Epithelial cell biology最新文献

Various experiments indicate that normal growth and differentiation of murine epithelia in vivo are dependent on dermal influences, and the growth-enhancing effects of various connective tissue elements have also been demonstrated in vitro. Such effects were examined by the in vitro growth of murine epidermis on various substrata (collagen, lens capsule) at the air/medium interface with various connective tissue elements or cells apposed to the undersurface of the supporting matrix. Patterns of epithelial growth, morphology and differentiation were examined by histology, electron microscopy, gel electrophoresis and antibody staining. Growth and differentiation varied with differing substrata but good patterns of growth and morphology, and appropriate expression of cytoplasmic and cell surface differentiation markers, were found only in specimens grown in association with dermal elements. It is concluded that diffusible factors of dermal origin facilitate epithelial growth and differentiation.

In order to establish a set of epidermal species markers, normal human skin, murine skin and human skin transplanted to nude mice were stained with monoclonal antibodies directed to cell membrane-bound carbohydrates, a basement membrane component and a structure in the cell nucleus. Three epidermal species markers were identified. Two markers stained exclusively human epidermis: LH7.2 detects type VII collagen and stained the basement membrane of human epidermis in unfixed frozen sections, while LP4N stained cell nuclei in human epidermis in methanol-acetone-fixed frozen sections. The third marker, HH14, stained exclusively murine epidermis. HH14 defines the histo-blood group H carbohydrate antigen and stained spinous cell membranes of murine epidermis in both frozen and formalin-fixed sections.

Keratins form the intermediate filaments in almost all epithelial cells in vertebrates. The keratins of the rabbit corneal epithelium have previously been shown to be composed of a major keratin pair, formed by an acidic keratin K12 and a basic keratin K3, together with a minor keratin pair (acidic K14 and basic K5). Using electrophoretic and immunological analyses with two monoclonal antibodies, AK12 and AE5, that are monospecific for K12 and K3 respectively, we showed that the acidic keratin K12 is highly conserved throughout all the species studied except the trout, whereas the basic keratin K3 shows interspecies variations and was unequivocally identified only in mammals. Furthermore, in dog and human cornea K3 is present in small amounts compared with K12, while in mouse it is undetectable, suggesting that, in those species, the acidic keratin K12 may interact with the basic keratin K5 to form the corneal intermediate filaments.

The patterns of keratin expression of taste buds in murine oral mucosa were examined using a panel of antibodies with various specificities for cytokeratins. The patterns for taste buds differed markedly from those of the surrounding epithelium but no regional differences in the staining patterns of the taste buds themselves were detected. The taste buds and the ducts of von Ebner's glands were strongly stained by monoclonal antibodies (mAbs) against cytokeratins (K) 8, 18 and 19, those typically expressed by simple epithelia. Merkel cells, which are also present in the oral epithelium and may correspond to the type III cells in taste buds, stained for K8 and K18 but not K19. None of the mAbs against simple epithelial keratins stained the stratifying epithelium of the trench wall or of the oral mucosa. Antibodies with specificity for keratins that are expressed as differentiation products of the mucosal epithelium did not stain taste buds. The staining pattern of the epithelium of the trench wall indicated that it expressed some keratins typical of stratifying epithelia but lacked the full pattern of differentiation of the adjacent mucosal epithelia. Staining within the taste buds was not homogeneous but no clear differences of keratin staining could be directly related to the subtypes of cells constituting them. The markedly differing patterns of keratin expression between taste buds and the adjacent epithelium raises questions about the type of inductive signals that produce and maintain these patterns.

