• Imaging Science in Dentistry
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• 제38권3호
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• pp.125-132
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• 2008

Purpose : To investigate the effects of irradiation on transforming growth factor ${\beta}_1$ (TGF-${\beta}_1$) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Materials and Methods : Cells were cultured in alpha-minimum essential medium ($\alpha$-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with $\alpha$-MEM supplemented with 10% FBS, 5 mM $\beta$-glycerol phosphate, and $50\;{\mu}g/mL$ ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-${\beta}_1$ mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. Results : The amount of TGF-${\beta}_1$ mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy. and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P < 0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Conclusion: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-${\beta}_1$ mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

• Korean Journal of Animal Reproduction
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• 제21권2호
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Journal of Periodontal and Implant Science
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• 제37권1호
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• pp.115-124
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• 2007

Silica is known as a promising osteoconductive material, and polycaprolactone is a bioactive and degradable material. The purpose of this study was to monitor the differentiation of MC3T3-E1 cells cultured on the layer-built silica/poly caprolactone non-woven fabric produced by electrospinning. Non-woven fabric (silica, polycaprolactone, PSP, SPS) was made by electrospinning and they were inserted in the 48 well cell culture plate. MC3T3-E1 cells were prepared by subculture. Cells were seeded to each well $1{\times}10^5$ concentration per well. Dulbecco's modified eagle medium with 10% FBS and 1% antibiotic-antimycotic solution was used. Confocal laser scanning microscope was taken 4 hours after incubation (95% air. 5% $CO_2$, $37^{\circ}C$). Cell proliferation was monitored by spectrophotometer on 1, 7, 14 days, and the morphology of the growing cells was observed by field emission scanning electron microscope. To monitor the differentiation of osteoblasts on the materials, MC3T3-E1 cells were incubated in 48 well culture plate after seeding with the density of $1{\times}10^5$ concentration. Then ELISA kit & EIA kit were used on to assess osteocalcin and osteopontin expression respectively. The other conditions were the same as above. MC3T3-E1 cells were proliferated well on all of the materials. There were no statistical differences among them. The osteopontin expression of silica, PSP, SPS was significantly higher than other groups on day 3 (p/0,05), but after that time, there were no statistically signigicant differences. The osteocalcin expression was significantly higher in silica and PSP than other groups on day 14. These findings show that PSP was as good as silica on the effect of osteoblast differentiation. The PSP non-woven fabric may have the possibility as bone graft materials.

• Herbal Formula Science
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• 제14권2호
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• pp.76-85
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• 2006

Objectives : We Investigated the effect of Dioscoreae Rhizoma(山藥) on the change of the corticosterone and the rectal temperature(直腸溫渡) of the mice induced by starvation stress(創戰 스트레스). Methods : After administration of Dioscoreae Rhizoma (0.25g/kg, 0.5g, 1.0g/kg, 3g/kg) three times, mice were starved. The corticosterone and rectal temperature were measured after 36.5 hours starvation stress. Results : The plasma cortiosterone levels in the S-2, S-3 and S-4 group were decreased significantly comparing with the control group (P<0.01) after 36.5 hours starvation stress. and rectal temperature was decreased in the control goup comparing with the normal group, but there is no significant change in the Dioscoreae Rhizoma treated group. Conclusion : it might be recognized that Dioscoreae Rhizoma has preventive-effect against starvation stress and also it might be needed further study in various viewpoints. Objectives : This study was disegned to elucidate the short term effect of Rossa rugosae Radix on proliferation. differentiation and maturation of 3T3-L1 Preadipocyte. Methods: 3T3-L1 preadipocytes obtained from Korean Cell Line Bank were cultured in a D ulbecco’ s modified eagle medium(MEM) culture solution containing 10% fetal bovine serum(FBS) and various concentrations of aqueous extract of Rossa rugosae Radix.. The short term effect of the extract of Rossa rugosae Radix on proliferation. differentiation and maturation of 3T3-L1 preadipocytes were investigate after treatment for 24 hours by measuring MTT. Oil Red 0 and latate dehydrogenase activity.. Results: The Rossa rugosae Radix extract inhibited significantly the proliferation of 3T3-L1 preadipocytes and tended to increase latate dehydrogenase activity in the media of differentiated 3T3-L1 preadipocytes & matured 3T3-L1 preadipocytes. the extract also inhibit the lipid accumulation of differentiated 3T3-L1 preadipocytes & matuered 3T3-L1 preadipocytes. Conclusions: These results demonstrated that the Rossa rugosae Radjx extract inhibited the proliferation. differentiation and maturation of 3T3-L1 preadipocytes. suggesting that Rossa rugosae Radix has anti-obesity effect: however further in vivo study is needed to demonstrate its pharmacological effects.

