The mechanism of the disease such as artherosclerosis is easily elucidated by the comparison among cells isolated from each aorta of knockout mouse and wild type mouse, respectively. This study was aimed at effectively harvesting the endothelial and smooth muscle cells from 4~6 weeks old wild type C57BL/6J mouse aorta. The tunica adventitia was completely removed to get the aortic tissues only consisting of the tunica intima and the tunica media under the stereoscope. These aortic tissues were treated with type I collagenase or type II collagenase solution, respectively, and then the endothelial or smooth muscle cell was isolated. CD31 marker of the endothelial cell and alphasmooth muscle actin marker of the smooth muscle cell were identified with confocal microscope. The percentages of the labelled cells by each marker represented the extent of purification of endothelial or smooth muscle cells, respectively, for harvested cells according to the collagenase solutions. 70~80% of culture vessel was covered with the endothelial cells 10 days after the treatment of the type I collagenase solution, while 40~50% of culture vessel covering with the cells after the treatment of the type II collagenase solution. 70~80% of culture vessel was covered with the smooth muscle cell regardless of the type of the collagenase solution on the 13th day. Percentages of the CD31 positive cells after the treatment with the type I or the type II collagenase solution was 91.1+/-.865%** and 86.4+/-.641%, respectively (**p <0.05, n=5). Percentages of the alphasmooth muscle actin labelled cells after the treatment with the type I or the type II collagenase solution were 87.9+/-.713% and 86.6+/-.778%, respectively, and these values were not significantly different. Taken together, the aortic tissues using the type I collagenase solution comparing with using the type II collagenase solution were much more effective in the isolation of the endothelial cells
Actins ; Adventitia ; Animals ; Aorta ; Collagenases ; Endothelial Cells ; Glycosaminoglycans ; Mice ; Mice, Knockout ; Muscle, Smooth ; Muscles ; Myocytes, Smooth Muscle ; Tunica Intima ; Tunica Media

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.
Actins* ; Cell Proliferation ; Cytoplasm ; Fibronectins* ; Golgi Apparatus ; HeLa Cells ; Heterochromatin ; Humans ; Immunohistochemistry ; Laminin* ; Paclitaxel*

The aims of this study were to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells, and were to develop in vitro system which could provide a tool for the study of ischemia-reperfusion injury. Kupffer cells were isolated following sequential collagenase digestion of the liver by perfusion and enrichment of a nonparenchymal cell fraction by a double-densities gradient centrifugation step using Percoll and were selected by allowing them to adhere to culture vessel for 2 h at 37 degrees C under 5% CO2. The purity of obtained Kupffer cell was about 90% assessed by the phagocytosis of 3 micrometer latex beads. This method for Kupffer cell isolation resulted in yields of 1~5 x10(7) Kupffer cells per liver and Kupffer cells were preserved in maintenance cultures for 10 days. The phagocytic capacity of cultured Kupffer cells was measured according to the amount of latex beads incorporated into the cytoplasm. Larger round Kupffer cells in the culture had higher phagocytic capacity compared with smaller round or irregular shaped Kupffer cells. The different phagocytic capacity of Kupffer cells which was dependent on size and shape in vivo was well preserved during culture. The experimental group of Kupffer cells in culture were sequentially treated with ischemia and reperfusion at 1h and 30 min. The ratio of Kupffer cells having latex beads in their cytoplasm was significantly increased compared with control (p<0.01). This result was able to explain the Kupffer cells' activation after ischemia-reperfusion injury in vivo. In conclusion, Kupffer cells in this culture well resembled the cells in vivo and this in vitro model could provide a valuable tool for the study of Kupffer cells with a key role in pathophysiology of ischemia-reperfusion injury.
Animals ; Cell Separation ; Centrifugation ; Collagenases ; Cytoplasm ; Digestion ; Ischemia ; Kupffer Cells* ; Liver ; Microspheres ; Perfusion ; Phagocytosis ; Rats ; Reperfusion ; Reperfusion Injury*