A culture system is presented in which a biologically more relevant substrate in the form of a collagen membrane and a biologically more relevant diffusion gradient system for nutrient delivery to the cells are provided. Five established human colon cancer cell lines (Caco-2, DLD-1, Widr, HCT 116 and HCT 8) were cultured in this system and three of them showed increased differentiation compared with the same cells cultured on the usual cell culture substrates of plastic and glass and with cells grown in an anchorage-independent manner. Caco-2 cells grow on plastic as a monolayer of large pleomorphic cells with scant mucin production. When cultured in a gradient diffusion system in a sandwich of type I/IV collagen the Caco-2 cells showed the highest degree of morphological and biochemical differentiation as evidenced by cellular alignment, glandular formation and mucin production. Similarly DLD-1 cells exhibited the greatest degree of morphological and biochemical differentiation in a gradient system in a type I/IV collagen sandwich. HCT 116 and HCT 8 cells, however, showed little change in differentiation phenotype under any of the 11 culture conditions tested. Widr cells grew in a type I/IV collagen sandwich in a gradient diffusion system as multilayered sheets of cells with intercellular spaces, suggestive of a moderate increase in differentiation phenotype. As judged by morphology and mucin production a range of differentiation capacities is exhibited by the five cell lines tested under the optimal differentiating culture conditions, with the order from most able to differentiate to least able to differentiate being Caco-2 > or = DLD-1 > or = Widr > HCT 116 > or = HCT 8.

The morphogenesis and functional differentiation of mammary epithelium depends on signalling from systemic hormones and on cues from the local tissue microenvironment. With regard to the latter, regulatory cues are mainly provided by two subcompartments of the mesenchyme/stroma [fibroblastic and adipocyte] and the subjacent basement membrane. During embryogenesis, fibroblastic mesenchyme determines the sexual phenotype of the gland while adipocyte mesenchyme controls mammary-specific ductal morphogenesis. In the juvenile animal, adipocyte stroma continues to support ductal expansion while fibroblasts negatively regulate ductal outgrowth via interactions with the epithelium possibly involving TGF-beta mediated deposition of collagen I and chondroitin sulphate. In the adult, evidence from culture studies show that the signals required for the induction of tissue-specific differentiation during pregnancy and maintenance of function during lactation arise primarily from basement membrane. Beta-casein synthesis is induced in single mammary epithelial cells embedded within a basement membrane matrix via an integrin-dependent pathway. Further support for a critical role for basement membrane in the functional differentiation of the gland comes from studies in involution where degradative loss of basement membranes correlates with loss of functional activity in the epithelium. Thus the extracellular matrix in conjunction with certain cytokines plays a central role in coordinating mammary epithelial development. The findings discussed give further credence to a modal where mammary epithelium, together with certain elements of the subjacent microenvironment, form a dynamic and reciprocally interactive functional unit that regulates tissue specific gene expression in the mammary gland.

Trichocytes, i.e. precursor cells of the hair cortex and medulla, isolated from plucked human scalp hair follicles (HF) were propagated on feeder layers of post-mitotic human dermal fibroblasts (HDF). Cell isolates from five HF routinely yielded about 0.5-1 x 10(5) cells within 3 weeks. When grown as 'surface epithelia' in vitro (on dermal equivalents exposed to air), trichocytes organized into stratified epithelia largely reminiscent of epidermis with regard to both tissue architecture and localization of epidermal differentiation products (keratins K1 and K10, involucrin, filaggrin). However, when HDF in the collagen matrix were replaced by dermal papilla cells (DPC) epidermoid differentiation was largely prevented while still allowing growth and stratification. Epidermal differentiation (keratinization) was virtually complete when trichocytes grown on collagen gels with HDF were transplanted onto nude mice; this was apparent by tissue organization, expression of K1 and K10 and the nearly regular epidermal localization of involucrin. In addition, the deposition of basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) at the epithelium-collagen interface further increased and was more regular in transplants than in vitro. Cells embedded in Matrigel together with HDF developed large spheroidal structures with inward-directed differentiation and all the epidermal markers found in 'surface' cultures, while only small keratinizing spheroids formed without HDF. In this system co-culture of trichocytes with DPC suppressed almost completely epidermal keratinization. Although typical hair proteins were not detectable, our data clearly demonstrate that: (1) bona fide trichocytes inherit the options for alternative directions of differentiation, and (2) external (in part mesenchymal cell-mediated) influences play a pivotal role in this determination.