• Proceedings of the KSAR Conference
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.69-69
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• 2001

The cell cycle phase in which donor nuclei exist prior to nuclear transfer is an important factor governing developmental rates of reconstituted embryos. It was suggested that quiescent G0 and cycling G1 cells could support normal development of reconstituted embryos. In a quest of optimized donor nuclei treatment prior to nuclear transfer, this study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells when cultured under a variety of culture treatments and the cell cycle change with the lapse of time after trypsinization. This was archived by measuring the DNA content of cells using flow cytometry, Cultured fetal fibroblast cells, adult skin and muscle cells, and cumulus cells were divided by 3 culture treatments; 1) grown to 60-70% confluency (cycling), 2) serum starved culture, 3) culture to confluency. Trypsinized cells were fixed by 70% ethanol and stained with propidium iodide. For one experiment, trypsinized cells were resuspended in DMEM＋10% FBS and incubated for 1.5, 3 and 6 h with occasional shaking before ethanol fixation. Cell cycle phases were determined by flow cytometry enabling calculation of percentages of G0＋G1, S and G2＋M. The majority of cells were in G0＋Gl stage regardless of origin of cells. Cultures that were serum starved or cultured to confluency contained significantly (P<0.05) higher percentages of cells in G0＋G1 (89.5-95.4%). For every cell lines and culture treatments, percentages of cells in existing in G0＋G1 increased with decreasing of the cell size from large to small. In the serum starved and confluency groups, about 98% of small cells were in G0＋G1 Serum starved culture contained higher percentages of small-sized cells (38.5-66.9%) than cycling and confluent cultures regardless of cell lines (P<0.05). After trypsinization of fetal fibroblast and adult skin cells that were serum starved and cultured to confluency, the percentages of cells in G0＋G1 significantly increased by incubation for 1.5(95.7-99.5%) and 3.0 h (95.9-98.6%). The results suggest that the efficient synchronization of bovine somatic cells in G0＋G1 for nuclear transfer can be established by incubation for a limited time period after trypsinization of serum starved or confluent cells.

• Food Science and Biotechnology
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• 제17권2호
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• pp.247-250
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• 2008

The $CHCl_3$, EtOAc, and n-BuOH fractions showed a marked inhibition of 5% fatal bovine serum (FBS)-induced cell proliferation. The $IC_{50}$ values of the chloroform fractions from leaf, stem, and root as well as the n-BuOH and EtOAc fraction from root on cell proliferation were $1.2{\pm}0.4$, $17.2{\pm}6.4$, $81.8{\pm}33.2$, $40.8{\pm}8.0$, and $237.1{\pm}85.6\;{\mu}g/mL$, respectively. On the other hand, the EtOAc fractions, and the $CHCl_3$ fraction significantly inhibited collagen-, arachidonic acid-, U46619-, and thrombin-induced platelet aggregations. The $IC_{50}$ values of EtOAc fraction from leaf, and the $CHCl_3$ and EtOAc fraction from stem were $214.1{\pm}12.2$, $134.3{\pm}2.5$, and $42.6{\pm}7.0\;{\mu}g/mL$ with collagen, $312.4{\pm}7.5$, $158.9{\pm}1.7$, and $82.2{\pm}2.7\;{\mu}g/mL$ with arachidonic acid, $31.1{\pm}2.4$, $48.7{\pm}0.3$, and $29.7{\pm}1.1\;{\mu}g/mL$ with U46619, and $36.7{\pm}2.4$, $69.1{\pm}11.3$, and $34.2{\pm}0.1\;{\mu}g/mL$ with thrombin, respectively. Taken together, these data provide new evidence that fractions from Allium victorialis var. platyphyllum (AVP) are able to inhibit vascular smooth muscle cell (VSMC) proliferation and platelet aggregation, which may be a novel resource for the development of anti-atherothrombotic agents.

