Biomedical subjects

P P Parnigotto

Publications and source records attributed to P P Parnigotto.

At least 19 recordsLinked to original sources

PubMed2006

Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue.

Expression analysis by reverse transcriptase (RT)-PCR indicates that human adipose tissue is not likely to perform de novo synthesis of steroid hormones from cholesterol because the mRNAs of cytochromes P450scc and P450c17, and of the steroidogenic-related proteins, steroidogenic acute regulatory protein and steroidogenic factor 1, were not detected. Instead, our data support an intracrine role of adipose tissue, in which adrenal dehydroepiandrosterone sulfate (DHEA-S), the most abundant circulating androgen in man, is selectively uptaken, desulfated, and converted into bioactive androgens and estrogens. Three organic anion-transporting polypeptides-B, -D, and -E, presumably involved in DHEA-S transmembrane transport, were demonstrated at the mRNA level. While sulfotransferase expression was not found, the occurrence of steroid sulfatase (STS), converting DHEA-S to DHEA, was established at the mRNA, protein and catalytic activity levels. The 5'-rapid amplification of cDNA ends analysis showed that STS transcription in adipose tissue is regulated by the use of two promoters which differ from the prevalent placental one. The adipose transcripts contain a distinct untranslated first exon, 0a or 0b, followed by a common partially translated exon 1b, and nine other exons that are also shared by the main placental transcript. The presence of an upstream open reading frame in the new transcript variants could lead to an N-terminal divergence restricted to the cleavable signal peptide and thus not interfering with the catalytic activity of the mature STS protein. The adipose transcripts are also present in the placenta as minor isoforms. Western blotting revealed the characteristic approximately 64 kDa band of STS in both the placenta and adipose tissue. The specific enzymatic activity of STS in adipocytes was 118 pmol/10(6) cells per hour, about 50-100 times lower than in the placenta. A similar rate of [3H] DHEA-S uptake plus desulfation was measured in preadipocytes and adipocytes, equivalent to 40-45 pmol/10(6) cells per hour. Thus, an excessive accumulation of fat may out-compete other peripheral organs that are also dependent on intracrine DHEA-S utilization, especially when the adrenal production is low or declining with aging.

3' Untranslated Regions↗

PubMed2003

Effects of some vanadyl coordination compounds on the in vitro insulin release from rat pancreatic islets.

Many lines of evidence indicate that vanadium inorganic salts possess insulin-mimetic and insulinotropic properties. However, they are poorly absorbed, so high oral doses are required to achieve effective plasma concentrations with possible undesirable toxic side-effects ensuing. Various organically-chelated vanadium compounds have been synthesized that are more potent than inorganic vanadium salts in their insulin-like effects due to their greater bioavailability. Unfortunately, little is known about the possible insulin secretagogue action of organic vanadyl coordination compounds. Hence, we investigated the effect of [VO(metformin)2]H2O, [VO(salicylidene-ethylenedimmine)2] and [VO(pyrrolidine-N-dithiocarbamate)2](VODTC) on insulin release from isolated rat pancreatic islets, and compared it to that of vanadyl sulfate (VOSO4). Of the three coordination compounds, only VODTC was found to exert insulin secretagogue action. VODTC, within concentrations ranging from 0.1 to 1.0 mM, enhanced both basal and glucose (11 mM)-stimulated insulin release. The effect involves calcium channels, since it was not appreciable in Ca2+-free medium. The stimulating action of VODTC required the presence of the whole metal-chelator complex inasmuch as the chelator DTC alone was ineffective. VOSO4 was unable to bring about any significant rise in insulin release from isolated islets. Taken together, our findings indicate that VODTC may be considered a potential elective pharmaceutical tool in the therapy of diabetes, especially of type 2, through its concomitant stimulatory effect on insulin secretion and insulin-mimetic action.

Animals↗

PubMed2002

Experimental abdominal wall defect repaired with acellular matrix.

