RESUMO
The presence of peroxisomes and their enzymic content were investigated and compared in healthy and neoplastic human colon epithelial cells using cytochemical studies at the ultrastructural level as well as biochemical analyses. Catalase-positive organelles were found to be more numerous in normal than in colonic neoplastic cells. Biochemical assays revealed that no D-aminoacid oxidase or L-alpha-hydroxyacid oxidase activity was detected in normal or tumor tissues. The specific activities of catalase, fatty-acyl CoA oxidase and enoyl-CoA hydratase/3 hydroxyacyl-CoA dehydrogenase (the so-called peroxisomal bifunctional enzyme of the beta-oxidation system) were found to be diminished in carcinoma cells compared with the control tissue. The fall in catalase activity correlated well with tumor stage according to Dukes, suggesting that this peroxisomal enzyme could be used as a potential prognostic marker.
Assuntos
Adenocarcinoma/ultraestrutura , Neoplasias do Colo/ultraestrutura , Microcorpos/enzimologia , Proteínas de Neoplasias/análise , Neoplasias Retais/ultraestrutura , 3-Hidroxiacil-CoA Desidrogenases/análise , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Oxirredutases do Álcool/análise , Catalase/análise , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , D-Aminoácido Oxidase/análise , Enoil-CoA Hidratase/análise , Humanos , Isomerases/análise , Pessoa de Meia-Idade , Complexos Multienzimáticos/análise , Estadiamento de Neoplasias , Enzima Bifuncional do Peroxissomo , Prognóstico , Neoplasias Retais/enzimologia , Neoplasias Retais/patologiaRESUMO
Two human colon carcinoma cell lines, HT29 and Caco-2 were co-cultured with fetal rat or human skin fibroblasts. Their morphological features, ultra-structural characteristics at the heterologous cell interface, and the deposition of basement-membrane molecules [laminin, type-IV collagen, heparan sulfate proteoglycan (HSPG)] at the epithelial-stromal junction were analyzed. The 2 cell lines behaved differently. HT29 cells did not spread on the fibroblasts and grew as clusters, while Caco-2 cells formed a monolayer over the fibroblastic feeder layer. Only the latter carcinoma cells exhibited cytoplasmic processes towards the fibroblasts and, after 5 days in co-cultures, a structured basement membrane (BM). The immunocytochemical analysis of the BM constituents revealed the absence of the molecules studied at the sites of heterologous contacts in the case of HT29 cells. In contrast, in the co-cultures comprising Caco-2 cells, laminin and type-IV collagen were progressively deposited in a polar fashion at the epithelial-fibroblastic interface which, however, remained devoid of HSPG molecules. Together with earlier data indicating a dual origin of the BM molecules located at the epithelial-fibroblastic interface in normal intestine, the present study shows that the cancer cells as well as the fibroblastic ones under the influence of carcinoma cells display an altered capacity to synthesize and/or secrete BM molecules. The extent of such abnormalities correlates with the differentiation of the cells. Finally, these modifications occur concomitantly with alterations in cell interactions which vary among cell lines.