Adult rodent (rat and mouse) prostatic ducts (PR) were recombined heterospecifically with fetal urogenital sinus mesenchyme (UGM) (mouse UGM plus rat PR or rat UGM plus mouse PR), and the resultant tissue recombinants were grafted under the renal capsule of male athymic mice. For recombination with mouse UGM the ductal tips of each of the 4 rat prostatic lobes [ventral (VP), lateral type 1 (L1), lateral type 2 (L2), and dorsal prostate (DP)] were used. For recombination with rat UGM the ductal tips of each of the 2 mouse prostatic lobes [ventral (VP) and dorso-lateral prostate (DLP)] were used. After 1 month of growth in vivo, the DNA content of UGM+PR recombinants increased substantially (6.1- to 76.8-fold increase) over the combined DNA content of the isolated UGM and PR prior to grafting. Immunocytochemical, polyacrylamide gel electrophoretic and Western blot analyses demonstrated that irrespective of the initial source of the adult prostatic duct, the epithelium of UGM + PR recombinants continued to express its normal lobe-specific secretory proteins as well as secretory proteins specific to other prostatic lobes. For example, rat ventral and lateral type 2 prostate do not normally express DP-1, a rat dorsal-prostatic-specific protein, but after recombination with mouse UGM the induced prostatic epithelium expressed DP-1 as well as C3, a rat ventral-prostatic-specific protein. Sensitive reverse transcriptase-polymerase chain reaction techniques (RT-PCR) verified the expression of mRNA for C3 and DP-1 in such tissue recombinants. Analogous results were obtained for UGM + PR tissue recombinants constructed with prostatic ductal tips from rat L1, L2 and DP and mouse VP and DLP. These findings demonstrate that adult rodent prostatic epithelium retains a responsiveness to its connective tissue environment, and that fetal UGM can permissively induce prostatic ductal growth and morphogenesis while instructively inducing the expression of a new spectrum of prostatic secretory proteins.

The differentiation of human nasal surface epithelial cells in primary three-dimensional (3D) culture was studied. The dissociated cells were seeded on type I and IV collagen gel and grown in a serum-free medium supplemented with hormones and growth factors. During the first days of culture, epithelial cells were infrequently differentiated. Detachment and retraction of collagen by the cells generally occurred after 8-10 days of culture, allowing the formation of a floating collagen gel. This induced the differentiation of epithelial cells on 3D cord-like structures consisting of a collagen core surrounded by well-differentiated cells. Under scanning and transmission electron microscopy, we observed the formation of a pseudostratified respiratory-type epithelium consisting of columnar mature ciliated cells and secretory cells, epithelial cells in the process of ciliogenesis, and small pyramidal basal cells. The videomicroscopic analysis of the ciliated cells showed that the mean ciliary beating frequency (12.2 +/- 1 Hz) was close to the values obtained on polyp explants (11.7 +/- 0.8 Hz). Immunocytochemical localization of secretion with mucin-specific antibodies showed the expression of mucous cell function. In addition, the epithelial cells within the cord-like structures maintained a differentiated morphology and active beating of ciliated cells for more than 35 days in primary culture. Conversely, when the cells were grown on a collagen gel attached to plastic, they remained more flattened and the number of differentiated cells was lower. These results suggest that human upper airway epithelial cell differentiation in culture, as assessed by mucociliary function, is enhanced by the 3D organization of the cells around the floating collagen gel substrate.

Genes of the ras family of dominant transforming genes are frequently activated by specific point mutations in common human epithelial malignancies and also in many experimental tumour models. Testing of the hypothesis that ras activation is a critical event in tumorigenesis involves reconstruction of genetic events in vitro using appropriate epithelial models. This article reviews experimental transformation of human and rodent epithelial cells by ras oncogenes in vitro, alone and in combination with cooperating oncogenic events.