• Journal of Embryo Transfer
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• 제25권3호
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• pp.189-193
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• 2010

We investigated the cleavage rate and blastocyst yield for each culture condition to enhance tolerance of cryo-preservation of bovine IVF embryo with relatively lower cryo-tolerance compared to in vivo embryo. The cleavage rate and blastocysts yield for CR1aa, IVMD, IVD, CR1aa+10% FBS were 73.2, 69.3, 72.8, 68.5% and 44.1, 30.8, 33.3, 48.0%, respectively. The values did not differ among each treatments without serum. For embryo vitrification, In vivo and In vitro blastocysts were exposed to VS1(10% glycerin, 0.1 M glucose, 0.1 M sucrose, PEG 1%) for 5 min, and VS2 (10% glycerin, 10% EG, 0.2 M glucose, 0.2 M sucrose, PEG 2%) for 5 min and then VS3 (10% glycerin, 30% EG, 0.3 M glucose, 0.3 M sucrose, PEG 3%) for 1 min. The exposed embryos were then loaded into the 0.25 ml plastic straws and then plunged into liquid nitrogen. The straws were held for period of 1 to 2 weeks before thawing. In embryo viability, no differences in blastocyst re-expansion rates were found between in vivo and in vitro embryos. whereas expansion-BL rates was significantly higher for in vivo-derived embryos (72.7%) when compared to in vitro-derived embryos (51.4%), respectively (P<0.05). In conclusion, our results indicate that combined use of CRIaa culture medium with vitrification might enhance tolerance of cryopreservation for bovine IVF embryo production.

• Journal of Periodontal and Implant Science
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• 제35권3호
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• pp.623-634
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• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.124-124
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• 2003

The successful development of embryos cloned by nuclear transfer (NT)have been dependent on a wide range of known factors including cell cycle of donor and recipient ooplast, oocyte quality, NT procedure and oocyte activation. The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from 26 mm follicles of slaughterhouse ovaries and cultured for 22 h in NCSU #23 medium supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 0.5 g/mL LH, 0.5 g/mL FSH and 10 ng/mL EGF. The COCs were further cultured for an additional 22 h in the same medium at $39{\cird}C$ in an atmosphere of 5% $CO_2$ in air, without hormonal supplements. Primary cultures of fibroblasts isolated from a female fetus on day 40 of gestation were established in DMEM + 15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM +0.5% FCS for 5 days in order to arrest the cells in G0/Gl. After enucleation, oocytes were reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 KV/cm, 30 sec) in 0.28 M mannitol containing 0.01 mM $CaCl_2$ and 0.01 mM $MgCl_2$. Eggs were then divided into three treatment groups, control (without further treatment, Group 1), eggs cultured in 10 g/ml cycloheximide (CHX) for 5 h (Group 2), and eggs cultured in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 5 h (Group 3). The eggs were then cultured in sets of 30 in 60 I drops of NCSU#23 supplemented with 4mg/ml BSA (essentially fatty acid free) until day 7 at $39{\circ}C$ in a humidified atmosphere of 5% $CO_2$. On day 4 the culture were fed by adding 20 I NCSU #23 supplemented with 10% FBS. Development rates into blastocysts were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls ($27.6 \mu 2.7% vs. 20.1 \mu 4.1%$, respectively), but rates did not differ in Group 2 compared to control ($23.8 \mu 5.7%$). Total cell number in Group 3 blastocysts was however significantly higher (P<0.05) than in Groups 1 and 2 ($44.6 \mu 2.4 vs. 19.9 \mu 1.9 and 21.9 \mu 2.1$, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.21-28
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• 2002

Objective: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). Methods: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and $x^2$. Results: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). Conclusions: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.