• Restorative Dentistry and Endodontics
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• v.48 no.2
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• pp.18.1-18.10
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• 2023

Objectives: This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs). Materials and Methods: The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 ㎍/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05. Results: CTHRC1 doses of 5, 10, and 20 ㎍/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs. Conclusions: CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

• Development and Reproduction
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• v.11 no.3
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• pp.167-177
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• 2007

In the present study, we isolated three human adult stem cells including adipose tissue-derived stem cells(HAD), umbilical cord-derived stem cells(HUC), and amnion-derived stem cells(HAM) and analysed their characteristics. And we examined whether HAD could be used as therapeutical cells for the heart diseases. Both HAM and HUC appeared very similar morphology but HAD was different. Doubling time of HUC was most fast, but total doubling numbers of HUC was same with HAM. Total doubling numbers of HAD was much more than others. Expression patterns of genes and proteins of three human adult stem cells were very similar. Also they were differentiated into adipocytes, osteocytes, and chondrocytes. In addition, they expressed many cardiomyocyte-related genes. But expression pattern of genes is a little different. When HAD were cultivated in the presence or absence of various combinations of BMP and FGF after 5-azacytidine expose for 24 h, expression of Cmlc-1, and ${\alpha}1c$ genes was significantly increased. However, expression of troponin T, troponin I and Kv4.3 genes was not changed. Based on these observations, it is suggested that HAD, HUC, and HAM might be used as potentially therapeutical cells for clinical application.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.286-290
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• 2009

The transcription of mRNA of avian influenza virus is regulated temporally during infection. Therefore, the measurement of transcript level in host cells should be performed before viral release from host cells because errors can occur in the analysis of the transcript levels if the viruses released from the infected cells re-infect cells. In this study, the timing of viral release was determined by measuring the level of viral RNA from viruses released from H9N2-infected chicken fibroblast cell line UMNSAH/DF-1 by semi-quantitative RT-PCR. The viral genomic RNA was isolated together with mouse total RNA which was added to the collected medium as carrier to monitor the viral RNA recovery and to use its GAPDH as an internal control for normalizing reverse transcription reaction as well as PCR reaction. It was found that viral release of H9N2 in the chicken fibroblast cell line UMNSAH/DF-1 took between 16 and 20 h after infection. We measured all 8 viral mRNA levels. Of the 8 transcripts, 7 species of viral mRNAs (each encoding HA, NA, PB1, PB2, NP, M, NS, respectively) except PA mRNA showed robust amplification, indicating these mRNA can be used as targets for amplification to measure transcript levels. These results altogether suggest that the method in this study can be used for screening antiviral materials against viral RNA polymerase as a therapeutic target.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.445-456
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• 2002

The purpose of this study were to determine that dexamethasone(Dex) induces differentiation of periodontal ligament(PDL) cells to osteoblastic cells and to investigate expression of matrix Gla protein(MGP), which is one of bone matrix protein. The isolated human PDL cells and gingival fibroblasts were prepared and cultured. The fourth or sixth sub-passage cells were used in this experiments. control group, ascorbic acid and ${\beta}$-glycerophosphate treated group, ascorbic acid, ${\beta}$-glycerophosphate and l00nM Dex treated group, ascorbic acid, ${\beta}$-glycerophosphate, and 5 ${\mu}M$ Dex treated group were made for study. The results were as follows: Cellular morphological change of PDL cells according to time was investigated. At first, the cells exhibited confluent monolayer of spindle or polygonal appearance. The multilayer of cells were seen after 7 days of treatment. After 14 days, the cells lost polarity and were densely packed. The mineralized nodule formation was seen at 21 days in the only Dex treated PDL cell groups. In the gingival fibroblast groups and no Dex treated PDL cell groups, the mineralized nodule was not seen. The mineralized nodule formation of 5 ${\mu}M$ Dex treated group was higher than 100 nM Dex treated group. Alkaline phosphatase(ALP) activity was higher in the Dex treated PDL cell groups of 14 and 21 days than 0 and 7 days. MGP was expressed in the control and all experimental groups and the expression was constant at 0,7,14,21 day. The above results confirm that Dex is affected to differentiation of the PDL cells to osteoblastic or cementoblastic cells and has dose-dependent effect for mineralization. And, MGP is expressed in the PDL cells and is not affected to mineralization of PDL cells.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.430-436
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• 2010

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.123-129
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• 2011

The pig has been considered to serve as an appropriate model of human disease. Therefore, establishment of porcine embryonic stem cell lines is important. The purpose of the present study was to further work in this direction. We produced porcine parthenogenetic embryos, and separately aggregated two of each of two-cell ($2{\times}2$), four-cell ($2{\times}4$), and eight-cell ($2{\times}8$) embryos derived by parthenogenesis. After culture for 4 days, the developmental ability of the aggregates and total blastocyst cell numbers were evaluated. The percentage of blastocysts was significantly higher in both $2{\times}4$- and $2{\times}8$-aggregated embryos ($58.3{\pm}1.9%$ and $37.2{\pm}2.8%$, respectively) than in the control or $2{\times}2$-aggregated embryos ($23.6{\pm}1.1%$ and $12.5{\pm}2.4%$, respectively). Total blastocyst cell numbers were increased in the $2{\times}4$- and $2{\times}8$-aggregated embryos (by $44{\pm}3.0%$ and $45{\pm}3.3%$, respectively) compared with those of control or $2{\times}2$-aggregated embryos ($30.5{\pm}2.1%$ and $30.7{\pm}2.6%$, respectively; p<0.05). The levels of mRNA encoding Oct-4 were higher in both the $2{\times}4$- and $2{\times}8$-aggregated embryos than in the control. When blastocysts derived from $2{\times}4$- aggregated embryos or intact normal embryos were cultured on mouse embryonic fibroblast feeder cells to obtain porcine stem cells, blastocysts from aggregated embryos formed colonies that were better in shape compared with those derived from intact blastocysts. Together, the data show that aggregation of porcine embryos not only improves blastocyst quality but also serves as an efficient procedure by which porcine embryonic stem cells can become established.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Journal of Korean society of Dental Hygiene
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• v.16 no.3
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• pp.471-477
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• 2016

