Isolation of Endothelial Cells and Smooth Muscle Cells from Rat Aort / 영남의대학술지

BACKGROUND: Atherosclerosis has emerged as the leading cause of death in developed countries. At present, human umbilical vein endothelial cells (HUVEC) are most commonly used for the investigation of Endothelial cells (EC). However, HUVEC are not found in arteries but only in veins. Currently there are many reports on methods used to isolate EC;, most of these methods require special equipment to remove contaminating smooth muscle cells (SMC). MATERIALS AND METHODS: The method described here may be used to isolate not only ECs but also SMCs;,the approach presented here did not require special equipment. Rat aorta was treated with 2 mg/ml of type II collagenase solution for 45 minutes. The isolated cells from the aorta were incubated in medium G for a week;, only ECs could be separated. After the collagenase treatment, the rest of aorta was cut lengthwise, and left undisturbed to obtain SMCs in the culture dish for 10 days. To verify the purity of the isolated cells, we performed immunofluorescence and evaluated the results with transmission electron microscopy analysis. RESULTS: The immunofluorescence study demonstrated specific expression of CD31 and alpha-smooth muscle actin in the isolated ECs and SMCs, respectively. Cultured ECs and SMCs showed their own fine structure characteristics. CONCLUSION: These results suggest that this method for isolating ECs and SMCs may be especially useful for the study of atherosclerosis.

Isolation of Endothelial Cells and Smooth Muscle Cells from Rat Aort / 영남의대학술지

BACKGROUND: Atherosclerosis has emerged as the leading cause of death in developed countries. At present, human umbilical vein endothelial cells (HUVEC) are most commonly used for the investigation of Endothelial cells (EC). However, HUVEC are not found in arteries but only in veins. Currently there are many reports on methods used to isolate EC;, most of these methods require special equipment to remove contaminating smooth muscle cells (SMC). MATERIALS AND METHODS: The method described here may be used to isolate not only ECs but also SMCs;,the approach presented here did not require special equipment. Rat aorta was treated with 2 mg/ml of type II collagenase solution for 45 minutes. The isolated cells from the aorta were incubated in medium G for a week;, only ECs could be separated. After the collagenase treatment, the rest of aorta was cut lengthwise, and left undisturbed to obtain SMCs in the culture dish for 10 days. To verify the purity of the isolated cells, we performed immunofluorescence and evaluated the results with transmission electron microscopy analysis. RESULTS: The immunofluorescence study demonstrated specific expression of CD31 and alpha-smooth muscle actin in the isolated ECs and SMCs, respectively. Cultured ECs and SMCs showed their own fine structure characteristics. CONCLUSION: These results suggest that this method for isolating ECs and SMCs may be especially useful for the study of atherosclerosis.

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.

The Investigation of the Melanocytes in a Cultured Skin Equivalent Model / 대한해부학회지

Melanocytes grown in the pure monolayer culture lack the three-dimentional organization and the cellular interactions that exist in vivo. These can be partially overcome by growing melanocytes together with other epidermal cells in cultured skin equivalent models. In this study, skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in ratio 10 : 1 onto artificially constructed dermis. They were cultured in DMEM/F12 (1 : 1) for 4 days and then lifted to the air-liquid interface and maintained in DMEM/F12 (3 : 1) for 10 days. Histological and electronmicroscopic examinations of the cultured skin equivalants revealed a structure that closely resembled human interfollicular epidermis; 1. Melanocytes, were identified by positive staining with melanoma-specific antibodies (NKI/C3 and S-100 protein) and prominent cytoplasm with rich cell organelles, were located in the basal layer. 2. Melanocytes contained predominently early stage melanosomes and prominent Golgi apparatus. Mature melanins, were usually abundant in normal skin, were hardly seen both in melanocytes and in neighbouring keratinocytes. 3. Melanocytes were surrounded by keratinocytes but did not form intercellular junctions with them. 4. Keratinocytes were charaterized by microfilament bundles and intercellular junctions such as desmosomes and hemidesmosomes with neighbouring keratinocytes and artificial dermis. The melanocyte in the above skin equivalents had a strong resemblance to the one of normal human skin and therefore this model can be used as artificial skin for the transplantation and in the investigation of melanocytes' role to the UV stimuli.