In the surgical repair of congenital abdominal-wall defects (AWD), the ready availability of a non-immunogenic and non-prosthetic biomaterial that could guide the regeneration of normal tissue is a fascinating possibility. Biomaterials are already in use, but in our experience, an acellular matrix (ACM) can stimulate exact regeneration of the absent tissue. We explored the possibility of using an ACM to repair a muscular AWD in an animal model. Male New Zealand white rabbits (3-4 kg, n = 18) were anesthetized and the abdominal wall was shaved and scrubbed; a vertical incision was made in the left lower quadrant and a large patch of external-oblique muscle was resected (3 x 3 cm). The animals underwent reconstruction with homologous diaphragm acellular matrix (HDAM) grafts that were previously prepared using a detergent enzymatic method. The patches were evaluated histologically at 9 (n = 6), 40 (n = 6), and 90 (n = 6) days post-surgery in each group; moreover, 90 days post-surgery an electromyogram (EMG) (n = 6) of the implanted matrix was recorded. Histologic analysis demonstrated that the HDAM supported fibroblast migration, deposition of newly-formed collagen, and neovascularization. No signs of necrosis, or evidence of skeletal-muscle-cell ingrowth were detected. The EMG revealed minimum muscular electrophysiologic activity, probably due to muscle underlying the patch. The HDAM we employed was thus not able to produce reconstruction of the skeletal muscle, and was progressively remodeled into fibrous tissue. Since the ultimate reason for failure of muscle regeneration is a lack of myogenesis, future studies will use ACMs preconditioned by various regulators of myoblast proliferation and differentiation.

Abdominal Wall↗

PubMed2001

Essential amino acids increase the growth and alkaline phosphatase activity in osteoblasts cultured in vitro.

An inadequate protein intake seems to be involved in the pathogenesis of osteoporosis. Moreover, protein from animal sources appears to protect against hip fracture, while protein from vegetable sources, which present low levels of essential amino acids, has no effect. In this preliminary work, the growth, the alkaline phosphatase activity and the collagen synthesis were evaluated in osteoblast cultures obtained from calvaria of newborn Sprague-Dawley rats and incubated with lysine, threonine, methionine, triptophan and arginine. Our results have shown that the essential amino acids can modulate the growth and the differentiation of osteoblasts cultured in vitro, confirming the relationship between osteoporotic hip fracture and inadequate protein intake. The compounds have mainly enhanced cell growth and alkaline phosphatase activity, and, to a lower degree, collagen synthesis. In summary, the essential amino acids can stimulate bone formation and could represents useful agents for the prevention and therapy of osteoporosis.

Alkaline Phosphatase↗

PubMed2001

Prostaglandin F2 alpha can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro.

The effects of PGF2 alpha on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF2 alpha at concentrations equal to or lower than 10(-6) M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10(-5) M PGF2 alpha induced a decrease in cell growth under all culturing conditions. PGF2 alpha did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF2 alpha induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF2 alpha and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE2.

Animals↗

PubMed2000

Experimental defect in rabbit urethra repaired with acellular aortic matrix.

Urethral reconstruction following failed hypospadias repair or post-traumatic chronic stricture requires adequate amounts of tissue. Many surgical techniques utilizing different types of biological tissues have been attempted: (a) vascularized skin flaps from the prepuce, scrotum or penile shaft; (b) full-thickness free skin grafts; (c) vesical or buccal mucosa grafts; (d) ureter; artery; vein and appendix tissue. More recently, biodegradable polymers have also been used as delivery vehicles of urothelial cells in animals. It has been demonstrated that the implant of an acellular tissue matrix in the bladder can guide the regeneration of urothelium, blood vessels, smooth muscle and nerves. The aim of this study was to create an experimental model of urethral defect, and then repair it by implanting homologous acellular aortic grafts as urethral substitutes. An acellular matrix was obtained by detergent enzymatic treatment of rabbit thoracic aorta. The growth of urethral epithelium was verified in vitro, and homologous acellular vessels were then implanted in rabbits, bridging a previous surgical urethral defect. The outcome of reconstructive surgery was evaluated histologically at 10 days, 3 weeks, 3 and 12 months. As the time after surgery increased, the neourothelium became less thick, signs of inflammatory response disappeared, and the orientation of collagen fibrils and smooth muscle fascicles resembled that of a normal urethra. The implants displayed abundant vascularization, and the luminal surface started to become irregular. Acellular blood vessels may represent a promising approach to urethral defect therapy for different reasons: (a) unlimited availability, (b) readily obtainable in different lengths and gauges, (c) the potential for being organized as tissue bank, and (d) that just one simple surgical procedure is needed. Nevertheless, before this technique can be applied in humans, it must be tested in more species and animals.