Assuntos
Adenocarcinoma/fisiopatologia , Membrana Basal/fisiologia , Proteoglicanas de Sulfatos de Condroitina/análise , Colágeno/análise , Neoplasias do Colo/fisiopatologia , Heparitina Sulfato/análise , Laminina/análise , Adenocarcinoma/patologia , Animais , Membrana Basal/citologia , Membrana Basal/ultraestrutura , Adesão Celular , Comunicação Celular , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Neoplasias do Colo/patologia , Replicação do DNA , Epitélio/ultraestrutura , Feto , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteoglicanas de Heparan Sulfato , Humanos , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica , Ratos , Pele/citologia , Fenômenos Fisiológicos da PeleRESUMO
Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Adenocarcinoma/análise , Proteoglicanas de Sulfatos de Condroitina/análise , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colo/análise , Colo/metabolismo , Pólipos do Colo/análise , Pólipos do Colo/metabolismo , Neoplasias Colorretais/análise , Glicosaminoglicanos/análise , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/análise , Humanos , Imuno-Histoquímica , Mucosa Intestinal/análise , Mucosa Intestinal/metabolismo , Reto/análise , Reto/metabolismoRESUMO
Cytodifferentiation of smooth muscle cells has been analyzed immunocytochemically during rat intestinal development and in chimaeric intestines by using monoclonal antibodies reacting specifically with smooth muscle actin species (CGA7 [10] and anti-alpha SM-1 [40]). As development proceeds, the various intestinal muscle layers differentiate in the following order: (1) cells expressing smooth muscle actin appear within the mesenchyme of the 15-day fetal rat intestine, in the circular muscle-forming area, the differentiation of cells in the presumptive longitudinal muscle layer starting with a 48-h delay; (2) smooth muscle fibers appear within the connective tissue core of the villi shortly after birth, in parallel with a progressive formation of the muscularis mucosae, which becomes clear-cut only in the course of the 2nd week after birth; (3) a distinct cell layer in the innermost part of the circular muscle layer arises during the perinatal period. Thereafter, the fluorescence pattern remains unchanged until the adult stage. Chimaeric intestines were constructed by the association of 14-day fetal intestinal epithelium and cultured fetal rat or human skin fibroblasts. These fibroblastic cells did not express actin at the time at which they were associated. The immunocytochemical analysis of smooth muscle actin in the hybrid intestines, which had developed as intracoelomic grafts for 12 days, revealed that the skin fibroblastic cells had been induced by the intestinal epithelial cells to differentiate into smooth muscle cells. Such a result was also obtained with allantoic endoderm. It was not obvious in cocultures of intestinal epithelium with skin fibroblastic cells. However, when intestinal epithelial cells were cocultured with intestinal mesenchymal cells, actin expression was stimulated in the latter cell population.
Assuntos
Actinas/metabolismo , Sistema Digestório/embriologia , Desenvolvimento Embrionário e Fetal/fisiologia , Fibroblastos/citologia , Intestino Delgado/citologia , Músculo Liso/metabolismo , Pele/citologia , Actinas/farmacologia , Actinas/fisiologia , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Sistema Digestório/citologia , Sistema Digestório/metabolismo , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Endoderma/citologia , Endoderma/metabolismo , Endoderma/fisiologia , Células Epiteliais , Epitélio/embriologia , Epitélio/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Imunofluorescência , Intestino Delgado/embriologia , Intestino Delgado/fisiologia , Mesoderma/citologia , Mesoderma/metabolismo , Mesoderma/fisiologia , Músculo Liso/citologia , Ratos , Ratos EndogâmicosRESUMO
The intestinal epithelium represents an attractive biological model of differentiation from stem cells to highly differentiated epithelial cells, not only during particular developmental events depending upon the vertebrate species considered but also throughout adult life. The ontogenic maturation of the intestinal epithelium arises from both a programmed expression of specific genes and epigenetic influences mainly due to epithelial and mesenchymal interactions and hormonal participation. In the present paper we review the structural and functional changes that occur in the amphibian, avian and mammalian intestine during embryonic and/or post-embryonic development. Furthermore, we review the data concerning the mechanisms which control the cytodifferentiation of the intestinal epithelium.
