A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells

Summary A “sequential culture step” system was devised to study osteoclast differentiation from newborn porcine bone marrow cells. Nonadherent cells were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in low-serum medium supplemented with L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adherent cells mainly composed of M-CSF-dependent macrophage/osteoclast progenitors, but devoid of stromal-like cells, were further cultured in medium supplemented with L9-CM and CM derived from serum-free cultures of fetal rat calvarial bones. This phase was characterized by a rapid induction of mono- and multinucleated (pre)osteoclast-like cells, positive for cytochemical TRAP activity, but negative for nonspecific esterase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] stimulated osteoclast generation, whereas calcitonin treatment significantly inhibited this process. The osteoclastic nature of the cells was confirmed by the occurrence of extensive, characteristic bone resorption on dentin slices, which was associated with release of type I collagen N-telopeptides from the bone matrix into the culture medium. The presence of a DNA synthesis inhibitor (HU) during the first 3 d of culture completely inhibited osteoclast formation, whereas HU treatment during the last phase did not affect production of multinucleated osteoclast-like cells. Likewise, a specific antibody directed against M-CSF during the first preculture period, completely abolished osteoclast formation. Adding the antibody during the last phase of the culture, however, strongly inhibited multinucleated osteoclast formation, accompanied by a significant increase in a mononuclear TRAP-positive, NSE-positive (osteoclast precursor) cell fraction. These results indicate that M-CSF is essential for progenitor proliferation as well as for (pre)osteoclast maturation and/or fusion into multinucleated cells, but also suggest that additional soluble (bone-derived) factors are involved as cofactors in the differentiation process to committed mononuclear osteoclast precursors. The porcine marrow culture approach provides a suitable model system to investigate specific soluble osteoclast-inducing factors affecting different stages of osteoclast development.     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

Dominant expression of porcine Calbindin-D9k in the uterus during a luteal phase

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue.     

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)‐1[[(2 S)‐1‐(1‐oxo‐4‐phenylbutyl)‐2‐pyrrolidinyl carbonyl]‐2‐pyrrolidinecarbonitrile (KYP‐2047). We found that KYP‐2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side‐chain cleavage enzyme (CYP11A), 3β‐hydroxysteroid dehydrogenase (3β‐HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP‐2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP‐regulated progesterone synthesis, U0126‐EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126‐EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126‐EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3β‐HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.     

Regeneration of fertile wheat (Triticum aestivum) plants from isolated zygotes using wheat microspore culture as nurse cells

A new method for growing isolated wheat zygotes by co-cultivation with microspores isolated from the same species has been developed. Although the mortality of isolated zygotes within 6 h after the transfer into the nurse culture was relatively high (70%) in this method, the majority of the structures which survived developed into fertile plants (61%). Zygotic structures grown in these cultures were morphologically not as regular as embryos grown in planta, but they resembled them closely in cytological properties and developmental pattern.     

Differentiation of cotton fibers from single cells in suspension culture

Summary A cotton cell suspension culture has been developed that provides unique opportunities for plant biologists to investigate early developmental events regulating cotton fiber properties, plant cell elongation, and cell wall biogenesis. The suspension culture was derived from cells of cotton (Gossypium hirsutum L.) ovule callus. These cells undergo the stages of fiber development previously described for in vivo fiber development. Fibers range in length up to 11 mm and have secondary walls. Supported by the U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Laboratory, New Orleans, Louisiana, and Cotton Incorporated, Raleigh, North Carolina.     

细胞渗透性神经酰胺可抑制大鼠离体黄体细胞甾体激素生成并诱导细胞凋亡

本文旨在观察神经酰胺对离体孵育的大鼠黄体细胞孕酮分泌及细胞凋亡的影响,以PMSG－hCG处理的雌性Wistar大鼠为模型,分离制备黄体细胞,将外源性细胞渗透性神经酰胺与黄体细胞共同孵育,分别用放免法和流式细胞仪分析神经酰胺对黄体细胞孕酮生成和凋亡的影响,同时还检测了一氧化氮合酶（NOS）活性和一氧化氮（NO）水平的变化,结果显示,神经酰胺可以剂量相关方式抑制hCG－诱导的孕酮分泌,而对基础孕酮没有显著影响,离体孵育12h的大鼠黄体细胞存在自发性凋亡,5umol/L神经酰胺能显著增加亡率（P＜0．05）,流式细胞仪分析可见增强的凋亡蜂,实验还发现,50umol/L神经酰胺能明显促进NOS活性（P＜0．01）和NO生成（P＜0．01）,结果提示,神经酰胺可能通过调节甾体激素生成和细胞凋亡而作为一种重要的信息分子参与黄体退化等卵巢的生理过程。     