Animals↗

PubMed2000

Production of epidermal growth factor in human prostatic cells cultured in vitro.

The Epidermal Growth Factor (EGF) plays an important role in the regulation of in vitro growth of prostate cells inducing a strong mitogenic effect. Nevertheless in our previous study we observed that the treatment of human hypertrophic prostate cell line U285 with exogenous EGF produces a restricted effect on the cellular growth rate. This phenomenon could be due to the capacity of the cells to produce EGF. In this study we aimed to verify this hypothesis by evaluating the presence of mRNA of EGF and EGF receptor (EGF-R) and of their translation products in U285 cells, before and after the treatment with suramin and exogenous EGF. Moreover we studied the effects exerted by these substances on the proliferative rate of the cells U285 after different treatment protocols. The presence in the cells of mRNA for EGF and EGF-R and of their translation products was demonstrated by means of reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods respectively. The modification of growth rate induced by these drugs was studied by FRAME Cytotoxicity Test. The operative modalities adopted to carry out these growth assays tended to 1) focus the effects of suramin in relation to in vitro cellular growth phase; 2) verify the reversibility of its effects; 3) ascertain if it was possible to antagonize the action of suramin by adding exogenous EGF. The results obtained from the RT-PCR showed the presence, in the control cells and in the treated ones, of mRNA coding for EGF and EGF-R. The immunocytochemical analysis indicated that 20% of the control cells are EGF positive, and 83% are EGF-R positive, confirming the results obtained with RT-PCR. Moreover, these stainings showed that the treatment with EGF does not significantly modify the percentage of cells marked by the anti-EGF antibody, while treatments with suramin and suramin plus EGF double this percentage. None of the treatments modifies the percentage of EGF-R positive cells. The growth assays showed that the exposition to highest doses of suramin in the first 24 h of cultures causes a decrease (p < 0.05) of the cellular proliferation during the following 48 h and 72 h and that these effects are irreversible. Moreover, a contemporaneous exposition of the cells to EGF and suramin at seeding strengthens the cytotoxic action of the last drug. To sum up, the demonstration of the presence in the U285 cells of mRNA coding for EGF and EGF-R and of the corresponding proteins, confirms the hypothesis that these cells can produce EGF. Moreover, the cytotoxicity experiments allowed a focusing of the role of the endogenous EGF in the regulation of the U285 cells proliferation and confirmed the importance of biological events that take place in U285 cells during the first 24 h of culture.

Analysis of Variance↗

PubMed2000

Short bowel syndrome: experimental approach to increase intestinal surface in rats by gastric homologous acellular matrix.

BACKGROUND/PURPOSE: In this preliminary work the authors used homologous acellular matrix obtained by the gastric wall to increase the small bowel surface in Sprague-Downey rats; through this experimental model the authors verified that homologous acellular matrix can support cell migration and the reconstruction of the intestinal wall. METHODS: A tract of about 2 cm of tubular gastric acellular matrix was inserted with bilateral anastomosis in an isolated ileal loop, which was located in endoabdominal position through a short subcutaneous tunnel. Twelve animals were analyzed at each of the time-points ranging from 1 to 6 weeks after surgery. RESULTS: Histologic evaluation showed that the implanted matrix can be reintegrated in the normal small bowel in a period ranging between 3 and 6 weeks from surgery. The implanted matrix was organized with 4 different tonacae from the third week after the surgery, without interruption at the site of the anastomosis. CONCLUSIONS: To date, the authors do not have a demonstration of the function of the ileal loop reconstructed with this technique; based on these results the authors are engaged in an experimental trial of restoration of intestinal viability with the ileal prosthesis after 3 weeks to study its function.

Animals↗

PubMed1999

Heterocyclic derivatives of all-trans retinoic acid: in vitro effects on fibroblast/keratinocyte growth and differentiation, and in vivo effects on guinea-pig skin.