Assuntos
Diferenciação Celular , Intestino Delgado/embriologia , Vertebrados/embriologia , Animais , Matriz Extracelular/fisiologia , Intestino Delgado/citologia , Microvilosidades/fisiologia , Modelos Biológicos , Músculo Liso/citologia , Músculo Liso/embriologiaRESUMO
Are the basement membrane (BM) molecules involved in epithelial-mesenchymal cell interactions known to be instrumental in intestinal development and differentiation? Several findings argue in favor of this assumption. First, quantitative and/or qualitative changes in type IV collagen, laminin-nidogen and heparan sulfate proteoglycan (HSPG) are obvious at the phases of intensive morphogenesis. Second, BM molecules deposited at the epithelial-mesenchymal interface are of dual origin: HSPG being produced by the epithelial cell population, while others like type IV collagen and laminin are mainly produced by the mesenchymal compartment. Third, the formation of the BM requires an actual contact between the epithelial and mesenchymal cells and always precedes the expression of differentiation markers in the epithelial cells. These data suggest that BM molecules display an instructive role in intestinal cell interactions.
Assuntos
Membrana Basal/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Colágeno/biossíntese , Heparitina Sulfato/biossíntese , Intestino Delgado/metabolismo , Laminina/biossíntese , Glicoproteínas de Membrana , Proteínas de Membrana/biossíntese , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Proteoglicanas de Heparan Sulfato , HumanosRESUMO
We report the relative frequency of sucrase-isomaltase (SI) antigen expression in human colonic adenocarcinoma (22/57), in peritumoral mucosa taken next to the tumor (31/41) or distant from it (29/42) as well as in 21/23 polyps. Our results are based on indirect immunofluorescence with a monoclonal antibody (MAb) specific for human intestinal SI. A regular and intense expression of SI occurred only in 6 tumor specimens. In the remaining 16 SI-positive tumor samples, labelling was heterogeneous, i.e., scattered over more or less extensive areas. A similar irregular staining pattern was also found in polyps and in peritumoral mucosa, irrespective of its distance from the tumor. Electron microscopic examination of 19 carcinomas mostly revealed altered brush-border membrane features, irrespective of histological SI staining pattern. Brush-border enzyme activities of sucrase, alkaline phosphatase and maltase showed no difference between tumor specimens and peritumoral mucosa, but aminopeptidase was depressed in the former. Sucrase activity was extremely low (mean values 1.1 to 1.8 mU/mg protein) and rose only exceptionally to 17.5 mU/mg prot.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias Colorretais/enzimologia , Complexos Multienzimáticos/análise , Complexo Sacarase-Isomaltase/análise , Adenocarcinoma/ultraestrutura , Pólipos do Colo/enzimologia , Neoplasias Colorretais/ultraestrutura , Humanos , Imuno-Histoquímica , Mucosa Intestinal/enzimologia , Microvilosidades/enzimologiaRESUMO
The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS. Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures. The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.
Assuntos
Dexametasona/farmacologia , Endoderma/metabolismo , Fibroblastos/metabolismo , Glicosaminoglicanos/biossíntese , Animais , Diferenciação Celular , Células Cultivadas , Sulfatos de Condroitina/biossíntese , Cromatografia por Troca Iônica , Endoderma/citologia , Epitélio , Fibroblastos/citologia , Heparitina Sulfato/biossíntese , Ácido Hialurônico/biossíntese , Intestinos , Ratos , PeleRESUMO
The effects of a protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and of a calmodulin antagonist calmidazolium (CZ), on a human colonic cancer cell line HT-29 were analyzed. HT-29 cells are undifferentiated in standard culture conditions (HT-29 G+) and display an enterocytic differentiation when cultured in glucose-deprived medium (HT-29 G-). Early effects of TPA and CZ on the localization of cytoskeletal proteins (caldesmon, alpha-actinin and vinculin) and on cell proliferation were examined. Differentiation of the cells was assessed after 4 weeks on the basis of ultrastructural and functional characteristics of enterocytic polarity, presence of apical brush borders, expression of brush border membrane antigens (Caco 5/50 and sucrase-isomaltase), and segregation of calmodulin to the brush border cytoskeleton. TPA treatment of HT-29 G+ or G- cells induced early morphological and cytoskeletal alterations: the cells rounded up and lost their stress fibers with the associated caldesmon, alpha-actinin, and vinculin. TPA did not modify the differentiation of G- cells, but induced in G+ cells the expression, although limited, of enterocytic differentiation characteristics. Addition of CZ to HT-29 G- cells enhanced their differentiation state but did not provoke any early morphological or cytoskeletal alterations. No effects of CZ on HT-29 G+ cells were obvious. The results suggest that protein kinase C, the TPA receptor, is involved in the triggering of HT-29 G+ cell differentiation whereas calmodulin-dependent functions would be implicated in HT-29 G- cell maturation.