Anti-tumor Effect of Green Tea Catechin on Cancer Cell Lines / 대한해부학회지

In this research, the author investigated antitumor effects of green tea catechin on cancer cell lines in various concentrations and durations. Additionally, antitumor mechanism of catechin associated with apoptosis and necrosis, especially their onset and duration were invesigated. Cancer cell lines, L1210 (lymphocytic leukemia), L929 (fibrosarcoma), HepG2 (hepatoblastoma) were used. In each group, intensity of morphological changes and cell damage was observed under inverted, light, confocal and electron microscopes, and MTT and flowcytometric analysis, gel electrophoresis were used to elucidate the effects of catechin after exposure to 1, 10, 100 and 500 microgram/ml catechin until 72 hours. In all cancer cell lines, catechin induced cellular injury and inhibition of proliferation in concentration- and duration-dependent manner. The effects of catechin were the strongestt in L1210 cells and L929 and HepG2 cells in order. Dual phenomena, of apoptosis and necrosis, were shown after catechin treatment. In necrotic cells, cellular swelling, cell organelles destruction, nuclear disintegration and random DNA fragmentation were observed. In apoptotic cells, apoptotic bodies, nuclear and cytoplasmic condensations, periodic DNA fragmentation were seen. In L1210 cells, necrotic and apoptotic cells were co-existed earlier, after exposure to catechin 100 microgram/ml and then apoptosis predom-inated later. In the same concentration, catechin induced apoptosis of L929 cells. but after exposure to 500 microgram/ml catechin, They were involved with apoptosis and necrosis simultaneously. On the other hand, HepG2 cells were damaged less than other cell lines but were involved with necrosis and inhibition of G2/M phase after treatment with 500 microgram/ml catechin. These results suggested that anti-tumor mechanism of catechin, associated with apoptosis, necrosis and cell cycle arrest, were quite different according to cancer type, concentration and duration of catechin treatment. Therefore, much more research would be essential for clinical application of catechin and this study would be the basic source for further study of green tea.

Effect of beta-amyloid Peptide on Fine Structure of Cardiac Myocytes in Culture / 대한해부학회지

Several predetermined concentrations of beta-amyloid peptide, (betaA) were administered to the rat cardiac myocyte cultures for three days to determine the effects of betaA. Stainings with congo red and crystal violet were used to evaluate the deposition of betaA in the cardiac myocytes and MTT assay was used to elucidate the cytotoxic effects of betaA by anlaysis of cell viability. Beating rates and morphological changes were investigated with inverted microscope and TEM was used to study the fine structures. Administration of 0.5 microgram/ml of betaA to cardiac myocytes induced the reduction of beating rate, however, it did neither affect the viability nor fine structures. No significant differences in cell viability or fine structures were noted in the experimental groups which were exposed to 5 microgram/ml or higher concentration of betaA. Deposition of betaA was confirmed in the cytoplasm of betaA treated cardiac myocytes with congo red and crystal violet amyloid stains. The viability of cardiac myocytes exposed to betaA was found to be reduced significantly (19%) compared to control cultures with the MTT assay. Cardiac myocytes treated with betaA presented a reduced cytoplasmic area that appeared very condensed under inverted microscope. Mitochondrial abnormalities in betaA treated cardiac myocytes included their significant enlargement, vacuolization, disorganization or paucity of cristae, paracrystalline inclusion, and accumulation of amorphous material in mitochondrial space. Mitochondrial abnormalities were present sometimes in betaA treated cardiac myocytes without disorganization of myofibils or degeneration of other cell organelles. To understand the mechanism involved in amyloid deposit and its role in pathogenesis of the diseases such as Alzheimer and inclusion body myositis (IBM), a need for in vitro model is imperative. This model of betaA treated cultured cardiac myocytes represent a amyloidosis model, and it offers several advantages for future studies of betaA to help elucidate the pathogenesis of amyloid diseases. For example, cardiac myocytes can be easily accessible, and since cardiac myocytes can be cultured for quite a long time, it is possible to study morphological and physiological changes consequent to amyloid deposits.

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.

The Characteristics of Proliferation and Differentiation of Psoriatic Keratinocytes in Culture / 대한피부과학회지

