Distribution Patterns of Involucrin in the Stratum Corneum of the Normal and Psoriatic Artificial Skins / 대한해부학회지

Cornified envelope is highly insoluble structure formed beneath the plasma membrane during terminal differentiation of keratinocytes and is stabilized by cross linking of various proteins, including involucrin, loricrin, and cornifin. Psoriasis is a chronic skin disease characterizing inflammatory reaction and hyperproliferation of keratinocyte. There are some differences in involucrin immunolabelling in stratum corneum between normal and psoriasis epidermis. Labelling was convergent to cornified envelope in psoriasis skin but throughout cytoplasm in normal skin. To compare terminal differentiation patterns of normal and psoriasis keratinocytes, we reconstructed normal and psoriatic artificial skin by using primary cultured keratinocytes from normal and psoriasis skin and then performed immunogold labelling for involucrin in stratum corneum. Psoriatic artificial skin had thin and poorly organized corneal layer. Immunogold labelling for involucrin revealed same pattern of that in vivo by showing throughout cytoplasm in lower layer but convergent cornified envelope in upper layer. Compared with psoriatic artificial skin, normal artificial skin had well organized and thick stratum corneum. Involucrin labelling was throughout cytoplasm in most of corneal layer but convergent to cornified envelope in some uppermost cells. Even though some cells show convergent pattern in normal artificial skin, absolute number of this pattern was no lesser than in artificial psoriatic skin because of normal artificial skin had thick stratum corneum. This result showed there was no difference in involucrin distribution in terminal differentiation of normal and psoriasis keratinocytes in organotypic culture model. It is concluded that although well organized multiple corneal layers are formed in normal artificial skin, they can not reach to full maturation of cornified envelope, and difference of involucrin localization in cornified envelope of psoriasis epidermis is related with not peculiarities of the cells but rapid growing in vivo.

Osteogenesis of Recombinant BMP-7 Adenovirus-transduced Human Dermal Fibroblasts in Collagen Sponge / 대한해부학회지

As a preceding study to apply recombinant BMP-7 gene to the human, we investigated bone formation in immunodeficient mice by using tissue engineering and gene transplatation. Human dermal fibroblasts were transduced with AdBMP-7 and cultured with type I collagen solution to form collagen sponge. The collagen sponge containing AdBMP-7 transduced fibroblasts was transplanted into hypodermis of the mice and osteogenesis in the spongy was investigated by histochemical, electronmicroscopic, and radiologic methods at 1, 2, 4, 6, and 8 weeks. At one week after transplantation, there were fluent cells infiltration around the collagen sponge and capsular structure was formed with fibers arranged in concentric circles. New vessel formation was observed in the capsule and subcapsular area of the sponge, but there were nucleus condensation and obscure cell boundary in the cells of the central region. Lacuna containing eosinophilic structures were observed in the capsular structure at two weeks. This structures were enlarged with time and were confirmed to be bone tissue by showing positive reaction for Von Kossa stain. Cartilaginous structure was not observed in light microscopic level, but a few chondroblasts were observed in pericapsular area in electron microscopic observation. After 6 weeks, radiopaque shadows were observed at the region of transplantation. Cortical bone was formed in periphery of the sponge while marrow like structure was observed in central region; some trabecula bone, adipocytes, and well developed vessels. The percentage of bone formation in transplanted sponge at 1, 2, 4, 6, and 8 weeks were 0, 63, 88, 100, and 100% (n = 8), respectively. From these results, bone formation by BMP-7 transduced human dermal fibroblasts using collagen sponge scaffolds in immunodeficient mouse shows another potential way of human gene transplantation using recombinant BMP-7 adenovirus.

Change in the Beating Rate and Intercellular Adhesion Molecule of Cardiomyocytes in Culture / 대한해부학회지

This experiment was designed for the elucidation of relationships between the cell adhesion molecules and synchronous beating rates of cardiomyocytes isolated from ventricles of 3-day-old rats. These cells were grown on the culture vessel coated or non-coated with cardiogel for 1, 3, 5, and 7 days. The synchronous beating rate and morphologic changes of cells were investigated under the inverted microscope. Those changes of cardiomyocytes were observed by transmission elcectron microscopy (TEM). Connexin43, pan-cadherin, and alpha-sarcomeric actin were stained by indirect immunofluorescent (IF) technique. Western blotting were used for identifying unique bands of connexin43 and pan-cadherin in the regions of specific size. The synchronous beating numbers of cardiomyocytes in each group on the dish coated or non-coated with cardiogel were significantly different for 3, 5, and 7 days in culture. The maximum values of synchronized beating in the cells appeared on the day 5. The beating numbers of cells grown on coated dish comparing with non-coated dish were significantly increased on the day 5 and day 7. The proliferation of cardiomyocytes increased markedly in the cardiogel-coated dish on the day 5, while the number of fibroblasts in non-coated dish were obviously increased on the day 7. In indirect IF studies, a normal redistribution of connexin43, pan-cadherin, and alpha-sarcomeric actin of the cells was expressed in day 3 throughout day 5. The reaction intensity of those proteins were increased or decreased in the proportion to the beating numbers. The lack of reaction was appeared in the fibroblasts consisting of monolayer on the day 7. Unique bands of connexin43 and pan-cadherin having a specific size were marked on Western Blotting. The structures such as gap junction, fascia adherence and desmosome compatible with intercellular adhesion in cardiac muscle were abundant on day 3 to 7. In conclusion, the amount or reaction intensity of connexin43 and pan-cadherin in cardiomyocytes were stronger simultaneously with the increase of synchronous beating numbers, particularly for 3 and 5 days. The maximum beating rates of cardiomyocytes reached on the day 5, while the maximum beating rates of them were markedly decreased owing to the proliferation of fibroblasts on day 7. TEM findings consisting of intercalated discs might be explained regardless of coating with cardiogel on why the close relationships between the beating rates and the connexin43 and pan-cadherin of those cells in culture exist.