Assuntos
Calmodulina/antagonistas & inibidores , Neoplasias do Colo/patologia , Imidazóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Antígenos de Neoplasias/análise , Calmodulina/análise , Proteínas de Ligação a Calmodulina/análise , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Imunofluorescência , Humanos , Fosforilação , Complexo Sacarase-Isomaltase/análiseRESUMO
The expression and immunocytochemical localization of three brush border cytoskeletal calmodulin-binding proteins, caldesmon, fodrin, and the 110 kDa subunit of the 110 kDa calmodulin complex, have been studied in human intestinal epithelial cells as a function of their ontogenic differentiation. At immature stages (fetal week 8), caldesmon and fodrin were present in undifferentiated intestinal epithelial cells. However, no 110 kDa protein was detectable except a 135 kDa immunoreactive species. The 110 kDa form appeared at week 12, when microvilli differentiate, and became prominent at week 14 simultaneously with the disappearance of the 135 kDa species. Finally at week 14, the calmodulin-binding protein pattern was identical to that found in adults. Immunocytochemical experiments revealed that at week 8, antibodies to caldesmon and fodrin gave a fluorescence lining at the periphery of the cells, whereas the 110 kDa immunoreactive species was hardly detectable. Then, as early as week 12 of gestation, with the three antisera, a bright fluorescence lined the apex of the cells, as in adults. In the colon, the events were delayed. This study demonstrates that the developmental pattern of the three calmodulin-binding proteins investigated, caldesmon, fodrin and the 110 kDa subunit, parallels the temporal differentiation of human intestinal brush borders and the proximo-distal morphological intestinal maturation.
Assuntos
Proteínas de Ligação a Calmodulina/análise , Feto/fisiologia , Intestino Grosso/citologia , Intestino Delgado/citologia , Microvilosidades/análise , Proteínas de Transporte/análise , Diferenciação Celular , Epitélio/análise , Imunofluorescência , Humanos , Imuno-Histoquímica , Intestino Grosso/análise , Intestino Delgado/análise , Proteínas dos Microfilamentos/análiseRESUMO
Calmodulin concentrations and localisation have been analysed as a function of development in human intestinal epithelial cells from normal and cystic fibrosis individuals. In normal fetuses up to eight weeks of gestation intestinal epithelial cells which were still undifferentiated were not immunoreactive and their calmodulin content was low. From eight weeks onwards there was a significant overall increase in calmodulin content concomitant with its segregation to the apical side of epithelial cells. At 14 weeks of gestation calmodulin concentrations and localisation closely resembled those of adults. The developmental pattern of calmodulin appeared to parallel the morphological and functional maturation of brush borders which occurs during the first trimester of pregnancy. In the intestinal epithelial cells from a 19 weeks cystic fibrosis fetus and a cystic fibrosis newborn infant neither calmodulin concentration, nor its localisation were affected. Similarly, brush border calmodulin binding proteins and enzymatic activities were similar in normal subjects and the cystic fibrosis intestine.
