Mesenchymal Stem Cells Decrease Oxidative Stress in the Bowels of Interleukin-10 Knockout Mice

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.

Superficial course of the medial plantar nerve: case report / 대한해부학회지

The medial and lateral plantar nerves are branched from the tibial nerve and move to the tip of the toes. A variation of medial plantar nerve was found on the left side of a 78-year-old Korean male cadaver. The tibial nerve was divided into the lateral and medial plantar nerves beneath the plantar flexor. The medial plantar nerve passed deep to plantar aponeurosis and superficial to the flexor digitorum brevis. It gave off a common plantar digital nerve and then divided into three proper plantar digital nerves near the metatarsal bases. In this article, we report a superficial course of the medial plantar nerve and describe its unique morphology and discuss the clinical significance of this variation.

Alterations of Colonic Contractility in an Interleukin-10 Knockout Mouse Model of Inflammatory Bowel Disease

BACKGROUND/AIMS: Inflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10(-/-)) mice. METHODS: Prepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining. RESULTS: The spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10(-/-) mice compared to IL-10(+/+) mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10(-/-) mice compared to IL-10(+/+) mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10(-/-) mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10(-/-) mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased. CONCLUSIONS: These results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.

Effects of Human Adipose-Derived Stem Cells in Regenerating the Damaged Renal Tubular Epithelial Cells in an Animal Model of Cisplatin-Induced Acute Kidney Injury

BACKGROUND: We conducted this experimental study to examine whether human adipose-derived stem cells (ADSCs) are effective in achieving a recovery of damaged renal tubular epithelial cells in an animal model of cisplatin-induced acute kidney injury using rats. METHODS: To examine the in vitro effects of ADSCs in improving nephrotoxicity, we treated mouse renal tubular epithelial cells with both ADSCs and cisplatin mouse renal tubular epithelial cells. And we equally divided 30 male white Sprague-Dawley (SD) rats into the three groups: the control group (intraperitoneal injection of a sterile saline), the cisplatin group (intraperitoneal injection of cisplatin) and the ADSC group (intraperitoneal injection of cisplatin and the hADSC via the caudal vein). At five days after the treatment with cisplatin, serum levels of blood urine nitrogen (BUN) and creatinine were measured from each SD rat. We performed histopathologic examinations of tissue samples obtained from the kidney. RESULTS: The degree of the expression of TNF-alpha and that of Bcl-2 were significantly higher and lower respectively, in cisplatin group (P<0.05). Serum levels of BUN (P=0.027) and creatinine (P=0.02) were significantly higher in cisplatin group. On histopathologic examinations, there was a significant difference in the ratio of the renal injury between cisplatin group and ADSC group (P=0.002). CONCLUSION: The ADSCs might have a beneficial effect in regenerating the damaged renal tubular epithelial cells.

Age-related NADPH Oxidase (arNOX) Activity Correlated with Cartilage Degradation and Bony Changes in Age-related Osteoarthritis

The purpose of this study was to investigate the age-related NADPH oxidase (arNOX) activity in patients with age-related knee osteoarthritis (OA). Serum and cartilage arNOX activities were determined using an oxidized ferricytochrome C reduction assay. Full-thickness knee joint cartilages obtained through total knee replacement surgery were graded according to the Outerbridge (OB) classification. Radiographic severity of OA was determined on Knee X-rays according to the Kellgren-Lawrence (K/L) grading system. Cartilage beta-galactosidase, HIF-1alpha, and GLUT-1 expression levels were evaluated as markers for tissue senescence, hypoxia, and glycolysis. Higher arNOX activities occurred with higher levels of cartilage beta-galactosidase, HIF-1alpha, and GLUT-1 (P = 0.002). arNOX activity in cartilages with surface defects (OB grade II, III) was higher than in those without the defects (OB grade 0, I) (P = 0.012). Cartilage arNOX activity showed a positive correlation with serum arNOX activity (r = -0.577, P = 0.023). Serum arNOX activity was significantly higher in the OA subgroup with bilateral ROA than in the OA with no or unilateral ROA (2.449 +/- 0.81, 2.022 +/- 0.251 nM/mL, respectively, P = 0.019). The results of this study demonstrate that OA itself is not a cause to increase arNOX activities, however, arNOX hyperactivity is related to a high degree of cartilage degradation, and a high grade and extent of ROA in age-related OA.

