• Journal of Periodontal and Implant Science
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• v.26 no.1
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• pp.216-229
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• 1996

Interferon(IFN) is a sort of glycoproteins that are produced by activated lymphocyte, monocyte and fibroblast. IFN has anti-viral effects, immuno-defensive mechanism and regulating properties to the several kinds of cells that includes affect on the bone formation and resorption. The effect of IFN on the osteoclast & other tissue cells has been studied in a number of researchers with the limited reports on the osteoblast. The purpose of this study was to evaluate the effects of IFN on the osteoblastic function. The MC3T3/El cell(Mouse osteoblast) was incubated in ${\alpha}-minimum$ essential medium containing 10% FBS. To detect the cytotoxic effect of $IFN-{\gamma}$ on osteoblast, the cells were cultured in 96-well plate to which $IFN-{\gamma}$ of various concentrations were added for 2 days. After staining with trypan blue, total cells and living cells were counted under microscope. To determine the activity of alkaline phosphataset(ALP), various concentrations of $IFN-{\gamma}$ were treated to culture medium, and biochemical assay was performed. $IFN-{\gamma}$ and $IFN-{\gamma}$ plus cycloheximide were added to culture medium separately and then ALP activity were determined. To detect the effect of the $IFN-{\gamma}$ on the bone formation of osteoblast, long-term culture was performed, and calcified nodule formation were observed using von Kossa's staining. After the addition of $IFN-{\gamma}$ with various concentrations to the medium, no cytotoxic effect of $IFN-{\gamma}$ was detected at any concentration. The significant increase in ALP activity of osteoblast were found the concentration of $IFN-{\gamma}$ 500-2500U/ml and the culture time of 24-48 hours respectively. The enhancement of ALP activity by $IFN-{\gamma}$ of osteoblast was decreased significantly by the treatment of cycloheximide. After long-term culture of osteoblast, the nodule formation was found to be increased in number and density by the addition of 500 U/ml $IFN-{\gamma}$. These results suggest that $IFN-{\gamma}$ was affected on the bone formation of osteoblast. Forthemore this kind of study or $IFN-{\gamma}$ to osteoblast will be held continuously.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.1-7
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• 2000

Objective: Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. Also, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration (oviduct - glucose: 0.5 mM, pyruvate: 0.32 mM, lactate: 10.5 mM; uterus - goucose: 3.15 mM, pyruvate: 0.1mM, lactate: 5.87 mM, respectively). This study was conducted to examine the effect of these energy sources added in DMEM with glutamine on the mouse embryo development. Methods: There was used ICR female mouse. Two cell embryos of mouse are collected by method of 'flushing'. Flushing fluid was used Ham's F-10 added to 20% FBS. The collected 2 cell embryos were cultured in media such as Control (only DMEM), group A and B (DMEM supplemented with 0.5 mM and 3.15 mM glucose), and group C and D (DMEM supplemented with 0.1 mM and 0.32 mM pyruvate), and group E and F (DMEM supplemented with 5.87 mM and 10.5 mM lactate). All experimental media supplemented with 20% hFF, respectively. Pattern of embryo development was observed to interval at 24hr during 96hr. Results : The media with glutamine added glucose (group A: 51.0%; group B: 48.4%) was significantly (p<0.05) higher than other experimental group in development into the morula stage after 24 hr in culture, but not significantly different compared with control and the rate of development into the blastocyst was significantly (p<0.05) low in the both of pyruvate (group C: 7.9% group D: 6.8%) and lactate (group E: 7.1%, group F: 7.1%) treatment group after 48 hr in culture. Development into the blastocyst and hatched balstocyst after 72 hr in culture revealed similarly in control (81.9%) and glucose treatment group (group A: 83.3%, group B: 82.8%). However, development into the hatched and attached blastocyst after 96hr in culture revealed significantly (p<0.05) development in the glucose treatment group (group A: 82.3%, group B: 78.5%) than control (63.2%), and its of pyruvate (group C: 34.1%, group D: 34.1%) and lactate (group E: 25.9%, group F: 33.3%) treatment group were significantly (p<0.05) lower than control similar to previous observations. Conclusion : The glucose added to the DMEM with only glutamine, as energy source, was highly to the rate of development compared with control, but the other energy sources were not, synthetically. Above refer to, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration. Thus, further studies are will examine continuously to effects by interaction of different energy sources in the mouse embryo development, and these results will provide to foundation on the human embryo culture.

