• Journal of Biomedical Engineering Research
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• v.42 no.1
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• pp.18-24
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• 2021

Vibration is a mechanical cue that can be applied to adipose tissues for the purpose of treating obesity. However, the exact correlation between vibration and other anti-adipogenic pathways, such as development of cytoskeleton and apoptosis, remains unknown. The objective of this study was to investigate the unknown anti-adipogenic effects of vibration with varied frequencies on preadipocytes. 3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% calf serum at 37 ℃ with 5% CO2 in a humidified incubator. Vibration was generated using Arduino Uno microcontroller and vibration motor module with 1 V DC, and applied to preadipocytes for 3 days. Frequency conditions were set to 20, 55, and 90 Hz. Then, the expressions of p38 pathway, ROCK-1, α-actinin, Bax, Bcl-2, caspase-9, 8, and 3 were analyzed with western blot. As a result, p38 pathway was inhibited in 55 and 90 Hz while ROCK-1 and α-actinin were expressed in 20 Hz. Caspase-3, a terminal apoptotic factor, was activated in 20 Hz via extrinsic pathway rather than intrinsic pathway. Results suggest that various frequencies of vibration can inhibit adipogenesis via different pathways which sheds light on future mechanotransduction applications of vibration for the treatment of obesity.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• The Journal of Korean Obstetrics and Gynecology
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• v.20 no.1
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• pp.50-60
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• 2007

Purpose : This investigation was undertaken to evaluate the anti-proliferation, in hexane, chloroform, ethyl acetate, butanol and water fraction from extract of Gyulyupsanbyonbang(GYSB) using MCF-7 human breast cancer cells. Methods : GYSB was added to distilled water(1500ml) and was boiled then filtered. The residue was suspended in distilled water and extracted with hexane, chloroform, ethyl acetate, butanol and water. MCF-7 cells were cultured in RPMI1640 complex badge, NIH3T3 was cultered in 37$^{\circ}$C, 5% moisture incubator of carbon dioxide with Dulbecco's Modified Eagle Medium(DMEM) supplemented with 10% fetal bovine serum and antibiotics. Cell cytotoxicity test about cancer cell was measured used MTT assay. Results: When it synthesizes a result, hexane and butanol fraction had shown anti-proliferation effect and safety together, and those anti-proliferation effect operating selectively appeared. Ethyl acetate fraction had anti-proliferation effect however, it was not selective. The Chloroform and water soluble fraction did not almost appear anti-proliferation effect. Conclusion : I can conclude that GYSB have anti-proliferation effect and safety together on MCF-7 cells. It suggest that GYSB may be useful for brest cancer patients.

• Development and Reproduction
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• v.15 no.4
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• pp.281-289
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• 2011

The objective of this study was to investigate the effective cryoprotectants for the cryopreservation of porcine mesenechymal stem cells (pMSCs). In order to understand the effectiveness of various cryoprotectants on pMSCs, we studied the most commonly used cryoprotectants; dimethyl sulfoxide (DMSO), ethylene glycol (EG), DMSO and EG. pMSCs were isolated from bone marrow matrix of piglet (2 month) and characterized by alkaline phopshatase (AP) activity, colony forming, and differentiation to adipocyte. In slow cooling cryopreservation, the pMSCs were exposed to cell medium containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% DMSO, 1.5M EG and 5% DMSO/0.75M EG, respectively, and freezed to $-1^{\circ}C$/min from $25^{\circ}C$ up to $-80^{\circ}C$ in a cryo-container. The proportion of viable cells and the growing rates in fresh pMSCs were significantly (P<0.05) higher than those of other groups, but did not differ between the cryopreserved groups. The expression of Sox-2 and Nanog gene was increased by extending culture time in cryopreserved groups. The expression of Bax gene in cryopreserved groups was similar with fresh pMSCs. Moreover, the gene expression of adipocyte-specific marker as well as chondrogenic/osteogenic factors in cryopreserved groups was similarly to fresh pMSCs. Taken together, our results suggested that all these cryoprotectants of 10% DMSO, 1.5M EG and 5% DMSO/0.75M EG could be used for cryopreservation of the pMSCs.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.5
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• pp.341-345
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• 2010