Stem Cells in Inflammatory Bowel Disease: New Potential Therapeutic Target

Understanding of the pathophysiology of inflammatory bowel disease (IBD) is constantly evolving and, recently, a number of biologic agents that selectively target specific molecules or pathways to correct the imbalance of the gut immune system have been developed. Among them, an antibody to tumor necrosis factor (anti-TNF) is the first developed drug which has dramatically improved the management of patients with IBD. However, more than one-third of IBD patients do not respond to medications, and there is the problem of antibody formation. Therefore, enormous efforts have been made into the development of novel anti-cytokines and stem cell injection as an alternative to has been made. However, the efficacy and safety of stem cell treatment are under investigation. Some studies have reported very promising data; however, others have shown conflicting results. In addition, most trials involved a very small number of subjects and did not compare stem cell treatment with anti-TNF. The present paper reviews the function and therapeutic mechanism of stem cells for the treatment of IBD.

Individual Variation in Growth Factor Concentrations in Platelet-rich Plasma and Its Influence on Human Mesenchymal Stem Cells / 대한진단검사의학회지

BACKGROUND: The objective of this study was to explore whether individual variations in the concentration of growth factors (GFs) influence the biologic effects of platelet-rich plasma (PRP) on human mesenchymal stem cells (HMSCs). METHODS: The concentrations of 7 representative GFs in activated PRP (aPRP) were measured using ELISA. The effects of PRP on the proliferation and alkaline phosphatase (ALP) activity of HMSCs were examined using several concentrations of aPRP from 3 donors; the relationships between the GF levels and these biologic effects were then evaluated using 10% aPRP from 5 subgroups derived from 39 total donors. HMSCs were cultured in DMEM with the addition of aPRP for 4 or 12 days; then, DNA content and ALP activity were measured. RESULTS: The quantity of DNA increased significantly at a 10% concentration of aPRP, but the ALP activity was suppressed at this concentration of aPRP. The GF concentrations varied among donors, and 5 subgroups of characteristic GF release patterns were identified via cluster analysis. DNA levels differed significantly between groups and tended to be higher in groups with higher concentrations of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factors (PDGFs). DNA quantity was positively correlated with TGF-beta1 concentration, and was negatively correlated with donor age. ALP activity was negatively correlated with PDGF-BB concentration. CONCLUSIONS: The varying GF concentrations may result in different biologic effects; thus, individual differences in GF levels should be considered for reliable interpretation of the biologic functions and standardized application of PRP.

Adult Mesenchymal Stem Cells for Cell Therapy in Clinical Application / 영남의대학술지

Human bone marrow-derived mesenchymal stem cells (MSCs) are a rare population of undifferentiated cells that have the capacity of self renewal and the ability to differentiate into mesodermal phenotypes, including osteocytes, chondrocytes, and adipocytes in vitro. Recently, MSCs have been shown to reside within the connective tissue of most organs, and their surface phenotype has been well analyzed. Many reports showed that transplanted MSCs enhanced regeneration as well as functional improvement of damaged organs and tissues. The wide differentiation plasticity of MSCs was expected to contribute to their demonstrated efficacy in a wide variety of experimental animal models and in human clinical trials. However, new findings suggest that the ability of MSCs to alter the tissue microenvironment via secretion of soluble factors may contribute more significantly than their capacity for differentiation in tissue repair. This review describes what is known about the cellular characteristics and differentiation potential of MSCs, which represent a promising stem cell population for further applications in regenerative medicine.

Gene Expression Changes of Drug-resistant Saos-2 Cells Induced by Anticancer Drug / 대한해부학회지

During the treatment of cancers, especially with anticancer drugs, the recurrence of cancer is the most important factor for survival rate. The most common cause of the recurrence is the resistance of cells to anticancer drugs. To explore and analyze the changes of gene expression during the induction of resistance by anticancer drugs in human osteogenic cancer cell line Saos-2. The drug resistance was induced with adriamycin, cisplatin or vincristine at 10(-7) M concentration of each and cDNA microarray was performed. Total RNA was purified from Saos-2, adriamycin-resistant (Saos-2AdR), cisplatin-resistant (Saos-2CpR) and vincristine-resistant (Saos-2VcR) and expressed genes were investigated with a Affymetrix Human HG-U133Plus2.0 GeneChip(TM). The genes of anticancer drug resistant cells that showed more than 2.5 fold expression change than Saos-2 were selected for differential expression. Four hundred seventeen genes were selected for Saos-2 vs Saos-2AdR. Two thousand five hundred thirty six genes were selected for Saos-2 vs Saos-2CpR. Two hundred twenty five genes were selected for Saos-2 vs Saos-2VcR. Eighty seven genes were selected for common differential expression. The results showed that many genes were changed in expression during the acquiring of resistance to anticancer drugs but most of genes were not in common among the three anticancer durg-resistant Saos-2. This means the different anticancer drug takes the different mechanism for acquiring resistance to anticancer drug even we use same cells.