Change in Expression of Keratin and Proto-oncogene Induced by Beta-propiolactone in HaCaT Cell / 대한해부학회지

To investigate the relationship between the morphologic changes and the expression of keratin and proto-oncogene induced by Beta-propiolactone (BPL), we assessed on the expression of keratins (K8, K10, K13) and proto-oncogenes (c-fos, c-jun, c-myc) in human HaCaT cell line. The cells were treated with 0, 0.1, 1 microgram/ml BPL for 2 or 6 hours. Morphologic studies revealed that BPL changed the cells into fibrocyte-shaped, caused highly lobulated nuclei and reduced desmosomes in their number. Findings of immunofluorescence and Northern blotting indicated that BPL consistently decrease expression of K10 representing a normal differentiation marker of keratinocytes, while increasing expression of K8 and K13 associated with a pathologic differentiation. This reagent also up-regulated expression of c-fos and c-jun, and down-regulated expression of c-myc. Together with staining for each keratin or proto-oncogene and DNA content in flow cytometry, BPL increased K8 expression dramatically at S-G2-M phase. The induction of c-Fos at S-G2-M phase appeared within 2 hours, and c-Jun gradually occurred. However, c-Myc was inhibited regardless of phases of cell cycle. In conclusion, these changes caused by BPL demonstrate a close relationship between the morphologic evidence and the altered expression of each keratin and proto-oncogene.

Effect of Paclitaxel on the beta-actin, Fibronectin, Laminin and Fine Structure in HeLa and L929 Cells / 대한해부학회지

The aim of this investigation was to elucidate the changes in the cytoplasmic distribution of beta-actin, fibronectin, and laminin through mainly the morphological changes occurring in HeLa and L929 cancer cell treated with paclitaxel. Whether or not paclitaxel regulates cell proliferation was assessed with MTT assay. Possible influence on the distribution of beta-actin, fibronectin, and laminin in these cells was confirmed with immunocytochemistry and analySIS Auto software program. The changes in cell morphology were observed under inverted and electron microscope. The MTT assay showed that 1 micrometer and 10 micrometer concentrations of paclitaxel inhibited HeLa cell growth approximately by 20% to 80% and L929 cell by 27% to 44% in a dose- and time-dependent manner. The distribution of these proteins was changed from the condensed around nucleus and strong reaction to the weak throughout cytoplasm. The morphological changes indicated that paclitaxel changes the location or number of protein synthesis apparatus: damages of RERs, Golgi complex, and increase of heterochromatin. These data suggest that the growth inhibition and morphological changes of tumor cells induced by paclitaxel might be modulated by the rearrangement and decreased production of beta-actin, fibronectin, and laminin in cytoplasm.

Effect of Lectin on Fine Structure and Extracellular Matrix Protein of Cancer Cell Lines / 대한해부학회지

Lectins are glycoproteins that bind specifically to carbohydrates. Considerable interests in the lectins were encouraged by several reports that certain members of the family bind to the extracellular matrix proteins (ECM), such as fibronectin and laminin. However, the relations between lectin and ECM protein remain unclear. To elucidate the relations of lectin-matrix-cell, we treated three cancer cell lines, HeLa, L929, and EATC with ConA and PHA-P at low dose (4 microgram/ml) and high dose (20 microgram/ml) for 1, 3, 5 days. 1. Whether or not lectins significantly regulate the cell proliferation was evaluated by MTT assays. 2. Whether the amount of fibronectin and laminin which of cancer cells can be influenced by lectins was confirmed by immunocytochemical staining. 3. Whether, in turn, the lectins which can change the morphology were observed under inverted and electron microscopes. ConA and PHA-P inhibited cell proliferation rate of all cell lines in a dose- and time- dependent manner. The amount of fibronectin and laminin considerably reduced in the three cell lines after the lectins treatment in a dose- and time-dependent manner. The cancer cell lines showed various morphological changes such as cell aggregation, irregular-shaped cellular processes, rounded cells, cytoplasmic vacuolation, swollen RERs, dilation of mitochondria, margination of chromatin and cell death. In conclusion, our results showed ConA and PHA-P caused damages of the three cancer cell lines, but the effect of PHA-P was much stronger than ConA. Taken together, the present data strongly indicate that ConA and PHA-P influence the cell proliferation rate, reduce the amount of fibronectin and laminin and induce cell injuries of HeLa, L929, and EATC cell lines. Our results also suggest that the cancer cell proliferation and the morphological changes might be modulated by the specific interaction between lectins and ECM proteins associated with the cell surface.