PURPOSE: To determine whether the analysis of abnormally high signal intensities in ischemic tissue, as revealed by diffusion-weighted MR imaging (DWI) can be used to evaluate reversible brain lesions in a cat model of acute ischemia. MATERIALS AND METHODS: Ten cats were divided into two groups of five (Group I and Group II), and in all animals the middle cerebral artery was temporarily occluded. Group I underwent T2-DWI 30 minutes after occlusion, and Group II 120 minutes after occlusion. In both groups, DWI was performed one hour and 24 hours after reperfusion (at one hour, non-T2-weighted; at 24 hours, T2-weighted). Both occlusion and reperfusion were monitored by 99m TC-ECD brain perfusion SPECT. All animals were sacrificed 24 hours later and their brain tissue was stained with TTC. Signal intensity ratios (SIR, signifying average signal intensity within the region of interest divided by that in the contralateral, nonischemic, homologous region) of the two groups, as seen on DWI were compared. The percentage of hemispheric lesions occurring in the two groups was also compared. RESULTS: SIR after occlusion of the middle cerebral artery was 1.29 in Group I and 1.59 in Group II. Twenty-four hours after reperfusion, SIR in Group I was higher than in Group II (p<0.01). After occlusion and reperfusion, the percentage of hemispheric lesions in Group I was less than in Group II. For the latter, the percentage of these lesions revealed by TTC staining and T2-weighted imaging was 48% and 59%, respectively, findings distinctly different from those for Group I. In addition, in group I, infarction was revealed by neither TTC staining nor T2-weighted imaging (p<0.01). CONCLUSION: The use of DWI to evaluate signal intensity ratios can help determine whether or not brain injury after temporary cerebral ischemia is reversible.
Animals* ; Brain Infarction* ; Brain Injuries* ; Brain Ischemia ; Brain* ; Cats ; Infarction ; Ischemia ; Magnetic Resonance Imaging* ; Middle Cerebral Artery ; Models, Animal* ; Perfusion ; Reperfusion ; Tomography, Emission-Computed, Single-Photon

Keratinocyte-derived factors are involved in regulating the proliferation, differentiation or melanogenesis of melanocytes. To investigate the effects of keratinocyte-derived factors on the skin pigmentation, human melanocytes and keratinocytes were cultivated in the forms of pure melanocyte or keratinocyte culture system, co-culture system (melanocytes and keratinocytes were cultivated together in a vessel but they were separated with semipermeable membrane) or mixed culture system (melanocytes and keratinocytes were cultivated together in a vessel in mixed form). The authors studied the cellular features (dendritogenesis and area) and the tyrosinase activities and observed the electron microscopic structures for melanosome transfer. Melanocyte in co-culture system increased in the area (p.0.05) but not in the dendritogenesis compared with the melanocyte in pure culture system. The tyrosinase activities of co-culture system on the 2nd and 4th day revealed higher compared with the ones of the pure and mixed culture system (p.0.05). In the co-culture system, the tyrosinase activities were gradually decreased with the lapse of time and vice versa in the pure and mixed culture system. On the 6th day culture, all of the three melanocyte culture systems showed the same tyrosinase activities. In spite of high tyrosinase activities in the medium, there were no tyrosinase activities in keratinocytes with the co-culture system. In transmission electron microscopic findings, there were scant of melanosomes in keratinocytes with the co-culture and mixed culture systems. In conclusion, keratinocyte-derived factors modulate the activities and melanogenesis of melanocytes but there were no effects on melanosome transfer to keratinocytes.
Coculture Techniques ; Humans ; Keratinocytes* ; Melanins ; Melanocytes* ; Melanosomes* ; Monophenol Monooxygenase ; Skin Pigmentation

The aims of this study were to verify the hypoxia-reoxygenation injury of primary cultured Kupffer cells and the effect of propofol against the hypoxia-reoxygenation injury through quantitating lactate dehydrogenase (LDH) release and superoxide dismutase (SOD) activity.The sequential treatments with hypoxia and reoxygenation induced significant increasement of LDH release (P.0.01) and decresement of SOD activity(P.0.05) in primary cultured Kupffer cell. The level of LDH release and SOD activity after sequential treatments with hypoxia and reoxygenation were restored to the control level by the propofol treatment in the concentration of 0.5 and 5 microgram/mL. Propofol in concentration of 50 microgram/mL induced significant increasement of LDH release (P.0.01) on both normal culture and hypoxia-reoxygenation culture of the Kupffer cell. As hypoxia and reoxygenation procedures and propofol treatment were concurrently added to the cultured Kupffer cell, propofol treatment in the concentration of 50 microgram/mL decreased significantly the SOD activity (P.0.01). In conclusion, propofol in this hypoxia-reoxygenation model could provide a valuable clue for the study of liver transplantation and of propofol.
Anoxia ; Kupffer Cells ; L-Lactate Dehydrogenase ; Liver Transplantation ; Propofol* ; Superoxide Dismutase* ; Superoxides*

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.
Actins ; Animals ; Blotting, Western ; Cell Adhesion Molecules ; Connexin 43 ; Desmosomes ; Fascia ; Fibroblasts ; Gap Junctions ; Microscopy ; Myocardium ; Myocytes, Cardiac* ; Rats