Effects of Dexamethasone Withdrawal on Proliferation and Differentiation of Mesenchymal Stem Cells / 대한해부학회지

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.

Effect of Propofol on Kupffer Cell Superoxide Dismutase Activities and Cytoprotections during Hypoxia-Reoxygenation / 대한해부학회지

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.

The Effects of Keratinocyte-derived Factors Involved in Transforming the Morphologies of Melanocyte and in Translocating Melanosomes to Keratinocyte / 대한해부학회지

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.

Effects of Dexamethasone on Proliferation of Mesenchymal Stem Cells in Long Term Culture / 체질인류학회지

Homeostasis of the body is maintained by balance of cell renewal, differentiation, and death. Dexamethasone has been used long time in field of MSCs research as differentiation inducer while, in contrast, there has been little work on the effect of dexamethasone on apoptosis and proliferation of MSCs. To determine the effect of dexamethasone on apoptosis and proliferation, MSCs were cultivated with (Dex) or without (Con) dexamethasone for 3 weeks. During this period weekly cell count, DNA assay, mitosis, apoptosis as well as alkaline phosphatase assay observation were recorded. DNA and cell number of Dex group was lower than Con group in early period but exceed at 3th week. There is no significant difference in mitosis between both groups whereas apoptosis frequency in Dex group was lower than that of in Con group. These results indicate that dexamethasone influences cell proliferation of MSCs in long term confluent culture by suppression of apoptosis.

Fenretinide Induced Apoptosis in Human Neuroblastoma Cell Lines / 체질인류학회지

Retinoids play an important role in growth, reproduction and differentiation. Recently, retinoids have been used to both protect and treat from various animal models of carcinogenesis. In this study the effect of N-(4-hydroxyphenyl) retinamide (fenretinide) on viability of human neuroblastoma cell lines were evaluated. For the evaluation of apoptosis of human neuroblastoma cell lines by fenretinide. MTT assay, cytoplasmic DNA fragmentation, TUNEL stain, and Western blot analysis were performed. In MTT assay, fenretinide inhibited the proliferation of CHP134, IMR32 and SH-SY5Y but not in PC12 cells. Cytoplasmic DNA fragmentation was induced by treament of fenretinide (10 micrometer) for 48 h in IMR32 cells. PARP cleavage was detected by Western blot analysis after 16 h of treatment of fenretinide in CHP134, IMR32 and SH-SY5Y. These fenretinide effects on growth inhibition and increased apoptosis followed to the time dependent manner. The fenretinide treatment did not affect the phosphorylation of MAP kinases (ERK, JNK, p38). There was no change of Bcl-x and Bad expression after treatment of fenretinide (1 micrometer) in neroblastoma cell lines. Pretreatement of PD98059, SB203580, LY294002, or genistein also did not affect fenretinide-induced PARP cleavage in neuroblastoma cell lines. From these results, the fenretinide-induced apoptosis is due to the PARP cleavage which occured MAP kinase signal cascades independently.

Morphological Study for Osteoinduction by Human Fibroblasts Transduced with rhBMP-7 Adenovirus / 대한해부학회지

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.

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.

Numerical Changes of Melanocytes in Artificial Epidermis Reconstructed by Keratinocytes and Melanocytes Coculture / 대한해부학회지

To follow up the change of melanocytes to keratinocytes ratio in stratified epidermis reconstructed by mixed culture of these cells, the author investigated the ratio of melanocyte to keratinocyte in the basal layer of reconstructed epidermis after induction of stratification for 1 week with cocultured melanocyte and keratinocyte in various ratio. Initial mixing ratios of melanocyte to keratinocyte were 1 : 10, 1 : 20, 1 : 40, 1 : 80. Post ratios were changed to 1 : 6.7, 1 : 10.7, 1 : 12.6, 1 : 13.8 respectively after 1 week. Eight time of initial gap reduced to two times and melanocyte to keratinocyte ratio converged to 1 : 10. To know the regulation mechanism, the author investigated the ratio of basal melanocytes to suprabasal melanotytes. The ratios were nearly same by showing 0.142, 0.140, 0.118, 0.104 respectively. These results suggest that the ratio of melanocyte to keratinocyte in reconstructed stratified epidermis in vitro was not related with initial mixing ratio and was regulated by proliferation of melanocytes controled by corresponding keratinocytes.

Role of Cardiac Transcription Factor Nkx2.5 on Cardiomyoplasty Model in vitro / 대한해부학회지

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.

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.