We investigated the biological effects of five all-trans retinoic acid derivatives, bearing heterocyclic ring systems in the side chain. Growth assays performed on submerged human fibroblast and keratinocyte cultures revealed that (E)4-[2-(5-terbuthyl-thiophen-2-yl)propenyl]benzoic acid (compound 5) is the best compound among the studied derivatives for it exhibits a weaker antiproliferative activity and induces, like all-trans retinoic acid does, a significant increase in fibroblast and keratinocyte growth. The morphological and morphometrical analyses of submerged human fibroblast cultures and human epidermis reconstructed in vitro showed that the compound 5 behaves similarly to all-trans retinoic acid: it induces a decrease in all the cell parameters of submerged fibroblast cultures, and modulates the differentiation of keratinocytes in in vitro reconstructed epidermis. Compound 5 induces thickening of epidermis in vivo, one of the most remarkable pharmacological effects of retinoids on skin, but compared to all-trans retinoic acid, it induces a weaker irritation on guinea-pig skin in terms of both erythema and scaling. Compound 5 could then represent a promising candidate for the treatment of certain dermatological diseases.

Animals↗

PubMed1998

Characterization and applications of human epidermis reconstructed in vitro on de-epidermized derma.

In recent years in vitro models have been developed to avoid the use of animals in cutaneous toxicological studies. Submerged human keratinocyte cultures in vitro could be so far employed as an alternative to animal testing and a good correlation between skin irritation and cytotoxicity has been demonstrated. Nevertheless, these submerged cultures are lacking in the stratum corneum which acts as a barrier to chemical toxicity, so that this type of culture is far from the in vivo situation. A better alternative method seems to be the use of in vitro reconstructed skin at the air-liquid interface that closely resembles the in vivo situation. In this work, in a first step we have characterized human epidermis reconstructed in vitro on de-epidermized derma (DED) after a two-week air exposure. Human skin reconstituted in vitro on DED was histologically similar to the in vivo skin. A stratified epidermis including the stratum corneum was obtained. The presence of basal lamina as well as of various important markers for epidermal differentiation (involucrin, K10 keratin, and filaggrin) were revealed. In a second step we have tested the cytotoxic and morphological effects of four surfactants on our model. A good rank correlation has been shown to exist between the irritation potency of surfactants on our model and reported ocular irritancy in vivo. From our results, in vitro reconstituted human skin could represent an attractive model for irritancy testing and could be an in vitro replacement for animal testing.

Child, Preschool↗

PubMed1998

Effects of prostaglandin E2 on growth, morphology, morphometry and keratin pattern of bovine corneal epithelial cells cultured in vitro.

In this work the relationship between the proliferation of bovine corneal epithelial cells and PGE2 has been studied. Our data indicate that PGE2 plays an important role in the growth of corneal epithelial cells. Actually, epithelial cells cultured on a keratocyte feeder-layer and exposed to indomethacin, a cyclooxygenase inhibitor, have shown a decrease in growth rate at drug concentrations which otherwise did not induce a reduction in the viability of the keratocytes as well as in epithelial cells in separate cultures. This effect has been reversed by an exogenous PGE2 addition to the culture media. Moreover, significant increases have been found in the growth of epithelial cells cultured in the presence of keratocytes, with basal medium and with conditioning medium after adding exogenous PGE2 at concentrations equal to or lower than 10(-6) M. Significant decreases in the dimensions of the corneal epithelial cells have been found only when PGE2 has been added to basal and to conditioning medium, suggesting that the autacoid maintains cell dimension and morphology. The appearance of keratins with high molecular weight (54 and 57 kDa) coupled with the tendency to stratification of the cells cultivated with media supplemented with PGE2, indicates that the autacoid could favour cell differentiation. The action of PGE2 on the corneal epithelial cells does not seem to be influenced by the presence of the fibroblasts and their products, since PGE2 has induced increases in cell growth and morphological variations, independent of cultural conditions and therefore also only in the presence of basal medium.

Animals↗

PubMed1998

Bovine corneal stroma and epithelium reconstructed in vitro: characterisation and response to surfactants.

In order to define safety profiles and proper handling procedures for new industrial products, it is essential to determine their potential for ocular irritation. The Draize test is normally employed but it involves using rabbits. There is today a great need for all researchers to limit the use of animals for laboratory experiments and to encourage the development and adoption of alternative in vitro methods to evaluate the potential toxicity of new products. This study proposes a three-dimensional model of bovine corneal stroma and epithelium that is not only easy to reproduce but may also be used in the toxicological field as an alternative to animal experimentation. The data presented here show that this model allows the growth of epithelium similar in features to in vivo epithelium. Basal cells are cube-shaped, whereas superficial areas are horizontally longer; desmosomes and 64 kDa keratin, as a marker for differentiation of corneal epithelial cells, are both expressed; the basal lamina is synthesised also. The 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay was carried out on the model to evaluate the toxicity of some surfactants: benzalkonium chloride, Triton X-100, sodium dodecylsulphate and Tween 20. Since the in vitro data fit very well the results of the Draize test in vivo as reported in the literature, the three-dimensional culture may be used to predict the potential cytotoxicity of surfactants.

