• Journal of Korean Neurosurgical Society
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• v.57 no.5
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• pp.329-334
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• 2015

Objective : To comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue. Methods : Degenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins ${\alpha}v$, ${\beta}1$, and ${\beta}3$ was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR). Results : Immunohistochemical staining showed that vimentin and integrin ${\alpha}v$ was broadly expressed in all tissues examined. By contrast, integrin ${\beta}1$ was weakly expressed only in 38.4% (5/13) of tissues. Integrin ${\beta}3$ was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin ${\alpha}v$ in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin ${\beta}1$ was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin ${\beta}3$ was variable. Conclusion : Spinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin ${\alpha}v$ and ${\beta}1$ in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.277-286
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• 2001

Improvement and possible commercialization of a home-made electroporation apparatus(home-made) were further tried to establish a simple and effective introduction of foreign gene into sperm followed by in vitro fertilization. Expressions of introduced pJJ9 and pNT plasmids were shown in all fertilized eggs with electroporated spermatozoa. In particular, with this gene transfer system all the fry showed a consistently transient expression in the syncytium of the yolk sac. This fact is important since some required, minute quantity of human proteins can be produced from the established transient expression on the yolk sac of all fry derived from in vitro fertilization with electroporated spermatozoa. To explore tissue-specific expression in fish, which we will use a similar system later, we targeted the nerve tissue to see whether tissue-specific promoter is working in fish properly. pNT plasmid containing a nerve cell-specific tubulin promoter gene demonstrated consistently exact targeted expressions among the developing nerve cells in later stages of embryos and hatched fry. Finally, liver-specific genes are now being cloned by using already selected primers for useful human protein gene fusion.

• Archives of Plastic Surgery
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• v.35 no.1
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• pp.1-6
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• 2008

Purpose: In tissue engineering, it is important that the scaffolds have high affinity with cells for making efficient use of cells. The authors studied the binding affinity of human adipose stem cells(ASCs) to micronized acellular dermal matrix(alloderm) using biotin and avidin linkages.Methods: Human ASCs were harvested from adipose tissue obtained by abdominoplasty. ASCs($1{\times}10^4$, $5{\times}10^4$, $1{\times}10^5$, $5{\times}10^5$, $1{\times}10^6$, $5{\times}10^6$ cells) were attached to micronized alloderm(1mg) in three groups; 1) control group in which no ASCs and alloderm was treated; 2) serum group in which alloderm was exposed to fetal bovine serum; and 3) biotin group in which biotinylated cells were attached to biotinylated alloderm. The binding affinities were determined 1 day after making ASC-alloderm complexes. The proliferation rates were determined by XTT assays in 4, 7, 14, and 21 days and scanning electron microscopic examination was performed in 7 and 21 days after culture of ASC-alloderm complexes.Results: The binding affinities of the biotin group were significantly increased in all cell concentrations. Maximum binding affinity was observed at $5{\times}10^4/mg$ of micronized dermal matrix in biotin group. The viabilities were lowest in biotin group in contrast to binding affinity, but the difference was not significant. SEM showed well attachment of cells to micronized dermal matrix in all groups. Conclusion: The use of avidin/biotin facilitated human ASCs attaching to micronized acellular dermal matrix. This attachment would not disturb adipose stem cells viabilities. The present study suggests that avidin/ biotin can be used as making efficient use of cells in adipose tissue engineering.

• Macromolecular Research
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• v.14 no.5
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• pp.565-572
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• 2006

Recombinant human bone morphogenic protein-2 (rhBMP-2), which is known as one of the major local stimuli for osteogenic differentiation, was immobilized on the surface of hyaluronic acid (HA)-modified poly$(\varepsilon-caprolactone)$ (PCL) (HA-PCL) scaffolds to improve the attachment, proliferation, and differentiation of human bone marrow stem cells (hBMSCs) for bone tissue engineering. The rhBMP-2 proteins were directly immobilized onto the HA-modified PCL scaffolds by the chemical grafting the amine groups of proteins to carboxylic acid groups of HA. The amount of covalently bounded rhBMP-2 was measured to 1.6 pg/mg (rhBMP/HA-PCL scaffold) by using a sandwich enzyme-linked immunosorbant assay. The rhBMP-2 immobilized HA-modified-PCL scaffold exhibited the good colonization, by the newly differentiated osteoblasts, with a statistically significant increase of the rhBMP-2 release and alkaline phosphatase activity as compared with the control groups both PCL and HA-PCL scaffolds. We also found enhanced mineralization and elevated osteocalcin detection for the rhBMP-2 immobilized HA-PCL scaffolds, in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.251-256
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• 1999

