Epithelial cell biology最新文献

A comparison has been made between different immunomagnetic techniques for separating normal human mammary epithelial cells based on the exclusive expression of EMA (epithelial membrane antigen) by luminal cells and CALLA (CD10) by myoepithelial cells. When cells labelled with antibodies to these antigens were incubated with Dynabeads, they rosetted myoepithelial but not luminal cells. However, both luminal and myoepithelial cells could be positively separated using the MACS system. Purity was established by analyzing the expression of CALLA and EMA using flow cytometry, and of cell-type specific cytokeratins using indirect immunofluorescence. Dynabead-separated myoepithelial cell populations were of high purity (> 98%) but the beads could not be removed from the cells. Luminal cell populations separated by the MACS method were also highly purified (> 95%), as were myoepithelial cell populations (> 90%). Using this immunomagnetic separation method, up to 10(7) cells of each type could be obtained from individual preparations.

Pokeweed (Phytolacca americana) lectin was found to stain the secretory granules in epithelial Paneth cells of small intestine in mice and rats. The distribution of Paneth cells stained with this lectin was identical to that obtained by another immunohistochemical marker for lysozyme. However, in comparison with other immunohistochemical markers, Pokeweed lectin is a more robust method for identifying Paneth cells in histological sections and for studying their secretory granules. Co-expression of the Pokeweed lectin binding sites in some oligomucous cells within the crypts suggested a close developmental link between these two cell types. Only one other non-epithelial cell type was stained by this lectin, and these were migratory lymphocytes found within the villus epithelium and lamina propria. Approximately 20% of these lymphocyte cells were also positive for the expression of CD3+. Pokeweed lectin was therefore used to study changes in the frequency of Paneth cells and intra-epithelial lymphocytes in normal and immunologically compromised animals (following infection with a parasite worm Trichuris muris and in a model of graft-versus-host rejection). This study confirmed that the population of Paneth cells turns over slowly even during conditions of inflammation.

We report a novel association of the calcium dependent cross-linking enzyme, transglutaminase (TGase) with the urea soluble reconstituted keratin filaments (RKF) isolated from the rat vaginal epithelial cells (VEC). This was ascertained by measuring the activity using 14C-spermidine incorporation and also by an increase in keratin filament aggregation by the addition of only TGase cofactor-calcium. These events were specifically inhibited by the treatment of calcium chelator, EDTA at a concentration > 2 mM as well as by pretreating the RKF with histamine, a TGase substrate inhibitor. The association was also exemplified by immunoblotting analysis where a specific and preferential polypeptide of molecular weight 58 kDa cross-reacted with TGase antibody amongst the other keratins. This phenomenon was not seen in the keratins isolated from skin, a non-targeting tissue for estradiol action.

Migration velocity estimates have been determined at each position along the crypt length for both the small and large intestine of the mouse at 6 different times of the day. Measurements also have been made of crypt circumference and length. Dramatic, and significant (P < 0.001), changes in migration velocity as a function of time of day were observed in the small intestine with a maximum 0.84 cell positions (cp) per hour at 0900 h and a minimum of -0.46 cp/h at 1700 h, although the negative velocity was probably artefactual. The 24-h mean velocity rose smoothly as a function of cell position to a peak of 0.45 cp/h at cell position 17 (around the top of the proliferative zone). Much more modest changes were seen in the percent of 3HTdR labelled cells (minimum 30.8%, maximum 38.3%, P < 0.001) and crypt circumference (minimum 16.9 cells, maximum 17.9 cells, P = 0.003). The migration velocity was rather less well determined in the large intestine with a peak in the 24-h mean velocity (0.26 cp/h) occurring at cell position 10. At this position significant circadian variation was detected (minimum -0.39 cp/h, maximum 0.75 cp/h, P = 0.006). Changes were seen in the percent of labelled cells (minimum 9.4%, maximum 22.3%, P < 0.001) and crypt circumference (minimum 18.3 cells, maximum 19.2 cells, P < 0.001). In both tissues it is suggested that the combination of the modest changes in cell proliferation rates in conjunction with the changes in crypt cell number can account for the large amplitude in variation of crypt output, and that the reservoir effects of changes in crypt geometry are an essential part of the process governing the maintenance of intestinal cell numbers.