Effects of Nocodazole on Fine Structures in Co-cultured Vascular Endothelial Cells and Cancer Cell Lines / 대한해부학회지

Endothelial cells were isolated from the aortic intima of Sprague-Dawley species, rat. These cells and each cancer cell line (HeLa, Hep G2, A549, L929 and NIH/3T3 cells) were co-cultivated in alpha-MEM with 3 micrometer or 30 micrometer nocodazole. To investigate the influences induced by nocodazole, the morphological changes were observed under inverted microscope and transmission electron microscope, the amounts of fibronectin produced by vascular endothelial cells and cancer cell lines and the activities of nitric oxide synthetase synthesized mainly by endothelial cells were analyzed in the aspects associated with fine structural changes. The vascular endothelial cells of control group at the 1st, 2nd and 3rd days extended the cell processes, which contacted with cells from all cell lines investigated, but the endothelial cells of nocodazole-treated groups didn't possess the processes. All cell lines in nocodazole-treated groups had a large number of micronucleated cells, but endothelial cells didn't show micronuclei. Compared with control group, the endothelial cells of nocodazole-treated groups at the 1st, 2nd and 3rd days showed the decrease of amounts of fibronectin because of the increase of heterochromatin area. The amounts of fibronectin increased in all cell lines of nocodazole-treated groups at the 2nd and 3rd days whereas the nuclear folding or the dilatation/numerical increase of rough endoplasmic reticulum didn't appear. The activities of nitric oxide synthetase heightened in endothelial cells of nocodazole-treated groups, and therefore the considerable changes in fine structures such as vesicles, lysosomes, liposomes, pyknosis and cell lysis occurred even though the extent of changes differed among the cell lines. Taken together, the materials such as fibronectin or nitric oxide synthetase produced by endothelial cells directly or indirectly acted on cancer cells, and the amounts of fibronectin and the vesicles, lysosomes, liposomes and cell lysis seemed to be much more increased or enforced. Therefore, co-culture system seemed to work better for the investigation of actions of nocodazole and the role of endothelial cells in cancer cells research. Also, the co-culture system was closer to the in vivo state and more favorable in studies for proliferation or metastasis of cancer cells.

Effects of Melatonin on Fine Structures and Extracellular Matrix Proteins of Cancer Cell Lines / 대한해부학회지

Melatonin could be used as an anticancer agent to suppress the proliferation of tumor cells and induce the differentiation of cancer cells. HeLa, HepG2, A549, L929, and NIH/3T3 cell lines were cultivated in alpha-MEM with 0.2 mM/2 mM melatonin. The influences of melatonin on quantitative changes of glycoprotein, fibronectin, laminin and actin related to the metastasis of tumor cells investigated with PAS or PAP at light microscopic level. To elucidate the possibility of antitumor actions of melatonin, the changes of cell organelles were observed under transmission electron microscope. Cell proliferation was suppressed after treatment with 2 mM melatonin for 2 or 3 days. Compared with control groups, the amounts of glycoprotein, fibronectin, laminin and actin in all cell lines at 1st, 2nd and 3rd day after treatment with 0.2 mM and 2 mM melatonin were generally increased. Heterochromatin in the nucleus formed clumps in all cell lines at 2nd and 3rd day after treatment with 0.2 mM and 2 mM melatonin. The numerical increase of rough endoplasmic reticulum and golgi complex observed in HeLa and L929 cells treated with 0.2 mM and 2 mM melatonin at 1st, 2nd and 3rd day. The number of lysosomes increased in HeLa, A549, and L929 cells treated with 0.2 mM and 2 mM melatonin at 3rd day. The number of vesicles increased in all cell lines treated with 0.2 mM and 2 mM melatonin at 1st, 2nd and 3rd day. Taken together, antimitotic effect of melatonin can be expected at least 2day after treatment with 2 mM melatonin. The production of fibronectin and laminin in all cell lines treated with 0.2 mM or 2 mM melatonin increased. Therefore, the increase of amounts of extracellular matrix proteins in the extracellular space can be expected. And the increase of amounts of actin connected to the extracellular matrix proteins through the integrin of plasma membrane seemed to strengthen cell attachment. In order to metastasize of cancer cells, it is important for them to secrete various enzymes to pass through the extracellular matrix proteins. Hence, it will be more difficult for the cells to metastasize into other regions due to the increase of the extracellular matrix proteins. It was postulated that the clumps of heterochromatin and the numerical increase of vesicles induced by treatment with 0.2 mM and 2 mM melatonin could be represented for the actions of melatonin as morpholgical criteria.

The cell cycle of the cardiac endothelial cell in short-term culture / 대한해부학회지

No abstract available.

In vitro proliferation of keratinocytes / 대한해부학회지

No abstract available.

The isolation and preservation of nuclei using polyethylene glycol, concanavalin A, and phytohemagglutinin / 대한해부학회지

No abstract available.