The present study was conducted to evaluate whether vitrification could be used for ovarian tissue preservation. The important issue here is that the vitrification is very simple, easy, and economical compared to the conventional cryopreserving method that using automatic freezing instrument. Human ovarian cortical tissues were cryopreserved by vitrification with 5.5 M ethylene glycol and 1.0 M sucrose as cryoprotectant. Three points of temperature ($4^{\circ}C$, room temperature, and $37^{\circ}C$) and two points of duration (5 or 10 minutes) for cryoprotectant treatment were examined to determine the best condition for vitrification of the human ovarian cortical tissues. After thawing, viability of the isolated primordial follicles was examined by dye-exclusion method. Histological appearance of tissues before and after the cryopreservation was evaluated. There was no toxic effect of the 5.5 M ethylene glycol on the primordial follicles. However, when the tissues were treated with cryoprotectant at $37^{\circ}C$ for 10 minutes and exposed to liquid nitrogen, it seems likely that there is certain deleterious effects on the viability of the primordial follicles. The highest viability of the primordial follicles was obtained with the treatment of cryoprotectant at room temperature for 10 minutes. Follicles and oocytes survived after freezing and thawing had the similar normal shapes as was seen in the specimens before cryopreservation. It would be useful to apply vitrification in establishing ovarian tissue banking for clinical purposes.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.28 no.6
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• pp.421-433
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• 2002

In order to elucidate the pathogenesis of cleft lip and palate, first of all, it is necessary to understand the developmental mechanisms of growth factors and extracellular matrix proteins in the tissues of cleft lip and palate. We have performed immunohistochemical studies on human cleft lip and palate tissues to elucidate the pathogenetic implications of cleft lip and palate. 16 specimens from postnatal human cleft lip and palate subjects and 17 specimens from autopsy of prenatal human cleft lip and palate were fixed in 10% buffered formalin, embedded in paraffin. The sections were routinely stained by hematoxylin and eosin, also stained by PAS, and followed by immunohistochemical stainings using the antiseras of growth factors and extracellular matrix proteins such as PCNA, S-100, c-erb-B2, MMP-3, MMP-10, HSP-70, transglutaninase-C, E-cadherin, VEGF, vWF. Both the prenatal and postnatal specimens of cleft lip and palate showed dysplastic proliferation of the basal cell layer, increased infiltration of melanocytes into mucosal epithelium, sebaceous gland hyperplasia ingrowing into the muscular tissue of lip and palate, and fatty infiltration into the submucosal deep connective tissue. The strong reactions of MMP-3 and HSP-70 were detected in the tissues of cleft lip and palate, especially increased in degenerating muscle bundles, while the immunostainings of PCNA and c-erb-B2 were weakly positive in the tissues of cleft lip and palate. These data suggest that the retrogressive tissue degeneration around the cleft areas persistently exist during the prenatal and postnatal period after cleft formation, and the sebaceous gland hyperplasia and fatty infiltration with the intense expression of MMP-3 and HSP-70 is closely related to the muscular degeneration around the cleft area.

• Journal of Periodontal and Implant Science
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• v.23 no.3
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• pp.622-634
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• 1993

Gingiva is remarkly sensitive to certain drugs. Especially, long term use of phentoin, dihydropyrydine (including nifedipine), cyclosporin and other drugs can be lead to pathologic changes in gingival tissue, especially in terms of proliferation of epithelium and connective tissue. Recent study in terms of proliferation of epithelium and connective tissue. Recent study is focused on the inhibition of drug-induced gingival hyperplasia by using medicaments. The purpose of this study was to investigate on the pharmacological effects of nifedipine, retinoic acid and glycyrrhetini acid to the activity in human gingival fibroblast. Human gingival fibroblasts were cultured from the healthy gingiva of orthodontic patients. Gingival fibroblasts were trypsinized and cultured in growth medium added $5{\mu}g/ml$ of nifedipine, $10^{+7}M$ of retinoic acid and glycyrrhetinic acid. The passage number of cultured fibroblasts were between fifth and eighth. The cell morphology was examined by inverted microscope and the cell acitivity was measured by the MTT assay. Nifedipine at the concentration of $5{\mu}g/ml$ was revealed significantly effective to increase the cell activity and lipopolysaccharide was cofactor to increase cell activity in the presence of nifedipine. However, retinoic acid was significantly effective on the globular change of cell morphology and loss of cell process regardless of the presence of nifedipine and LPS. Cell activity was significantly decreased by the glycyrrhetinic acid at the concentration of $10^-M$ regardless of the presence of nifedipine and LPS. These results suggested that the increased cell activity by nifedipine might be modulated by retinoic acid and glycyrrhetinic acid. Further study is needed to clarify on their toxicological effects during cellular modulation and mRNA expression change.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.310-315
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• 1994

