 1982; Accepted April 13, 1983)     

Cultivation of Excised Anthers in vitro--Effect of Nucleic Acids

Anthers of Allium cepa and Rhoeo discolor, excised at leptotene-zygoteneand diplotene-diakinesis, have been cultured in modified White'smedium supplemented with different concentrations of ribonucleicacid and desoxyribonucleic acid. The best development was obtainedwith ribonucleric acid in which there was a 100 per cent. survivalof the microspore mother cells up to the formation of 1-celledmicrospores. In R. discolor, the time taken by the microsporemother cells to form tetrads in vitri was half of that requiredin nature. All attempts to induce division of the microsporenucleus in vitro failed.     

Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells

The levels of ß1,2-N-acetylglucosaminyltransferase(GlcNAc-T) I and II activities in cultured cells from Bombyxmori(Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda(Sf-9 and Sf-21) were investigated. Apart from initial experimentswith Man     

Phosphorylation of a Protein (pp56) is Related to the Regeneration of Rice Cultured Suspension Cells

Short-term cultured suspension cells of rice (Oryza sativa L.)are capable of regeneration, but not in long-term culture. Forclarification of the mechanism of regeneration, protein phosphorylationin short-term and long-term cultured suspension cells was comparedby two dimensional- polyacrylamide gel electrophoresis. A 56kDa protein having an isoelectric point of 4.5 was phosphorylatedin vitro in short-term cultured suspension cells, but was notphosphorylated after regeneration. This protein in longtermcultured suspension cells remained phosphorylated after transferto the regeneration medium. However, using an antibody raisedagainst this protein from short-term cultured suspension cells,it was always detected in long-term and short-term culturedsuspension cells after transfer to the regeneration medium.The partial amino acid sequence of the HPLC-purified proteinshowed homology to a calcium-binding protein from maize. Thephosphorylation of the 56 kDa protein (pp56) appears to be associatedwith the regeneration of cultured rice cells. (Received December 11, 1995; Accepted June 3, 1996)     

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

The response ofH⁺-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa⁺-independentH⁺ extrusion was ATP dependent and K⁺ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa⁺-independentH⁺ extrusion in cultured medullarycells is mediated via H⁺-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H⁺-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H⁺-ATPase activity in innermedullary collecting duct cells. Upregulation ofH⁺-ATPase could play a protectiverole against cell death in severe intracellular acidosis.

     

The photoautotrophic culture of chlorophyllous cells

Photosynthesis in chlorophyllous cells in heterotrophic cultureswas investigated. Chlorophyllous cells from the amur cork-tree,scotch broom and tobacco, all of which had relatively high chlorophyllcontents (70 to 120 µg/g fresh weight) were selected throughoutcallus induction and cell subculture. When cultured under variouslight intensities, growth was stimulated by increases in lightintensity. This stimulation depended on the chlorophyll contentsof the cells. It disappeared on the addition of photosynthesisinhibitors (DCMU or 2-chloro-4,6-bis(ethylamino)-s-triazine).These phenomena indicate that photosynthesis accounted for athird to a half of cell growth under strong illumination. These heterotrophic cultures were then developed as autotrophiccultures. When these chlorophyllous cells were cultured withaeration using CO₂-enriched air in the light condition, thescotch broom and tobacco chlorophyllous cells grew photoautotrophically.Nearly the same amount of growth as with 3% sucrose in the darkwas observed in an autotrophic culture with aeration using aircontaining 1% CO₂. The green tobacco cells have been subculturedautotrophically for about one year. (Received November 28, 1977; )     

Developmental Pattern of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Caulogenesis and rhizogenesis were studied in cultured leafexplants of Nicotiana tabacum cv. Xanthi nc. using both lightand scanning electron microscopy. The timing of organ appearancewas also recorded. The patterns of development seen were comparedto each other and to that in explants grown on growth regulator-freemedium. Shoots first appeared after 12 d in culture and rootsafter 7 d. In caulogenesis nodules appear at the explant edgeand from these the shoots arise. The nodules are mainly derivedfrom palisade mesophyll cells, along with some spongy mesophylland bundle-sheath cells. The nodules form a continuous row alongthe edge of the explant and their initiation appears to be centredon veins. Shoots are produced indirectly. Roots are produceddirectly from bundle-sheath and vein parenchyma cells. Withoutplant growth regulators bundle-sheath cells still divide, althoughonly a few divisions were seen. Key words: Nicotiana tabacum, in vitro, caulogenesis, rhizogenesis     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )     

In Vivo and in Vitro Conversion of Teasterone to Typhasterol in Cultured Cells of Marchantia polymorpha