Animals↗

PubMed1998

Antiproliferative activity and phototoxicity of some methyl derivatives of 5-methoxypsoralen and 5-methoxyangelicin.

The in vitro antiproliferative activity and in vivo phototoxicity of some methyl derivatives of 5-methoxypsoralen and 5-methoxyangelicin, i.e. 4,4'-dimethyl-5-methoxyangelicin (compound I), 3,4'-dimethyl-5-methoxyangelicin (compound II), 4,4'-dimethyl-5-methoxypsoralen (compound III); and 3,4'-dimethyl-5-methoxypsoralen (compound IV), have been investigated. The effects of the compounds were evaluated in vitro on HL60 and A431 cells, using 5-methoxypsoralen as the reference compound. In both cell lines compound I, II and III showed better antiproliferative activity than compound IV and 5-methoxypsoralen. Scanning electron microscopy revealed that all the compounds induced the formation of blebs and blisters on a A431 cell surface. Significant variations in the nuclear area strictly related to the toxicity of the compounds have been shown in both cell lines. Skin irritancy in vivo was evaluated by mean of histopathological responses on guinea-pig skin. For each compound a damage index was determined by morphometrical analysis of empty spaces in the epidermis. Histopathology revealed skin phototoxicity of compounds which lacked erythemogenic activity by visual scoring. By coupling cytotoxicity data in vitro to skin sensitization ones in vivo, compound I proved a promising candidate for use in clinical trials since due to a high inhibitory effect on the growth of human cell lines coupled to low skin phototoxicity.

5-Methoxypsoralen↗

PubMed1997

Uptake and intracellular distribution of idarubicin in secondary cultures of normal and neoplastic urothelium.

This study analyzes the uptake and endocellular distribution of idarubicin (IDA) in normal and neoplastic urothelial secondary cultures in relation to the changes in concentration and time of exposure. The urothelial lines were isolated by Freshney's method from biopsy fragments taken from five patients with superficial bladder cancer. Pharmacological experiments were carried out on subcultures previously immunophenotypically characterized and did not exceed ten passages. The uptake and endocellular distribution of IDA was analyzed by densitometric image analysis on cells treated for 10, 20, 30 and 60 min and 2 h with scalar dosages from 10 ng/ml to 2430 ng/ml. Microscopic observations and densitometric analyzes revealed that in the cells treated with IDA, fluorescence was higher in the cytoplasm compared to the nucleus and increased with the change in dosage. Moreover, densitometric data showed that IDA uptake in the first 20 min was higher in the neoplastic cells, but after that period its behavior became heterogeneous at 30 and 60 min, while at 2 h there was an inversion of the trend. These results suggest that the in vitro cytotoxicity should be evaluated in order to verify whether the elevated uptake of IDA in the first 20 min of treatment is really correlated to a more elevated toxicity in the neoplastic cells with respect to the normal cells. This is presently under investigation.

Antibiotics, Antineoplastic↗

PubMed1997

Effects of DHT and EGF on human hyperplastic prostate cells cultured in vitro: growth, morphology and phenotype characterisation.

This work studies the effects of dihydrotestosterone (DHT) and epidermal growth factor (EGF) on the growth, morphology and phenotype characterisation of the U285 line obtained from human prostate hyperplastic tissue. Modifications of growth rate induced by these two substances have been evaluated by means of the neutral red assay formulated by Borenfreund and Puerner (1985) as well as by means of Kenacid blue assay described by Knox et al. (1986), culturing cells for 24, 48 and 72 hr with scalar doses of DHT (0.5, 1, 2, 5, 10 microM) and EGF (5, 10, 20, 100 ng/ml). An optical microscope connected to a computer aided system and a scanning electron microscope were used to monitor morphological changes induced by DHT and EGF. The immunophenotype characterisation of the treated and control cells was carried out by using a monoclonal antibody panel. Our results show that the expression of anti-cytokeratin 5+6+18, anti-cytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies varied in relation to the type of treatment undergone by the cells. Moreover, exogenous DHT does provoke a flattening of the U285 cells without modifying their rate of growth, while EGF both shortens the lag phase reactivating the quiescent cells and regulates the subsequent log growth phase, thus causing no cellular overgrowth.