The embryonic collecting duct epithelium of neonatal kidney undergoes profound functional changes during maturation. In its initial state as inductor epithelium it appears homogeneous, but differentiates into a heterogeneously composed collecting duct epithelium consisting of principal and intercalated cells. The mechanism of this terminal differentiation process is unknown. We used morphological and immunohistochemical methods to investigate the maturation of the collecting duct system in neonatal rabbit kidney and under organotypic culture conditions. The new perifusion culturing method allowed us to follow the differentiation of the ampullary collecting duct epithelium under conditions as close as possible to the situation within the organ. With this technique we were able to induce a differentiation process similar to that in the in situ situation. This process led to the appearance of a mixed cell population consisting of principal and intercalated-like cells, respectively. A continuous perifusion of the medium made it possible to stabilize the microenvironment under culture conditions and thus to maintain the heterogeneous composed collecting duct epithelium in a differentiated status over long periods of time.

This study aimed to assess the suitability of sheep tracheal epithelium as a model for studies of human airway ion transport. Ovine and human airway epithelium were mounted in Ussing chambers under short circuit conditions. Bumetanide (100 microM) reduced short-circuit current (Isc) by a mean of 21.3% +/- SEM 2.0, n = 8, in sheep, and 30.4% +/- 9.7, n = 3, in human airway epithelium. Acetazolamide (100 microM) decreased Isc by 10.6% +/- 1.2, n = 18, in sheep, and 5.8% +/- 2.9, n = 3, in human airways. Phloridzin (200 microM) reduced Isc by 4.7% +/- 0.8, n = 7, and 3.1% +/- 5.1, n = 3 in sheep and human tissue respectively. Amiloride (100 microM) decreased Isc by 42.9% +/- 3.5, n = 12, in sheep airways, whilst bathing the mucosal surface with Na(+)-free solutions reduced Isc by 67.4% +/- 4.2, n = 18. The sequential addition of acetazolamide, bumetanide, phloridzin, amiloride and mucosal Na(+)-free solutions totally inhibited the basal Isc in both sheep and human tissues, suggesting that Cl- and HCO3- secretion, Na(+)-glucose co-transport and amiloride-sensitive and -insensitive Na+ absorption contribute to the Isc. The similarities between the species suggest that sheep tracheal epithelium is a useful model for basal studies of airway ion transport, and may prove a valuable tool for further regulatory studies.

The mitosis inhibitory pentapeptide, pGlu-Glu-Asp-Ser-GlyOH (EPP), which was isolated from mouse epidermis extracts, belongs to a group of growth inhibitory peptides that all have pyroglutamyl at the N-terminal end. Earlier experiments with crude or partially purified skin extracts have shown that the inhibitory effect could be enhanced by beta-receptor agonists and by dibutyryl cAMP, and that beta-receptor blockade could neutralise it. We now show that treatment with the beta receptor blocker propranolol before or after EPP treatment of hairless mice significantly modifies the effect of EPP on mouse epidermal cell proliferation, as estimated by using a metaphase-arrest technique (Colcemid) to estimate the G2-M cell flux. The interaction between propranolol and EPP is complex; only the EPP-induced inhibition of the G2-M cell flux was modified by beta-receptor blockade, while the late (18-21 h) inhibition of the mitotic rate was unaltered. Propranolol alone was followed by a dose-related and transient increase in the epidermal mitotic rate. The phosphodiesterase inhibitor caffeine had no effect on its own on epidermal cell proliferation but counter-acted the late (18-21 h) EPP-induced inhibition.

