Background/Aims: Inflammatory bowel disease (IBD) is an autoimmune disease characterized by chronic inflammation mainly in the large intestine. The interleukin-10 knockout (IL-10 KO) mouse is a well-known animal model of IBD that develops spontaneous intestinal inflammation resembling Crohn’s disease. Oxidative stress is considered to be the leading cause of cell and tissue damage. Reactive oxygen species (ROS) can cause direct cell injury and/or indirect cell injury by inducing the secretion of cytokines from damaged cells. This study evaluated the effects of mesenchymal stem cell (MSC) on the progression of IBD. Methods: In this study, human bone marrow-derived MSCs were injected into IL-10 KO mice (MSC). Oxidative stress and inflammation levels were evaluated in the large intestine and compared with those in control IL-10 KO mice (CON) and normal wild-type control mice (Wild). Results: The levels of ROS (superoxide and hydrogen peroxidase) and a secondary end-product of lipid peroxidation (malondialdehyde) were considerably higher in the CON, while superoxide dismutase and catalase levels were lower in the MSC. Inflammation-related marker (interferon-γ, tumor necrosis factor-α, IL-4, and CD8) expression and inflammatory histological changes were much less pronounced in MSC than in CON. Conclusions MSCs affect the redox balance, leading to the suppression of IBD.

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

BACKGROUND: This study was performed to compare the clinical outcomes of intestinal Behcet's disease with a simple ulcer. METHODS: We analyzed the medical records of 52 patients that were suspected as having intestinal Behcet's disease. Of these patients, 27 patients (Group 1) met both the criteria of the International Study Group for Behcet's Disease and the Behcet's Disease Research Committee of Japan. Thirteen patients (Group 2) met only the latter criteria and the other patients (Group 3) did not meet any criteria. The efficacy of medical treatment was assessed by the presence of gastrointestinal symptoms and follow-up colonoscopic findings. RESULTS: The mean age for patients with a diagnosis of an intestinal lesion was 38.6+/-12.2 years. The sex ratio was 1.08:1 (M:F) and the mean follow-up duration was 35.2+/-39.5 months. A single, smaller than 5 mm, round and shallow ulcer with an erythematous margin that was located at the leocecal area showed most typical colonoscopic features for intestinal Behcet's disease. No significant differences were found in the clinical manifestations and colonoscopic findings among the three groups of patients. Nineteen (44%) patients achieved complere remission from a sumptomatic point of view and 10 (39%) patients were proved to be complete remission according to follow up colonoscopy after medical treatment. Eleven patients (21.2%) underwent surgery. The overall cumulative rates of a first surgery and re-surgery were 40.5% and 71.9% at 10 years. No statistical relationship was found in the response of medical treatment and the cumulative rate of surgery among the groups. CONCLUSIONS: The clinical course and outcomes of an intestinal simple ulcer are not different from that for intestinal Behcet's disease.
Colonoscopy ; Diagnosis ; Follow-Up Studies ; Humans ; Japan ; Medical Records ; Sex Ratio ; Ulcer*

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

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*

To evaluate availability of the BMP-7 adenovirus (AdBMP-7) as a gene therapy for osteoinduction, we investigated in morphological aspect at 1, 2, 4, 6 weeks after cells injection. Primary cultured human dermal fibroblasts, transduced with AdBMP-7, were injected into gastrocnemius muscle of the nude mice. One week after fibroblasts transplantation new tissue was observed in the muscle. Majority of new tissue was evaluated as cartilage and calcification in the matrix was confirmed by Von Kossa stain as well as electron microscopy. Two weeks after transplantation, spongy bone was built up and adipocytes were observed in intertrabecular spaces. Osteoblasts and osteoclasts were observed in the bony tissue surface. In the result of Von Kossa-Van Gieson stain, osteolysis was dominant in bony trabeculae. Bone marrow was established in 4th weeks with intertrabecular space filled up by hematopoietic cells. At the 6th weeks, the number of trabeculae decreased and thickness of the cortical bone was increased. A great part of bone matrix has laminar structure which run paralleled to surface and which included osteocytes and canaliculi. These data demonstrate that cell mediated AdBMP-7 for gene therapy initiate development of cartilage and calcification of matrix within 1 week and complete bone and bone marrow formation within 4 weeks, so then, could be made practical application for promotion of osteoinduction.
Adenoviridae* ; Adipocytes ; Animals ; Bone Marrow ; Bone Matrix ; Bone Morphogenetic Protein 7 ; Cartilage ; Fibroblasts* ; Genes, vif ; Genetic Therapy ; Humans* ; Mice ; Mice, Nude ; Microscopy, Electron ; Muscle, Skeletal ; Osteoblasts ; Osteoclasts ; Osteocytes ; Osteolysis

Despite therapeutic advance, the prevalence of ischemic heart disease continues to increase. Recently, cell transplantation of stem cell has been proposed as a strategy for cardiac repair following myocardial damage. However, low differentiation efficiency into cardiomyocyte and poor cell viability associated with transplantation have limited the reparative capacity of these cell. In this study, we engineered P19 embryonal carcinoma cells using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction of P19 in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. An P19 embryonal carcinoma cell line expressing GFP, MEF2c, Nkx2.5 was generated by gene transfection and clonal selection. Nkx2.5 overexpression induced connexin43 expression level decrease. Electron microscopy revealed myofibril organization and immunostaining with cTnT showed positive staining in P19-Nkx2.5, consistent with early stage cardiomyocyte. Connexin43 and N-cadherin was expressed between P19-MEF2c and cardiomyocyte, P19- Nkx2.5 and cardiomyocyte in co-culture. And beating rate of cardiomyocyte co-cultured with P19-Nkx2.5 increased much more than other group, even if P19-Nkx2.5 did not have synchronous contraction with cardiomyocyte. Additionally, P19-Nkx2.5 had a resistance against hypoxia. These result suggest that overexpression of Nkx2.5 induced differentiation of P19 into cardiomyocyte and would be electro-mechanical coupling with cardiomyocyte after transplantation. Futhermore, Nkx2.5 overexpression had protection potential to hypoxic injury. Therefore, P19 cell overexpressed Nkx2.5 would be promising cell source for further study of new therapy of myocardial disease and building up in vitro model.
Anoxia ; Cadherins ; Cardiomyopathies ; Cardiomyoplasty* ; Cell Survival ; Cell Transplantation ; Coculture Techniques ; Connexin 43 ; Embryonal Carcinoma Stem Cells ; Intercellular Junctions ; Microscopy, Electron ; Myocardial Ischemia ; Myocytes, Cardiac ; Myofibrils ; Plasmids ; Prevalence ; Stem Cells ; Transcription Factors* ; Transfection ; Transplants

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.
Adenoviridae ; Adipocytes ; Animals ; Bone and Bones ; Bone Marrow ; Bone Morphogenetic Protein 7* ; Chondrocytes ; Collagen Type I ; Collagen* ; Eosinophils ; Fibroblasts* ; Humans* ; Mice ; Osteogenesis* ; Porifera* ; Subcutaneous Tissue ; Tissue Engineering

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