Assuntos
Calmodulina/análise , Fibrose Cística/metabolismo , Doenças Fetais/metabolismo , Intestinos/embriologia , Adulto , Proteínas de Ligação a Calmodulina/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Idade Gestacional , Humanos , Recém-Nascido , Intestinos/análise , GravidezRESUMO
The production and deposition of extracellular matrix proteins and the cellular origin of type-IV collagen have been analysed immunocytochemically in cocultured or transplanted intestinal epithelial-mesenchymal cell associations. In the first experimental model, rat intestinal endodermal cells were cultured on top of confluent mono-layers of rat intestinal or skin fibroblastic cells. Under these conditions, interstitial matrix and basement membrane proteins were deposited within the fibroblastic layer over the whole culture period; interactions between the epithelial cells and the fibroblastic cell population, whatever their organ of origin, were required for the production of the basement membrane. In addition, its formation was progressive as assessed by the shift of a spot-like labelling to a continuous linear pattern at the epithelial-mesenchymal interface, and paralleled epithelial cell differentiation. In the second experimental model, chick-rat epithelial-mesenchymal recombinants developed as intracoelomic grafts were used, and the immunocytochemical detection of a basement membrane protein, type-IV collagen, was performed with species-specific antibodies. The major role of the mesenchyme in the deposition of type-IV collagen is supported by the fact that anti-chick but not anti-mammalian antibodies stained this antigen in chick mesenchyme-rat endoderm recombinants. These observations emphasize the role of tissue interactions in the formation of a basement membrane and show that the mesenchymal compartment is the principal endogenous source of type-IV collagen.
Assuntos
Membrana Basal/embriologia , Comunicação Celular/fisiologia , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Mesoderma/fisiologia , Animais , Membrana Basal/metabolismo , Comunicação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Embrião de Galinha , Técnicas de Cocultura , Endoderma/citologia , Endoderma/metabolismo , Endoderma/fisiologia , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Mucosa Intestinal/metabolismo , Mesoderma/metabolismo , Especificidade de Órgãos/genética , Especificidade de Órgãos/fisiologia , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
The complex morphogenetic events and the concomitant structural and functional differentiation of intestinal progenitor cells are dependent on tissue interactions. Several experimental models of hetero-species or -topic recombinants between epithelial and mesenchymal anlagen are described. They enabled us to elucidate the respective roles of these tissue components in morphogenesis, epithelial differentiation, and hormone-elicited responses. Among the mechanisms of tissue interactions, the possible mediation of permissive and instructive information via the extracellular matrix is postulated. Arguments in favor of this are provided by the observation of compositional changes in matrix molecules during intestinal development and differentiation. On the other hand, in vitro experimental data emphasize the role of actual contacts between epithelial and mesenchymal cell populations and the importance of the mesenchyme for basement membrane formation.
Assuntos
Intestinos/embriologia , Células-Tronco/citologia , Animais , Membrana Basal/citologia , Comunicação Celular , Diferenciação Celular , Embrião de Galinha , Células Epiteliais , Matriz Extracelular/fisiologia , Intestinos/citologia , Morfogênese , RatosRESUMO
Among the extracellular matrix components which have been suggested to be involved in developmental and neoplastic changes are glycosaminoglycans (GAGs). To try to correlate their amount and nature with the process of enterocytic differentiation, we studied glycosaminoglycan synthesis of human colonic adenocarcinoma cells (HT29 cell line) by [3H]glucosamine and [35S]sulfate incorporation. Enterocytic differentiation of the cells obtained in a sugar-free medium (for review, see A. Zweibaum et al. In: Handbook of Physiology. Intestinal Transport of the Gastrointestinal System, in press, 1987) resulted in a marked increase in total incorporation of labeled precursors (20-fold for [3H]glucosamine, 4.5-fold for [35S]sulfate) as well as in uronic acid content (5-fold); most of the synthesized GAGs were found associated with the cell pellet. Chromatographic and electrophoretic analysis of the labeled GAGs revealed that undifferentiated cells synthesized and secreted hyaluronic acid, heparan sulfate, and one class of chondroitin sulfate. Differentiation of HT29 cells because associated with the synthesis of an additional class of chondroitin sulfate (CS4) concomitant to a decrease in heparan sulfate which is no longer found secreted in the medium. Furthermore, the charge density of this latter GAG component varied as assessed by a shift of its affinity on ion-exchange chromatography.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Glicosaminoglicanos/biossíntese , Adenocarcinoma/patologia , Contagem de Células , Diferenciação Celular , Células Cultivadas , Cromatografia por Troca Iônica , Neoplasias do Colo/patologia , Glicosaminoglicanos/análise , HumanosRESUMO