To evaluate the maturation and differentiation state of cultured keratinocytes, the author investigated expression of differentiation markers in cultured keratinocytes. The specimens were divided into three experimental groups, 3rd passage keratinocytes cultured in serum free media (3rd SFM group), 6th passage keratinocytes cultured in serum free media (6th SFM group) and 3rd passage keratinocytes cultured in DMEM (DMEM group). CK14, marker of basal layer, expressed in all groups. The expression was localized and condensed in the SFM groups but spreade in the DMEM group. Most of the cells in both SFM groups were positive but a few cells in DMEM group were also positive. CK10, marker of initiation of differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. Involucrin, marker of terminal differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. CK16 and 17, markers of fast turnover of keratinocytes, were not expressed in SFM groups. Weak positive reactions were observed in DMEM group. With these results the authors concluded that the keratinocytes from 3rd passage to 6th passage, cultured in serum free media with calcium less than 0.1 mM, had highly homogeneous basal cell characteristics.
Antigens, Differentiation ; Calcium ; Culture Media, Serum-Free ; Humans* ; Keratinocytes* ; Keratins*

With potential of differentiation into many different lineages, mesenchymal stem cells have been candidate on cell therapy for recovery of injured body. Dexamethasone plays important role in mesenchymal stem cells differentiation and can derived into osteoblast, chondrocytes, adipocytes, and fibroblasts in vitro. There has been many studies on effect of dexamethasone for differentiation of MSCs with continuous exposure, but little work on the effect for deprivation during this progress. This result will be an important guild line for evaluation of transplanted MSCs after pretreatment with dexamethasone. In this study, dexamethasone was deprived by weekly withdrawal schedule in the process of differentiation induction by dexamethasone. During this period, expression of APase was evaluated as mark of osteoblast differentiation and number of BrdU incorporated cells were counted as index of proliferation. APase level of one or two week exposure groups decreased immediately after deprivation of dexamethasone and approached to control level at 4~5 week but three or four week exposure groups reached peak level at 3th week then decreased but still remained higher level than other groups. Dexamethasone exposure groups showed the trend of decreased in mitotic activity compared to control, but there were significant increase in mitosis after deprivation of dexamethasone. This pattern prominent in 6, 9, 12, 15 day exposure groups. These results showed that the effect of dexamethasone derived MSCs differentiation into osteoblasts is faint without full enough exposure and the period should be more than three weeks.
Adipocytes ; Appointments and Schedules ; Bromodeoxyuridine ; Cell- and Tissue-Based Therapy ; Chondrocytes ; Dexamethasone* ; Fibroblasts ; Mesenchymal Stromal Cells* ; Mitosis ; Osteoblasts

Recently, new treatments for human heart disease such as ischemia, infarction, cardiomyopathy, coronary heart disease have been developed. transplantation various kinds of cells from skeletal muscle, endothelium, mesenchyme, hemopoietic tissue to injured area after infarction were challenged. It's so called 'Cell Transplantation'. This therapeutic strategy already adopted and got a good result in clinical trial. But several limitations are still remained, including ethics, donor cell numbers, side effects, therapeutic efficiency. In this research, we investigated the formation of intercellular junction and synchronous contraction between cardiomyocyte and H9c2 cell line in co-culture to establish experimental model in vitro for cell transplantation. For this purpose, two kinds of cells, primary cultured cardiomyocyte and H9c2 (cardiomyoblast cell line) were used. Cultured cardiomyocytes had repetitive contraction-relaxation pattern along longitudinal axis both in single and coculture. But their contractions were slower, less regular, less strong in co-culture than in cardiomyocyte culture only. H9c2 cells did not contracted actively themselves, but moved toward cardiomyocyte passively coincided with contraction. In contact region between two kinds of cells, there was no signal after immunocytochemical staining labeled with connexin43 (gap junction), desmoplakin (desmosome), N-cadherin (adherent junction) even though they had membrane contact. Moreover, F-actin and striation were less developed. These results suggested that co-culture system interfere with remodelling of contractile apparatus, intercellular junction formation as well as contraction-relaxation. Furthermore cardiomyocyte could not induce H9c2 cells differentiation into cardiomyocyte. Therefore, much more research would be essential for clinical application of cell transplantation and this study would be the basic source for further study of new therapy of myocardial disease and building up in vitro model.
Actins ; Axis, Cervical Vertebra ; Cadherins ; Cardiomyopathies ; Cell Count ; Cell Line* ; Cell Transplantation ; Coculture Techniques* ; Connexin 43 ; Coronary Disease ; Desmoplakins ; Endothelium ; Ethics ; Heart Diseases ; Humans ; Infarction ; Intercellular Junctions* ; Ischemia ; Membranes ; Mesoderm ; Models, Theoretical ; Muscle, Skeletal ; Myocytes, Cardiac* ; Tissue Donors ; Transplants