Identification by full-scan GC-MS revealed that [²H₆]-teasteronefed to suspension cultured cells of Marchantia polymorpha wasconverted to [²H₆]3-dehydroteasterone and [²H₆]typhasterol.This indicates that the cells carry out a C3-epimerization inwhich teasterone is converted to typhasterol via 3-dehydroteasterone.In vitro enzymatic conversions of teasterone to typhasterolwere also investigated. A crude cytosolic solution preparedfrom Marchantia cells catalyzed not only the dehydrogenationof teasterone to 3-dehydroteasterone but also the reductionof 3-dehydroteasterone to typhasterol. The major 4-demethysterolin cultured M. polymorpha cells was 24-methylcholesterol, theprecursor of brassinosteroids. These results suggest that enzymessimilar to those involved in the early C-6 oxidation pathwayof the brassinosteroid biosynthesis are present in the liverwort. (Received March 19, 1999; Accepted June 28, 1999)     

Mineral nutrition, of cultured chlorophyllous cells of tobacco VII. Effects of the NH4/NO3 ratio and of Cl in the media on the growth and ionic balance of cells

NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (C—A) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO₃/liter as the sole N source. (C—A) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH₄ 10+NO₃ 42 in meq/liter,had lower growth rates and lower (C—A) values as comparedwith NG on media containing NO₃ as the sole N source. NG, cultured on media containing NH₄ 30+NO₃ 42 in meq/liter,had high growth rates and (A—C) values of 22 to 53 meq/100gof dry cells. In this case, the (A—C) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|C—A| contents [|C—A| being the absolute valuesof (C—A)]. (Received May 15, 1980; )     

Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

A series of myo-inositol phosphates including myo-inositol mono-to hexa-phosphates was observed during growth of cultured riceplant cells. We also found that ³²Pi and myo-[2-³H] inositolwere incorporated into all these myo-inositol phosphates. myo-Inositolphosphorylating activity, which depended on ATP and Mg²⁺, wasdetected in the soluble fraction from the cells, and the reactionproduct was identified as myo-inositol-2-phosphate. (Received January 21, 1980; )     

Cytological Analysis of Seedling Roots, Transformed Root Cultures and Roots Regenerated from Callus of Swainsona galegifolia (Andr.) R. Br.

The stability of chromosome number was investigated in culturesof roots from Swainsona galegifolia. Roots from germinated seedsor plants grown in vitro when cultured in liquid medium howed90% or more cells with the diploid number of 2n = 32. The remainingcells showed aneuploidy mostly below 32. The stability of chromosomenumbers was not affected by transformation with Agrobacteriumrhizogenes although when roots were transformed with A. rhizogenesLB 9042 the range of chromosome numbers in the few aneuploidcells present was higher than in roots for which strain A4 wasused. In contrast, roots regenerated from callus had only 15%of cells with 2n = 32 and showed a modal number of 18. Six rootcultures established from individual roots regenerated fromcallus showed a wide variation in number (8–83). Fivecultures had a modal number around 18, the sixth, a modal numberof 39 which is above the diploid number. The implication ofthe results for the production of secondary metabolites fromroot culture is discussed. Key words: Agrobacterium rhizogenes, callus cultures, chromosome number, root cultures, Swainsona galegifolia     

Ultrastructural Observations on Proliferating Storage Cells of Mature Cotyledons of Phaseolus vulgaris L. Cultured in vitro

Explants of mature cotyledons of Phaseolus vulgaris form callusrapidly when cultured in vitro with their adaxial surfaces embeddedin a solidified nutrient medium containing coconut milk, kinetinand 2,4-D. Proliferation is confined to the highly polyploidstorage cells and commences near the adaxial epidermis, whichis soon ruptured by the callus developing internally. Callusformation progresses to the abaxial tissue and within 3–4weeks sub-culturing is possible. The in vitro grown storage cells undergo thinning of their walls,loss of food reserves, hypertrophy, development of various new-wallsand nuclear activation leading to division. The induction ofnuclear and cell divisions within this mature storage tissuecontrasts with normal germination in which these cells undergorapid senescence after depletion of their food reserves. Nuclear division in early callus growth is apparently mainlyamitotic. It is preceded by the development of multiple nucleoli.The nuclear envelope also becomes more complex and deeply lobed;leading to formation of a nuclear isthmus and final separationinto two nuclei. No chromosomes are visible during nuclear fragmentation.Amitosis is accompanied by freely-forming walls, which may developadjacent to a nuclear isthmus and perhaps participate in nuclearfragmentation. Large labyrinthine wall bodies frequently occuron these walls. Mitoses are only observed in already dividedstorage cells. A cell plate forms between the two daughter nuclei,and microtubules are present at its margins in contrast to freely-formingwalls where none are evident. Phaseolus vulgaris L., bean, in-vitro culture, cotyledon, ultrastructure     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)