Microexplants of 14- or 15-day-old fetal rat intestinal endoderm, separated from mesenchyme by collagenase, were placed on culture dishes coated with different extracellular matrix components or on confluent monolayers of intestinal mesenchymal cells or of fetal skin fibroblasts. Only small variations in the attachment or spreading of the endodermal cells could be observed when they were cultured on the different acellular substrata, and their survival never exceeded one week. When cocultured with intestinal or skin fibroblasts, endodermal cells proliferated and the survival time was prolonged to 2 or 3 weeks. Furthermore, differentiation, as assessed by the polarization of the cells, occurred and was characterized by the maturation of apical brush borders and by the synthesis of microvillar digestive enzymes visualized immunocytochemically with monoclonal antibodies. Glucocorticoids accelerated structural differentiation and stimulated or induced brush border enzymes only in the coculture conditions. These experiments emphasize the role of a fibroblastic support without tissue specificity on the cytodifferentiation of intestinal endodermal cells. They also suggest a mesenchymal dependence on the hormonal response.
Assuntos
Endoderma/citologia , Intestinos/citologia , Animais , Diferenciação Celular , Células Cultivadas , Técnicas Citológicas , Endoderma/efeitos dos fármacos , Endoderma/enzimologia , Fibroblastos/fisiologia , Glucocorticoides/farmacologia , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Ratos , Ratos Endogâmicos , Pele/citologiaRESUMO
To investigate the role of epithelial-mesenchymal interactions on intestinal maturation, we cultured embryonic epithelial cells in several experimental conditions. Microexplants of 14-15 days fetal rat intestinal endoderm, separated from the mesenchyme by collagenase, were seeded on dishes coated with different extracellular matrix components (collagens I, III, IV, fibronectin, laminin) or on confluent monolayers of intestinal mesenchymal cells or of fetal skin fibroblasts. Only small variations in the attachment or spreading of the endodermal cells could be observed when they were cultured on the different substrata and their survival never exceeded one week. When cocultured with intestinal or skin fibroblasts, however, endodermal cells grew, formed a monolayer, survival time was prolonged up to two to three weeks, and differentiation occurred. This differentiation was assessed by cell polarisation, morphological maturation of apical brush borders, synthesis of microvillar digestive enzymes and of extracellular matrix molecules seen immunocytochemically. Finally, glucocorticoids which are known to stimulate or induce brush border enzymes, accelerated the morphological and enzymatic maturation only in the cocultures.
Assuntos
Endoderma/citologia , Intestinos/embriologia , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Epitélio/embriologia , Matriz Extracelular/fisiologia , Fibroblastos/citologia , Mesoderma/citologia , RatosRESUMO
The digestive tract and the gut as a paradigm represents an attractive system for the study of mechanisms involved in the differentiation of two types of progenitor cells: the endodermal cells during embryonic life and the undifferentiated crypt cells during epithelial renewal of the adult intestine. The morphological and functional events that accompany the differentiation processes of progenitor cells into the polarized epithelial cell types characteristic of the intestine appear comparable in both situations (1,2). During organogenesis of the gut, histological observations underlined a close relationship between epithelial cells and their underlying mesenchymal cells (3,4). Developmental biologists have emphasized experimentally the importance of interactions between the endoderm and mesenchyme during organogenesis of the digestive tract. In the adult intestine, gastroenterologists have focused their attention on a specialized mesenchymal cell type (the pericryptal fibroblasts) that displays, like epithelial cells, proliferative activities and migrating properties. The aim of this review is to provide current knowledge on epithelial-mesenchymal interactions during ontogenesis of the digestive tract and also to relate some experiments supporting the view of the perpetuation of epithelial-mesenchymal interactions beyond embryonic life.