Introduction: Skeletal homeostasis is normally maintained by the stability between bone formation by osteoblasts and bone resorption by osteoclasts. However, the correlation between the inflammatory reaction and osteoblastic differentiation of cultured osteoprogenitor cells has not been fully investigated. This study examined the effects of inflammatory cytokines on the osteoblastic differentiation of cultured human periosteal-derived cells. Materials and Methods: Periosteal-derived cells were obtained from the mandibular periosteum and introduced into the cell culture. After passage 3, the periosteal-derived cells were further cultured in an osteogenic induction Dulbecco's modified Eagle's medium (DMEM) medium containing dexamethasone, ascorbic acid, and $\beta$-glycerophosphate. In this culture medium, tumor necrosis factor (TNF)-$\alpha$ with different concentrations (0.1, 1, and 10 ng/mL) or interleukin (IL)-$1{\beta}$ with different concentrations (0.01, 0.1, and 1 ng/mL) were added. Results: Both TNF-$\alpha$ and IL-$1{\beta}$ stimulated alkaline phosphatase (ALP) expression in the periosteal-derived cells. TNF-$\alpha$ and IL-$1{\beta}$ increased the level of ALP expression in a dose-dependent manner. Both TNF-$\alpha$ and IL-$1{\beta}$ also increased the level of alizarin red S staining in a dose-dependent manner during osteoblastic differentiation of cultured human periosteal-derived cells. Conclusion: These results suggest that inflammatory cytokines TNF-$\alpha$ and IL-$1{\beta}$ can stimulate the osteoblastic activity of cultured human periosteal-derived cells.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

• The Journal of Advanced Prosthodontics
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• v.7 no.2
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• pp.172-177
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• 2015

PURPOSE. The purpose of this study was to evaluate cell toxicity due to ion release caused by galvanic corrosion as a result of contact between base metal and titanium. MATERIALS AND METHODS. It was hypothesized that Nickel (Ni)-Chromium (Cr) alloys with different compositions possess different corrosion resistances when contacted with titanium abutment, and therefore in this study, specimens ($10{\times}10{\times}1.5mm$) were fabricated using commercial pure titanium and 3 different types of Ni-Cr alloys (T3, Tilite, Bella bond plus) commonly used for metal ceramic restorations. The specimens were divided into 6 groups according to the composition of Ni-Cr alloy and contact with titanium. The experimental groups were in direct contact with titanium and the control groups were not. After the samples were immersed in the culture medium - Dulbecco's modified Eagle's medium[DMEM] for 48 hours, the released metal ions were detected using inductively coupled plasma mass spectrometer (ICP-MS) and analyzed by the Kruskal-Wallis and Mann-Whitney test (P<.05). Mouse L-929 fibroblast cells were used for cell toxicity evaluation. The cell toxicity of specimens was measured by the 3-{4,5-dimethylthiazol-2yl}-2,5-diphenyltetrazolium bromide (MTT) test. Results of MTT assay were statistically analyzed by the two-way ANOVA test (P<.05). Post-hoc multiple comparisons were conducted using Tukey's tests. RESULTS. The amount of metal ions released by galvanic corrosion due to contact between the base metal alloy and titanium was increased in all of the specimens. In the cytotoxicity test, the two-way ANOVA showed a significant effect of the alloy type and galvanic corrosion for cytotoxicity (P<.001). The relative cell growth rate (RGR) was decreased further on the groups in contact with titanium (P<.05). CONCLUSION. The release of metal ions was increased by galvanic corrosion due to contact between base metal and titanium, and it can cause adverse effects on the tissue around the implant by inducing cytotoxicity.

• Proceedings of the Ginseng society Conference
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• 2007.05a
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• pp.23-24
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• 2007