用于药物评价的体外心脏组织模型研究进展

候选药物对心脏的毒副作用是其在开发过程中被淘汰的重要原因之一.传统药物评价所采用的动物模型存在种属差异、成本高、效率低等缺陷.因此,近年来随着干细胞和生物打印等技术的快速发展,体外心脏组织模型的构建受到了越来越多地关注.本文追踪体外心脏模型构建的起源与发展,综述模型所利用的心肌细胞来源以及二维、三维模型构建的相关技术与方法,着重阐述心肌组织模型血管化的重要性及研究进展,并对该领域未来的发展方向进行展望,以期为体外心肌组织模型在药物评价方面的研究和应用提供新思路.     

A new three-dimensional culture of human keratinocytes: optimization of differentiation

Many attempts have been made to obtain reconstructed human epidermis comprised of keratinocytes and extracellular-matrix constituents (essentially collagen) in the presence or absence of fibroblasts. A simple model of cultured human keratinocytes grown at the air-liquid interface of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immunohistochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also intregrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes.In our culture system, the expression of the different integrin subunits (2, 3, 5, 6, 1) was studied as a function of the differentiation state in two different media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal calf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the 2 and 3 subunits in the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significant technological advance in culturing keratinocytes and is an easy-to-handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - EGF epidermal growth factor - FCS fetal calf serum - K-SFM keratinocyte serum free medium - MAb monoclonal antibody - NHEK normal human epidermal keratinocytes - PBS Dulbecco's phosphate-buffered saline     

Maize meiocytes in culture

A method for culturing isolated maize meiocytes has been developed to study dynamic meiotic processes. This culture method was used to monitor spindle formation and function in vitro using polarization, epifluorescence, and differential-interference contrast optics. It was found that before nuclear envelope breakdown, there are no obvious bipolar spindles; instead, they form during prometaphase, as chromosomes move to the metaphase plate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of isolated digestive-gland cells in the study of biochemical and physiological processes in gastropod molluscs

We describe a method for preparation and maintenance of isolated digestive-gland cells in the abalone, Haliotis kamtschatkana. Viability of the isolated cells was confirmed by the fact that 18 h after preparation the cells exhibited less than 5% staining with trypan blue and actively synthesized glycogen following the addition of glucose substrate. Use of the method in a 15-month study of metabolic activity of the digestive gland of H. kamtschatkana showed significant differences in oxygen consumption of isolated-cell preparations correlated with seasonal differences in somatic and gametogenetic growth, and with relative size of the digestive gland.     

Somatic embryogenesis in callus culture of Mussaenda erythrophylla cvs. Queen Sirikit and Rosea

Somatic embryogenesis was achieved from mid-rib and internodal calluses of Mussaenda erythrophylla L. cvs. Queen Sirikit and Rosea cultured on Murashige and Skoog basal medium containing 8.9 M BA+0.57 M IAA+10 mg l^-1 ascorbic acid. Clumps of somatic embryos were separated and grown into complete plantlets when transferred to 1/2 MS medium+37 M adenine sulphate with 2% (w/v) sucrose.     

Impact of culture vessel ventilation on the anatomy and morphology of micropropagated carnation

Dianthus caryophyllus cv. Nelken was cultured in vitro under different ventilation rates (0.11, 0.21, 0.68 and 0.86 changes h⁻¹). Ventilation modified the anatomical characteristics of shoots and leaves described for plants grown in non-ventilated vessels: the cuticle became thicker, there was a decreased cell size and intracellular space size. Also, there were more photosynthetic and supportive tissues, including thicker cell walls. Increased ventilation promoted in vitro hardening of micropropagated carnation shoots, and pushed the culture-induced phenotype closer to that of ex vitro acclimatized plants. Anatomical variability of in vitro-grown plants was demonstrated to be a consequence of ventilation. This revised version was published online in August 2006 with corrections to the Cover Date.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Morphological events in cultures of mechanically isolated maize mcirospores

Summary In tis androgenic response, maize is considered to be a recalcitrant plant. We used mechanically isolated microspores of maize genotype A18 to establish a responsive microspore culture of maize. Morphological events occurring during the first days of maize androgenesis in a microspore culture were observed and described, and some morphological markers for distinguishing between embryogenic microspores and nonembryogenic microspores were identified. It was found that the enlargement of microspores during the first days in culture and the ‘star-like’ organization of the cytoplasm inside the microspore are connected with reprogramming of the developmental pathway in maize microspores. Some differences were also found in the surface wall architecture of embryogenic microspores. Fertile plants were successfully recovered from microspore-originated structures.     