Assuntos
Sistema Digestório/embriologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem Celular , Sistema Digestório/citologia , Células Epiteliais , HumanosRESUMO
Calmodulin-binding proteins have been studied in presumptive rat jejunal epithelial cells and in purified rat brush borders during development. Incubation of nitrocellulose replicas with [125I] calmodulin revealed that, at immature stages (13-15 days of fetal life), only two calmodulin-binding bands were detectable with molecular masses of approximately 145,000 and 135,000 daltons. By fetal day 19, additional calmodulin-binding proteins of 240,000 and 110,000 daltons were observed. The 145,000- and 240,000-dalton calmodulin-binding bands contained polypeptides that were immunologically similar to caldesmon and to the alpha-subunit of the non-erythroid spectrin (fodrin) respectively. Antisera reactive with the 110K subunit of the microvillus 110K-calmodulin complex labelled a 135,000-dalton band which comigrated with one of the calmodulin-binding proteins. This 135,000-dalton immunoreactive polypeptide persisted until birth but was absent in brush borders isolated from adult intestine. In addition, the 110K antisera reacted with an approximately 110,000-dalton subunit by fetal day 19. At birth, numerous lower-molecular-mass 110K immunoreactive bands were also detectable. Immunocytochemical localization of the three calmodulin-binding proteins revealed that, at fetal day 14, caldesmon and fodrin displayed fluorescence lining the periphery of the epithelial cells, whereas staining with the 110K antisera was very weak. At fetal day 19, staining with the three antisera resulted in bright fluorescence localized in the apical part of the epithelial cells, in parallel to the differentiation of brush borders. At this stage, the apical staining of the calmodulin-binding proteins was similar to that of the adult.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Envelhecimento/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas Fetais/metabolismo , Jejuno/crescimento & desenvolvimento , Envelhecimento/fisiologia , Animais , Proteínas de Ligação a Calmodulina/fisiologia , Epitélio/metabolismo , Epitélio/fisiologia , Epitélio/ultraestrutura , Proteínas Fetais/fisiologia , Imuno-Histoquímica , Jejuno/embriologia , Jejuno/metabolismo , Microscopia Eletrônica , Peso Molecular , Ratos , Ratos EndogâmicosRESUMO
The culture of animal cells and tissues is a widely used technique in the field of cellular and molecular biology; one of the most interesting aspect being linked to the study of the mechanisms of cell differentiation. In the specific case of intestinal epithelial cells, various tissue culture technologies have proved to be important tools for the study of precise facets related to intestinal function, pathology and differentiation. Concerning this latter aspect, organ culture experiments have brought about interesting data on the hormonal or nutritional control of intestinal maturation. Nevertheless, the study of the precise mechanisms underlying epithelial proliferation and/or differentiation at the cellular level needs more adequate cell culture model systems. One of them has been described for two cell lines derived from human colonic adenocarcinomas, in which the cells can be induced to achieve enterocytic-like differentiation. Up to date, none of the continuous cell lines starting from normal undifferentiated cells have allowed generation of morphological or functional enterocytic polarity. In contrast, primary cell cultures which allow maintenance of a more physiological environment for the epithelial cells like contacts with their in vivo counterparts, mesenchymal cells or extracellular matrix molecules, have proved to be promising approaches.
Assuntos
Técnicas de Cultura/métodos , Intestinos/citologia , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Humanos , Enteropatias/fisiopatologia , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiopatologia , Intestinos/fisiopatologiaRESUMO
Calmodulin was immunocytochemically localized in the brush borders of rat intestinal epithelial cells from the tip to the base of the villi, from day 18 of fetal life up to the adult stage. The early (14th day) fetal cells, like the adult crypt cells, were not immunoreactive, although their calmodulin content was equal to that of the mature cells from the tips of the villi.