Objectives: Fluoride is widely used in the prevention and control of dental caries. The purpose of this study is to examine the biological effects of Sodium fluoride on the proliferation of oral normal cell in vitro(MDPC-23, HaCaT, HGF-1 cells). Methods: The proliferation of normal cells and the cyto-skeletal change of normal cells were assessed by WST-1 assay and F-actin stain assay. The statistical significances of the resulting data were analyzed using SPSS(Window 12.0). Results: The sodium fluoride(0-12 mM) treatment decreased the cell viability in a dose and time dependent manner: HaCaT(6 h): $100{\pm}0$, $98{\pm}0.39$, $82{\pm}2.68$, $75{\pm}0.83$, $69{\pm}1$, $67{\pm}1.42%$(p<0.005); HaCaT(24 h): $100{\pm}0$, $98{\pm}1.85$, $54{\pm}0.64$, $43{\pm}0.4$, $38{\pm}0.32$, $36{\pm}0.13%$(p<0.006), MDPC-23(6 h): $100{\pm}0$, $93{\pm}1.48$, $85{\pm}0.28$, $82{\pm}1.58$, $79{\pm}1.48$, $76{\pm}1.93%$(p<0.009); MDPC-23(24 h): $100{\pm}0$, $91{\pm}1.26$, $58{\pm}0.65$, $49{\pm}1$, $44{\pm}0.74$, $2{\pm}0.05%$(p<0.005), HGF-1(6 h): $100{\pm}0$, $97{\pm}2.93$, $89{\pm}5$, $71{\pm}5.42$, $58{\pm}4.82$, $43{\pm}3.47%$(p<0.009); HGF-1(24 h): $100{\pm}0$, $97{\pm}2.05$, $73{\pm}1.73$, $22{\pm}1.61$, $14{\pm}1.73$, $7{\pm}0.85%$(p<0.005). Thus, changes in cell morphology and disruption of filamentous(F)-actin organization were observed in higher concentration. Conclusions: These results suggest that higher concentrations of fluoride lead to a reduce the number of cells and morphology change of normal cell.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.4
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• pp.331-339
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• 2007

Background: Distraction osteogenesis(DO) is a useful method for treating cases demanding the generation of new bone. During DO, the angiogenic activity is crucial factor in the new bone formation. The aim of this study was to detect the autocrine growth activity in the cellular components of the distracted bone with observation of the co-expression of vascular endothelial growth factor(VEGF) and its receptors following the mandibular DO. Materials and methods: Unilateral mandibular distraction(0.5 mm twice per day for 10 days) was performed in six mongrel dogs. Two animals were killed at 7, 14, and 28 days after completion of distraction, respectively. Immediately after the animals were killed, the right mandibles were harvested en block. Immunohistochemical staining was processed for observation of the VEGF expression, and double immunofluorescent staining was also processed for detection of the co-expression of osteocalcin and VEGF's two distinct receptors(VEGFR-1 and VEGFR-2). Results: At 7 and 14 days after distraction, the expressions of VEGF were significantly increased in the osteogenic cells of the distracted bone. Up to 28 days after distraction, VEGF was still expressed moderate in the osteoblastic cells of distracted bone. The co-expressions of osteocalcin/VEGFR-1 and osteocalcin/VEGFR-2 were observed in the distracted bone at 7 and 14 days after distraction. In the double immunofluorescent staining, the co-expression' s level of osteocalcin/VEGFR-1 was more than that of osteocalcin/VEGFR-2. Conclusion: Taken together, this study suggested that VEGF plays an important role in the osteogenesis, and these osteoblastic cell-derived VEGF might act as autocrine growth factor during distraction osteogenesis. In the other word, the cellular components, such as osteoblasts and immature fibroblast-like cellsor mesenchymal cells in the distracted bone, might have autocrine growth activity during distraction osteogenesis.

• KSBB Journal
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• v.21 no.4
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• pp.292-297
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• 2006

One of the steps in angiogenesis is the degradation of the underlying basement membrane via proteases. Endothelial cells release proteinases to degrade the extracellular matrix for their sprouting in vivo. In this study, we examined the effect of water extracts of Phellinus linteusis(Phellinus extracts) and combination of Phellinus extracts and fibroblast growth factor(FGF-2) on cultured porcine pulmonary artery endothelial cells(PPAECs). Phellinus extracts induced sprouting of PPAECs, which was inhibited by MMPs and plasmin inhibitors, and induced the secretion of matrix metalloproteinase-3(MMP-3) and plasmin. At high concentration of Phellinus extracts($200{\sim}400{\mu}g/mL$), the active MMP-2 secretion was induced. It is therefore, suggested that Phellinus extracts induces the sprouting of cultured endothelial cells by means of increased active MMP-2 and plasmin secretion. Also, combination with Phellinus extracts and FGF-2 produced an enhanced effect on sprouting and secretion of active MMP-2, and MMP-3 and plasmin from PPAECs.