BACKGROUND: Psoriasis is a common, scaly erythematous disease of unknown etiology, marked by remissions and exacerbations of unpredictable onset and duration. Among many etiologic factors, psoriatic keratinocyte is found to play the most important role. OBJECTIVE: The purpose of this study is to evaluate the hypothesis that the mechanism(s) responsible for the abnormal proliferation of psoriatic keratinocytes may be located within the cell themselves. METHODS: Human epidermal keratinocytes were isolated from lesion(PL) and from uninvolved skin (PN) with chronic plaque-like psoriasis and from the normal skin(NN). Keratinocytes were passaged onto culture vessels without the feeder layer and maintained with serum free medium. Growth rates were measured in secondary cultures by MTT assay and ultrastructural findings of cell differentiation were evaluated with a transmission electron microscope. Results : 1 Keratinocytes from PL reached 50% confluency in one week compared to two weeks of PN and NN in primary cultures. 2. By the MTT assay, keratinocyte proliferation from PL showed a significantly faster rate than those from PN and NN(p0.05). 3. All of the three cell populations(PL, PN, NN) showed variable degrees of cell differentiation during secondary culturing in a serum-free medium. In the PL, however, small, compact basal cells were more prevalent than PN and NN. 4. When keratinocytes underwent differentiation by culturing in DMEM with serum, keratinocytes from PL formed more cell layers with incomplete formation of cornified envelopes suggests the presence of some unknown factors that induce or promote psoriasis. While keratinocytes from PN and NN were characterized by a complete codified layer as in normal skin. Conclusion : These results indicated that the characteristic hyperproliferation and the defective terminal differentiation of keratinocytes of PL were maintained throughout the culture period.

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.

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.

Expression Patterns of Cytokeratin and Involucrin in the Epidermis of the Artificial Skin Reconstructed by Cultured Cells / 체질인류학회지

To investigate differentiation and growth process of keratinocytes in organotypic cultured skin, we carried out immunohistochemical studies for cytokeratin (CK) 10, 14, 16, 17 and involucrin in the cultured skin. In normal skin CK14 and CK10 were detected in the basal and all suprabasal layer, respectively, whereas in artificial skin CK14 was detected up to the middle of spinous layer but CK10 expressed from the middle of spinous layer. The detection of involucrin in normal skin was from the upper spinous layer but found from lower spinous layer in the artificial skin. Both CK16 and CK17 did not expressed in in vivo skin but expressed weakly in the spinous layer of artificial skin. It is therefore concluded that the characteristics of basal cell were maintained in the several, lower layers of the sartificial skin. The growth and differentiation steps of the skin were similar to those of in vivo although differences were seen in the expression level.

Development of Psoriasis Model in Vitro / 대한해부학회지

Psoriasis is a disease caused by hyperproliferation of keratinocytes. Pathogenesis of psoriasis is still unclear, but many reports suggest that psoriatic keratinocytes themselves may have some factors of pathogenesis. The author developed artificial psoriatic skin by culturing keratinocytes of psoriasis skin over collagen lattice which was constructed with collagen and normal fibroblasts. After the keratinocytes had grown to full layers of stratification, expression patterns of differentiation marks and ultrastructural changes were investigated by immunohistochemistry and electron microscope. The results were very similar to those of psoriasis skin in vivo as follows. Cytokeratin (CK)10, marker of initiation of differentiation of keratinocytes, was expressed in the spinous layer. CK14, marker of basal cells of stratified squamous epithelium, was expressed in the basal and spinous layer. CK16 and CK17, markers of fast turnover of squamous epithelium, were expressed in the spinous layer. Involucrin, marker of terminal differentiation of squamous epithelium, was expressed weakely over the lower spinous layer. In immuno electron microscopical study, involucrin was expressed but confined to cornified cell envelops in the horney layer. Mitochondria, rER and ribosomes were abundant in the basal layer. They continued to appear in the upper spinous layer but intermediate filaments were scarce. Keratohyalin granules were visible in some parts of the granular layer zone, but the granules were smaller and fewer. In the horney layer, cells were thicker than normal and there were many lipid droplets within the cells. Intercellular spaces were enlarged at the basal layer but disappeared in the upper spinous layer. In these results, non systematic expression of differentiation markers and ultrastructural changes suggest that psoriasis is a disease caused by hyperproliferation of keratinocytes concurrent with unstable maturation and degeneration. Artificial psoriatic skin, in exclusion of systemic or dermal effects, showed very similar results with psoriasis skin in vitro. So it was concluded that psoriasis keratinocytes had some factors of pathogenesis and this kind of model on artificial psoriatic skin can be used for further studying of psoriasis.

Transition of Marker Enzymes of Rat Hepatocyte Organelles in Culture / 영남의대학술지

To investigate recovery, growth, and activity of hepatocyte in primary culture after cell separation, the authors followed up the marker enzyme activities of golgi complex, mitochondria and biologic membrane. Thiamine pyrophosphatase, the marker enzyme of golgi complex, activity approached the level of long term culture at 4th day. Succinate dehydrogenase, the marker enzyme of mitochondria, activity decreased with time, then it maintained constant level after 4th day. Alkaline phosphatase, the marker enzyme of biological membrane, activity increased from 3rd day, and after 5th day it showed strong reaction. These data suggested that hepatocytes were stabilized and recovered normal activity 4 day after cell separation. But the main secretory function was speculated to be reduced in culture.