Cell Division↗

PubMed1996

Morphological features and kinetics of primary cultures of BPH tissue supported by TV1 medium.

222 human prostatic biopsies were used to prepare cell cultures by means of a medium--colony formation permissive--containing fetal calf serum, called TV1. After 7, 14 and 21 days, the cultures were examined by optical and scanning electron microscopy. TV1 medium induces the formation and growth of two types of colonies, one mainly composed of epithelioid cells and distinguished by early growth; the second one made up exclusively of fibroblastoid cells which appear later in the culture. Epithelioid colonies, comprising three different cell types, appear to be arranged as a growth halo concentric to the bioptic fragment with a large central area, formed by a monolayer, and a pluristratified edge. Fibroblastoid cells weakly adhere to the substrate and form "satellite growth halos" separated from the primitive bioptic fragment. All the epithelioid cells were positive to cytokeratin LP34 Mab and negative to anti-smooth muscle-actin and anti-proline-4-hydroxylase antibodies. Fibroblastoid cells were only anti-proline-4-hydroxylase positive. The cell kinetics of epithelioid cells were also studied, revealing an extension of the S phase, in contrast to what happened with WAJC 404, and consequently a reduction of the percentage of cells entering mitosis. For this reason, the addition of fetal serum to the culture medium does not allow the use of prostate primary cultures for more than 14 days.

Actins↗

PubMed1996

Relationship between the proliferation of keratinocytes cultured in vitro and prostaglandin E2.

In the growth of keratinocytes "in vitro", PGE2 seems to play an important role. We have shown that in fibroblast-keratinocyte co-cultures, indomethacin, employed at concentrations which inhibit the PGE2 synthesis, reduced the proliferation of epidermal cells. This effect was reversed by an exogenous PGE2 addition to the culture media. To better understand the relationship between keratinocytes and the autacoid, we have tested PGE2 at various concentrations in different cultural conditions, that is, epidermal cells were grown on a 3T3-J2 feeder layer, without fibroblasts and with a 3T3-J2 conditioned medium. We observed an increase in keratinocyte proliferation induced by the autacoid alone in the presence of fibroblasts, while a severe inhibitory effect was relieved when dermal cells or the conditioning medium were absent. The lack of fibroblasts and their products in the culture medium modified the morphology of keratinocytes cultured in vitro. PGE2 induced significant morphological and morphometrical variations only if added to the conditioning medium. The autacoid decreased the expression of 66 kDa protein, if cells were grown in the presence of fibroblasts or with conditioning medium, whereas it completely inhibited this keratin and those of 60, 54 kDa if cells were cultured only with a basal medium. From morphometrical and electrophoretical data we can suppose that PGE2 inhibits cell differentiation. Thus PGE2 action on keratinocytes seems to be strictly related to the presence of dermal cells.

3T3 Cells↗

PubMed1996

Growth, morphology, morphometry and keratin patterns of bovine corneal epithelial cells cultured in vitro.

In this study, the effects of different culture systems on bovine corneal epithelial cells were analysed in order to better understand the influence of bovine keratocytes on epithelial cells. Growth, morphological, morphometrical analyses of cells and keratin patterns were evaluated. The aim was to improve the culture technique in order to obtain a good in vitro proliferation of these cells for their employment in clinical and toxicological situations. The bovine corneal epithelial cells were cultured under different conditions: on keratocyte or 3T3-J2 fibroblast feeder layers, with media conditioned either by the two feeder layers or with a basal medium. The epithelial cells cultured on a keratocyte feeder layer as compared to those grown under the other conditions, proved to have a higher growth rate as well as to be smaller in the cytoplasmic and nuclear area; moreover, after 21 days of culture they expressed 64-kDa keratin, designed as a marker for corneal epithelial cell differentiation. To sum up, the keratocyte feeder layer is the most effective for stimulating the growth and differentiation of corneal epithelial cells, resembling the in vivo situation. It might also be successfully employed for clinical and toxicological purposes.

3T3 Cells↗