Photography as a tool of research and documentation in plant tissue culture

Summary Scientific photography is an important facet of plant tissue culture. The aim of photography in plant tissue culture should be to illustrate clearly the developmental stages occurring in vitro. However, the photographic results presented in publications are often poor, and morphogenetic responses are often not clearly documented. Plant tissue culture is a very visual science, and the valuable tool of photography is often not used properly. If the morphogenetic responses are not well documented, an important part of the reserch is missed, and the report ends up having limited scientific value. Simple methods for improving the results of photography in plant tissue culture are discussed, along with photographic equipment, photomacrography, stereophotomicrography, suitable backgrounds for photography, use of a digital scanner, and the construction of photographic plates.     

Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

Summary A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65–75%) and goblet cells (25–35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5±0.5×10⁴ cells) were incubated with toxin dilutions (0.01–20 μg) for 1 h at 24° C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5–5 μg). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5–10 μg. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.     

Ultrastructural observations on human cerebral capillaries in organ culture

The use of an organotypic-in the strictly literal meaning of the word, nervous tissue culture device has allowed the identification and ultrastructural study of various types of developing capillaries in human cerebellum and olfactory bulb in vitro. Most capillaries were similar to those already described by other authors or by us, in human or animal embryos and fetuses. Large Type I Capillaries. Their luminal diameters were greater than 8 microns. The basement membranes were thin and discontinous. Numerous interendothelial junctions were either plate-like attachments or contained pentalaminar zones. Type II Capillaries. Their lumina were between 2 and 8 microns in diameter. The basement membranes were wider than those of type I capillaries and were sometimes continuous. The interendothelial junctional complexes of type II capillaries included pentalaminar portions. Many simple or complex vascular sprouts (type IV and V capillaries) had small or non-patent lumina. Their basement membranes were absent or very thin and discontinuous. Their interendothelial junctions were similar to those of type I capillaries. Some of the less frequently encountered capillary types seen in developing human nervous tissue were absent in culture. Some pathological features were seen-especially in long-term cultures-in type I and II capillaries containing degenerating blood cells or processes sometimes obviously related to histiocytic cells. They consisted mainly of an accumulation of microfilaments and modifications of the rough endoplasmic reticulum in the endothelial cells. These pathological changes did not modify the main characteristics of the capillaries. The origin of the vascular sprouts, the exact nature of the interendothelial junctions and the significance of the pathological changes are discussed. This model may prove useful for the study of cerebral vasculogenesis, the development of the blood-brain barrier and the physiological or pathological properties of the human brain capillaries in tissue culture.     

Tumorigenesis of rat mammary epithelial cells by N-nitroso-N-methylurea in anin vitro system: Characterization of the microtumors

Summary Chemical carcinogenesis is a lengthy process that involves the rather loosely defined stages of initiation, promotion, and progression. Several model systems of mammary carcinogenesis have been designed to elucidate the mechanisms of chemical carcinogenesis. Most of these systems have included animal models. While organ specific chemical carcinogenesis can be initiated in these systems, the subsequent stages of promotion and progression are difficult to study in detail. Investigations onin vitro carcinogenesis have shown transformation of mammalian cells in culture; the transformational event, however, is difficult to discern within the monolayer culture. We have recently reported the development of anin vitro carcinogenesis system that allows both the initiation as well as the progression of mammary cells in a collagen gel matrix culture system. The cells transformed by a chemical carcinogen develop into discernible microtumors with the three dimensions of a collagen gel culture. Isolation of these microtumors from the collagen gel an subsequent culture in monolayer has produced cells capable of colony formation in soft agar. The present study further characterizes these microtumors originatedin vitro by analysis of cell growth kinetics versus parallel control cells. In addition, flow cytometric and cytogenetic studies have been performed to investigate the chromosomal stability of these cells. It was also observed that the microtumors, producedin vitro from mammary epithelial cells of an inbred strain of rats, show the ability to form tumors upon transplantation into the fat pad of syngeneic hosts.