Purpose: 인삼이 항당뇨 활성을 가진다는 연구가 많은 연구자들에 의해 진행되었고, 이는 인삼의 구성 성분 중 ginsenoside에 기인한다는 보고가 있다. 본 연구는 ginsenoside의 항당뇨 작용기전을 in vitro에서 알아보고자 3T3-L1 지방세포에서 glucose uptake와 췌장 베타세포인 HIT-T15 세포에서 insulin 분비 효과를 확인하였다. 이를 위하여 인삼을 식초로 처리한 긴삼의 70% MeOH 분획으로부터 protopanaxadiol 계인 ginsenoside $Rb_2$, $Rg_3$ 그리고 protopanaxtriol 계인 $Rg_2$를 분리하여 본 실험에 사용하였다. Method: Ginsenoside $Rb_2$, $Rg_2$, $Rg_3$가 지방 세포에서 glucose uptake에 미치는 효과를 확인하기 위하여 3T3-L1 세포를 DMEM (Dulbecco's Modified Eagle's Medium) 배지에서 분화 유도시켰으며 3T3-L1 preadipocyte가 80% 정도 자라면 분화 유도 배지 (5% fetal bovine serum (FBS), 0.5 mM isobutylmethylxanthine (IBMX), 1 mM dexamethasone 그리고 $10{\mu}g/ml$ insulin가 포함된 DMEM)로 4일, $10{\mu}g/ml$ insulin가 포함된 DMEM으로 2일, FBS만 포함된 DMEM으로 2일 배양하여 총 8일 동안 분화를 유도하였다. 분화 유도된 3T3-L1 adipocytes 에 각각 $Rb_2$, $Rg_2$, $Rg_3$를 $20{\mu}M$로 처리하여 16시간 배양하여 low glucose DMEM에서 3시간 배양한 후에 $37^{\circ}C$에서 insulin 10 ng/ml 과 각각 $Rb_2$, $Rg_2$, $Rg_3$가 포함된 Krebs Ringer Hepes buffer(KRP buffer)에서 20분간 배양하였다. 2-deoxy-D-[$^3H$]-glucose를 넣고 10분 후에 차가운 PBS로 반응을 종결시켜 lysis buffer로 cell을 모은 후 scintillation counter를 이용하여 glucose를 측정하였다. Insulin 분비 효과는 HIT-T15 세포와 일차 배양한 흰쥐 소도세포(islets)를 사용하여 확인하였다. HIT-T15 세포는 24 well plate에 well 당 $2{\times}10^5$ 개씩 분주하여 24시간 동안 배양한 후 시료를 처리하였으며 소도 세포는 Sprague-Dawley rat의 췌장에 collagenase가 포함된 Hanks' Balanced Salt Solution(HBSS)을 주입하여 분리하고 islets을 얻었다. 분리한 소도세포를 $1{\sim}2$일 동안 배양하여 $Rb_2$, $Rg_2$, $Rg_3$가 각각 $20{\mu}M$의 농도로 첨가된 insulin 측정용 buffer인 Krebs-Ringer buffer (KRB+0.3% BSA, KRBB)에 $37^{\circ}C$에서 1시간 incubation 시킨 후 배양액으로 분비된 인슐린의 양을 측정하였다. 한편 ginsenoside의 인슐린 분비 촉진 기전을 알아보기 위한 실험에서는 ATP-sensitive $K^+$ channel opener인 diazoxide (0.5 mM)가 ginsenoside에 의해 촉진된 인슐린 분비를 억제하는지 살펴보았다. Result: glucose uptake assay 에서는 $Rg_2$가 가장 크게 glucose uptake를 증가시켰고 $Rb_2$, $Rg_3$는 그 활성이 크지 않았다. 한편 Insulin 분비 효과는 diol계인 $Rg_3$에서 용량 의존적으로 인슐린의 분비를 촉진시켰으며 $20{\mu}M$ 농도에서 대조군과 비교해 1.5배 이상의 분비 촉진 효과를 보였고 triol계인 $Rg_2$ 에서는 이러한 효과가 나타나지 않았다. $Rg_3$의 인슐린 분비 촉진 기전을 0.5 mM 의 diazoxide를 이용하여 확인한 결과 $Rg_3$에 의해 촉진된 인슐린 분비를 감소시켰다. 이로 미루어보아 $Rg_3$의 인슐린 분비 촉진 기전은 ATP-sensitive $K^+$ 채널의 봉쇄에 의한 것임을 확인할 수 있었다.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.109-122
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• 2001

Gingival fibroblasts(GF) and periodontal ligament fibroblasts(PDLF) are the major cellular components of periodontal soft connective tissues, but the precise molecular biological differences between these cells are not yet known. In the present study, we investigated the expression of S100A4, S100A2 calcium-binding protein and osteoblast-specific factor 2(OSF-2, Periostin) mRNA in GF and PDLF in vitro through the process of reverse transcription-polymerase chain reaction(RT-PCR) and Northern blot analysis in each. Human GF and PDLF were isolated from the gingival connective tissue and the middle third of freshly extracted healthy third molars. They were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum and cells in the third passage were used in the experiments. After extracting total RNA from cultured cells, RT-PCR and Northern analysis were performed using S100A4-, S100A2- and Periostin-specific oligonucleotide primers and subcloned cDNA probes in each. In PT-PCR and Northern analysis, the expression of S100A4 and Periostin mRNA in GF was slightly detectable. Interestingly, the expression of S100A4 and periostin mRNA in PDLF was much higher than that in GF. On the other hand, S100A2 mPNA was highly expressed in both GF and PDLF. Since there was a marked difference of S100A4 and Periostin expression between GF and PDLF in vitro, these data suggest that S100A4 and periostin could be used as a useful marker for distinguishing cultured gingival fibroblasts and periodontal ligament cells.