We have examined the proliferative organization of the ulcer-associated cell lineage (UACL), in a series of human ileal tissues involved by Crohn's disease, using antibodies to trefoil peptides and Ki-67. The UACL arises in conjunction with endodermal damage and is likely to play a role in subsequent tissue repair. Our study supports the view that the UACL initially forms through extrusion from the base of a parent intestinal crypt without indigenous mitotic activity. Eventually a duct forms extending to the gut surface and within this duct a proliferative zone develops. We also confirm that the trefoil peptides pS2 and hSP, which play a major role in the repair process, are spatially segregated within the UACL with a zone of overlap in the duct. Using triple antibody labelling techniques we have demonstrated that the zone of overlap of these peptides coincides with the proliferative region and provides evidence for a novel stem cell zone.

Cholera toxin catalyzed the transfer of ADP-ribose from [alpha-32P] NAD to 45kDa protein in pig epidermis. Western blot analysis using anti-Gs alpha antibody identified the 45kDa protein to be Gs alpha. In contrast to pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, the cholera toxin-catalyzed ADP-ribosylation was enhanced by the presence of Mg2+ in the reaction mixture. The cholera toxin-catalyzed ADP-ribosylation of the epidermal 45kDa membrane protein was significantly decreased, when samples were prepared from the cholera toxin-pretreated epidermis. The results, coupled with our previous report (Tsutsui and Iizuka 1990), indicate that pig epidermis contains functional G proteins (Gs and Gi), that affect the epidermal adenylate cyclase activity. Tape stripping-induced hyperproliferative epidermis showed an increased cholera toxin-catalyzed ADP-ribosylation of the 45kDa protein (Gs alpha) at 12-24 h following the tape stripping. Immunoblot analysis, however, showed no remarkable change in the level of Gs alpha compared with non-stripping controls. There was no significant difference in the level of the pertussis toxin-induced ADP-ribosylation of 40kDa protein (Gi alpha) in the tape-stripped epidermis. Immunoblot analysis showed no change in Gi content, either. Forskolin-induced cyclic AMP accumulation was markedly increased in the tape stripping-induced hyperproliferative epidermis. Cholera toxin-induced cyclic AMP accumulation was slightly increased, but this was not statistically significant. These results indicate that the alteration of Gs that is documented by cholera toxin-catalyzed ADP-ribosylation, is among the functional derangements of adenylate cyclase of tape stripping-induced hyperproliferative epidermis.

The distribution of IGF-I receptor is reported in the odontogenic epithelium and mesenchyme of the continuously erupting mandibular incisor of the rat by immunohistochemistry using a polyclonal antibody specific to the IGF-I receptor. Odontogenic epithelium is a unique odontogenic sequence in that all stages of the complex life cycle of the ameloblast are represented along the length of the enamel-forming aspect of the tooth. Pre-ameloblasts become post-mitotic before secreting enamel matrix. When the full thickness of the enamel has been formed, a remarkable transition in phenotype takes place in the ameloblast. It changes from a protein secretory cell to one active in maturation of enamel matrix by removal of water and protein from the increasingly mineralized matrix. The distribution and intensity of IGF-I receptor expression varied with the phenotypic stages of the ameloblasts. Diffuse cellular staining for IGF-I receptor was found during the active secretory phase of amelogenesis. However, towards the end of this phase, the staining was confirmed to granular or vesicular structures within the cytoplasm. These granular deposits gradually decreased as the ameloblasts made the transition towards enamel maturation. This transition is accompanied by programmed cell death (apoptosis) of approximately 25% of the ameloblasts and cells in this zone did not stain for IGF-I receptor. With the onset of enamel maturation, diffuse staining of the ameloblast layer was re-established gradually and staining remained evident right up to the reduced enamel epithelium, which joins with the oral epithelium. Strong IGF-I receptor immunoreactivity was observed in the stratum basale and stratum spinosum of the adjacent labial gingival epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)