• Development and Reproduction
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• v.10 no.4
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• pp.239-245
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• 2006

Experiments were conducted to determine the effects of beta-mercaptoethanol(${\beta}-ME$) supplements to the maturation medium on in vitro fertilization(IVF) and intracellular glutathione(GSH) concentration. Bovine cumulus-intact oocytes were matured in TCM-199 medium containing FBS, hormonal supplements, and ${\beta}-ME$(0, 25 and $50\;{\mu}M$) for 12h and 24 h. After culture, cumulus-free matured oocytes were co-incubated with frozen-thawed spermatozoa for 24h. Maturation rate increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant differences among treatment groups. Also, increases(p<0.05) in intracellular GSH concentration before and after fertilization were observed in $50\;{\mu}M\;{\beta}-ME$ supplements to the maturation medium. Male pronuclear formations after IVF was increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant difference among treatment groups. In conclusion, supplementing ${\beta}-ME$ into the maturation medium increased maturation rates, fertilization rates, and intracellular GSH concentrations.

• Development and Reproduction
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• v.4 no.2
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• pp.215-220
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• 2000

Most of endocrine disrupters (EDs) have been reported to exhibit estrogenic or anti-androgenic activity and thereby may disrupt reproductive development in human or wildlife. This study was performed to investigate the effects of estrogen (E$_2$), bisphenol (BP) and octylphenol (OP) on the mouse Leydig cell line (TM3). TM3 originated from testis of 11~13-daly-old BALB/c nu/＋ mice was cultured in DMEM supplemented with 10％ FBS alone or medium with estrogen (E$_2$), bisphenol (BP) and octylphenol (OP; 1 pM, 1 nM, 1 $\mu$M, 1 mM, respectively) for 48 hours. After culture, total cell number and viability were assessed by heamocyto-meter and trypan blue stain. Expression of cytochrome P450scc (CYPscc) mRNA whose product is involved in steroid hormone biosynthesis and estrogen receptor $\alpha$(ER $\alpha$) mRNA were detected by RT-PCR. As a result, treatment of TM3 with E$_2$, BP and OP(1 mM, respectively) significantly decreased the viability but not all of groups as high as 1 $\mu$M. Exposure of TM3 to OP significantly reduced the total cell number but not E$_2$ or BP. The expression of CYPscc mRNA was slightly reduced in BP (1 nM, 1 $\mu$M) and significantly decreased in OP (1 nM, 1 $\mu$M) treated TM3, except E$_2$ group. But the expression of ER $\alpha$ mRNA was sightly increased in all treated groups. In conclusion, BP and OP (high concentration) might inhibit steroidogenesis by decreasing the CYPscc mRNA expression in the mouse testis. These results suggest that BP and OP might impair spermatogenesis and subsequently disturb testicular function.

• Journal of Physiology & Pathology in Korean Medicine
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• v.33 no.6
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• pp.356-362
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• 2019

Scutellaria baicalensis (SB) has widely used in the treatment for various brain diseases in the field of Oriental medicine. Biofermantation of SB can make major chemical constituents of SB to pass blood-brain barrier easily and to have more potent anti-oxidant ability. There is a little information about the contribution of fermented SB (FSB) to the formation or maintenance of the neural plasticity in the hippocampus. The purpose of this study was to evaluate effects of FSB extract on hydrogen peroxide (H₂O₂) - induced impairments of the induction and maintenance of long-term potentiation (LTP), an electrophysiological marker for the neural plasticity in the hippocampus. From hippocampal slices of rats, the field excitatory postsynaptic potentials (fEPSPs) were evoked by the electrical stimulation to the Schaffer collaterals - commissural fibers in the CA1 areas and LTP by theta-burst stimulation by using 64 - channels in vitro multi-extracellular recording system. In order to induce oxidative stress to hippocampal slices two different concentrations (200, 400 μM) of H₂O₂ were given to the perfused aCSF before and after the LTP induction, respectively. The ethanol extract of FBS with concentration of 25 ㎍/ml, 50 ㎍/ml was diluted in perfused aCSF that had 200 μM H₂O₂, respectively. Oxidative stress by the treatment of H₂O₂ resulted in decrease of the induction rate of LTP in the CA1 area with a dose - dependent manner. However, the ethanol extract of FSB prevented the reduction of the induction rate of LTP caused by H₂O₂ - induced oxidative stress with a dose - dependent manner. These results may support a potential application of FSB to ameliorate impairments of hippocampal dependent neural plasticity or memory caused by oxidative stress.