The pharmacokinetics and tissue distribution of DA-3030 (recombinant human granulocyte colony-stimulating factor, rhG-CSF, recently manufactured by Dong-A research laboratory of Dong-A Pharmaceutical Company) were compared with reported data in the literature. After intravenous(i.v.) administration of DA-3030, at dose of 5, 10 and 100 $\mu\textrm{g}$/kg to rats, some pharmacokinetic parameters, such as terminal half-lives(1.05, 1.19 and 1.83 hr, respectively) and clearance (84.0, 54.8 and 45.5 mι/hr/kg, repectively), were dose-dependent. This could be due to the saturable metabolism of DA-3030 in rats. Similar results were also reported. After subcutaneous(s.c.) and intramuscular(i.m.) administrations of DA-3030, 10 $\mu\textrm{g}$/kg to rats, the extent of bioavailability(absolute bioavailability) were incomplete; the values were 23.3 and 18.2% after s.c. and i.m. injections, respectively, due to the degradation of DA-3030 by protease. After 7-consecutive day i.v. administrations of DA-3030, 10 $\mu\textrm{g}$/kg/day, to rats, the plasma concentrations and pharmacokinetic parameters of DA-3030 were not significantly different from those in single administration. In mice and dogs at DA-3030 dose of 10 $\mu\textrm{g}$/kg, the plasma concentrations of DA-3030 were also declined rapidly with terminal half-lives of 1.31 and 1.15 hr, respectively. DA-3030 was highly concentrated in the kidney after i.v. administration of DA-3030, 10 $\mu\textrm{g}$/kg, to rats, and the results were similar to those obtained using radiolabelled rhG-CSF in the literature. Above data indicate that DA-3030 has similar properties to rhG-CSF manufactured by other companies in view of pharmacokinetics.

• Molecules and Cells
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• v.21 no.1
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• pp.121-128
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• 2006

Maitotoxin (MTX) is known as one of the most potent marine toxins involved in Ciguatera poisoning, but intracellular signaling pathways caused by MTX was not fully understood. Thus, we have investigated whether intracellular reactive oxygen species (ROS) are involved in MTX-induced cellular responses in human umbilical vein endothelial cells. MTX induced a dose-dependent increase of intracellular [$Ca^{2+}$]. MTX stimulated the production of intracellular ROS in a dose- and time-dependent manner, which was suppressed by BAPTA-AM, an intracellular $Ca^{2+}$ chelator. Ionomycin also elevated the ROS production in a dose-dependent manner. MTX elevated transamidation activity in a time-dependent manner and the activation was largely inhibited by transfection of tissue transglutaminase siRNA. The activation of tissue transglutaminase and ERK1/2 by MTX was suppressed by BAPTA-AM or ROS scavengers. In addition, MTX-induced cell death was significantly delayed by BAPTA-AM or a ROS scavenger. These results suggest that [$Ca^{2+}$]-dependent generation of intracellular ROS, at least in part, play an important role in MTX-stimulated cellular responses, such as activation of tTGase, ERK phosphorylation, and induction of cell death, in human umbilical vein endothelial cells.

• Proceedings of the KOSOMBE Conference
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• v.1995 no.11
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• pp.14-17
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• 1995

An in vitro construct of three dimensional artificial skin equivalent has been engineered using human cervical epithelial cells and human foreskin fibroblasts with a matrix of bovine type I collagen. Two cell lines were established from cervical uteri cancer tissues which have the HPV(human papillomavirus)18 genome. These two cell lines came from the same origin but have slight differencies in growth rate and tumorigenicity. The organotypic raft culturing of epithelial cells were accomplished at air-liquid interface. The differentiation related characteristics were examined by immunohistochemistry using monoclonal antibodies against EGFreceptor, cytokeratin 5/6/18 as proliferation markers and against filaggrin, involucrin, and cytokeratin 10/13 as differentiation marker. We have obtained the stratification and the differentiation in the artificial skin equivalent, and differentiation-related proteins were expressed more in the C3-artificial skin, and proteins of proliferation were expressed more in the C3N-artificial skin, relatively. We found that reconstituted artificial skin have the same characteristics of differentiation proteins of original tissue or cells of human body.