• Analytical Science and Technology
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• v.28 no.5
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• pp.309-316
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• 2015

The analytical method of lead in plasma by ICP-MS was validated after securing environment within class 1,000 classification. We tested specificity and accuracy of within-run and between-run. According to measurement of the amount of suspended particulates in a clean room, 0.3~62 particles were detected in 0.3 µm size while 0.0~28.3 particles were observed in 0.5 µm size. Total suspended particulates met required environment with up to 90.3 particles. The MDL (Method detection limit) of the sample which has been fabricated using fetal bovine serum (FBS) blank was 1.77 ng/L, and LOQ (Limit of quantification) was 5.55 ng/L. The slope, intercept and correlation coefficient of the calibration curve were y=1.09×10⁻³x+4.88×10⁻² and r=0.9999, which showed good correlation. The specificity, within-run and between-run accuracy satisfied the standard at more than 50 ng/L. The average lead concentration in plasma of the general people, current workers and retired workers was 55.4 ng/L, 440 ng/L, and 132 ng/L.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.665-683
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• 2002

A novel glucanhydrolase from a mutant of Lipomyces starkeyi KSM 22 has additional amylase activity besides mutanolytic activity and has been suggested as promising anti-plaque agent. It has been shown effective in hydrolysis of mutan, reduction of mutan formation by Streptococcus mutans and removal pre-formed sucrose-dependent adherent microbial film and has been strongly bound to hydroxyapatitie. These in vitro properties of Lipomyces starkeyi KSM 22 glucanhydrolase are desirable for its application as a dental plaque control agent. In human experimental gingivitis　model and 6 month clinical trial, mouthrinsing with Lipomyces　starkeyi KSM 22 dextranase was comparable to 0.12% chlorhexidine mouthwash in inhibition of plaque accumulation and gingival inflammation and local side effect was negligible. This study was aimed to evaluate the cytotoxic effect of Lipomyces starkeyi KSM 22 glucanhydrolase on human gingival fibroblasts. Primary culture of human gingival fibroblasts at the 4th to 6th passages were used. Glucanhydrolase solution was made from lyophilized glucanhydrolase powder from a mutant of Lipomyces stakeyi KSM 22 solved in PBS and added to DMEM medium to the final concentration of 0.5, 1, and 2 unit. Cells were exposed to glucanhydrolase solution or 0.1 % chlorhexidine and the cells cultured in DMEM with 10% FBS and 1% antibiotics as control. After exposure, the morphological change, cell attachment, and cell activity by MTT assay were evaluated in 0.5, 1.5, 3, 6, 24 hours after treatment. The cell proliferation and cell activity was also evaluated at 2 and 7 days after 1 minute exposure, twice a day. The cell morphology was similar between the Lipomyces smkeyi KSM 22 glucanhydrolase groups and control group during the incubation periods, while most fibroblasts remained as round cell regardless of incubation time in the chlorhexidine group. The numbers of the attached cells in the glucanhydrolase groups were comparable to that of control and significantly higher than the chlorhexidine group. The numbers of the proliferated cells in the glucanhydrolase groups at 7 days of incubation were comparable to the control group and higher than the chlorhexidine group. The cell activity in glucanhydrolase groups paralleled with the increased cell number by attachment and proliferation. According to these results, Lipomyces starkeyj KSM 22 glucanhydrolase has little harmful effect on attachment and proliferation of human gingival fibroblasts, in contrast to 0.1% chlorhexidine which was cytotoxic to human gingival fibroblasts. Therefore this glucanhydrolase preparation is considered as a safe and promising agent for new mouthwash formula in the near future.

• The Journal of Internal Korean Medicine
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• v.36 no.4
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• pp.518-526
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• 2015

Objectives This study investigated the effect of purified safflower (Carthamus tinctorius Linne) and safflower seed (Carthamus tinctorius L. seed; CS) extract, using hot water and ethanol extract methods , on the osteogenic differentiation of MC3T3E1 cells.Methods The safflower and safflower seed were extracted with hot water and ethanol. The samples were concentrated by a rotary evaporator and then freeze-dried using a freeze-dryer. The MC3T3E1 cells were propagated and maintained in DMEM (Gibco) containing 10% FBS and a 1% antibiotic antimycotic solution. To induce osteogenic differentiation, the cells were treated for 14 days with DMEM with 10 mM β-glycerophosphate and 50 μM ascorbic acid. Extract doses were confirmed by the results of an MTT assay, and treatment of the extracts was performed in a differentiation medium every two days. The ALP staining and activity were tested after osteogenic differentiation for five days, and after 14 days, osteogenic differentiation was determined by alizarin red S staining. The mRNA expressions of osteogenic-related genes were quantified using quantitative real-time PCR.Results In the results of the MTT assay, all concentrations of safflower extracts had no toxicity in the MC3T3El cells. But in the groups of 100 ng/ml and 200 ng/ml concentrations of safflower seed extracts, the cell viability was significantly reduced by up to 40-50%. So we fixed the treatment concentration of the extract at 50 ng/ml. In the ALP and alizarin red S staining, all extract groups increased osteogenic differentiation compared with the control group. The water-safflower extract group showed the highest mRNA level of Alp, Runx2, and Dlx5 genes. The mRNA level of Ocn, an osteogenic gene related to late-stage differentiation, in the ethanol-safflower extract group increased the mineralization more significantly than in other groups.Conclusions These data suggest that the extract of safflower increases the osteoblastic differentiation activates of MC3T3E1 cells like the extract of safflower seed. The water-extract and ethanol-extract of safflower have effects on different stages of osteogenesis in MC3T3El. Not only safflower seed but also safflower will be useful therapeutic reagents for age-associated chronic diseases such as osteoporosis.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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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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• v.24 no.2
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• pp.303-320
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• 1994

Current acceptable methods For promotin gperiodontal regeneration are base on removal of diseased soft tissue, root treatment, guided tissue regeneration, inteoduction of new graft materials and biological mediators. Platelet-derived growth factor(PDGF) is one of polypeptide growth factor. PDGF has been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. The purposes of this study is to evaluate the effects of PDGF-AA, BB on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the first premolar tooth extracted for the orthodontic treatment and were cultured in DMEM/10% FBS at the $37^{\circ}C$, 5% $CO_2$ incubator. Author measured the DNA synthesis, total protein, collagen and noncollagenous protein synthesis and alkaline phosphatase activity according to the concentration of PDGF-AA and BB(0, 0.1, 1, 10, 100ng/ml). The results were as follows : The DNA synthetic activity was increased dose dependently by PDGF-AA and BB. The maximum mitogenic effect was at the 100ng/ml of PDGF-AA and 10ng/ml of PDGF-BB. The total protein, collagen and noncollagen systhesis was increased dose dependently by PDGF-AA and BB. The % of collagen was slightly decresed according to the concentration of PDGF-AA and BB. The effect of PDGF-AA and BB were not specific for collagen synthesis since it also increased noncollagenous protein synthesis. The effect of PDGF-AA and BB on alkaline phosphatase activity did not show any significant, meanwhile the alkaline phosphatase activity of 14 days group showed significnat increase. In conclusion, PDGF-AA and BB may have important roles in stimulation of DNA synthesis in human periodontal ligament cells, which means an increase in collagen-synthesizing cells, and may be useful for clinical application in periodontal regenerative procedures.