• Journal of Periodontal and Implant Science
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• 제30권2호
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• pp.389-405
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• 2000

The purpose of this study is to evaluate the effect of IGF-I for DNA synthetic activity and the mRNA expression of bone matrix protein, type I collagen and osteopontin in prolifetation and differentiation of MC3T3-E1 cells. To evaluate DNA synthetic activity, cells were seeded at $2{\times}10^4cells/ml$ in 24 well plates and to evaluate mRNA of type I collagen and osteopontin cells were seeded at $5{\times}10^5cells/ml$ in 100mm culture dishes. These cells were cultured in alpha-minimum essential medium(${\alpha}-MEM$) containing 10% fetal bovine serum at $37^{\circ}C$, 5% $CO_2$ incubator. For DNA synthetic activity test 1, 10, 100ng/ml IGF-I were added to the cells which had been cultured for 3 days before 24 hours. For type I collagen mRNA expression 1, 10ng/ml IGF-I were added to the cells which had been cultured for 5, 10 days and for osteopontin mRNA expression 0.1, 1, 10ng/ml IGF-I were added to the cells which had been cultured for 5, 15, 20 days. Cell proliferaton was measured by the incorporation of [$^3H$]-thymidine into DNA and expression for type I collagen and osteopontin were measured by northern blot analysis. The results were as follows : DNA synthetic activity were generally higher in experimental group than control group. Expressions of type I collagen mRNA were higher at 5 day group and much lower at 10 day group in the control groups. In the experimental groups, mRNA expressions were slightly increased when 1 ng/ml IGF-I were added to 5 day group and decreased in all experimental 10 day groups. Expressions of osteopontin mRNA were higher at 20 day groups and lower at 15 day groups than the control groups. In the experimental groups, mRNA expressions were incereased when 0.1, 1 ng/ml IGF-I were added to 5 day group and in all the 15 day groups, but decreased when 0.1, 1, 10 ng/ml IGF-I were added to 20 day groups. IGF-I stimulated DNA synthetic activity of MC3T3-E1 cells during proliferation stage significantly, did not greatly changed effects on type I collagen mRNA expression and stimulated osteopontin mRNA expression at 15 day especially. In conclusion, we suggests that IGF-I have a tendency of stimulation effect of DNA synthetic activity but do not stimulate type I collagen mRNA in proliferation stage of MC3T3-E1 cell cultures, and stimulate osteopontin mRNA in differentiation stage of MC3T3-E1 cell cultures.

• Journal of Periodontal and Implant Science
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• 제30권2호
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• pp.347-360
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• 2000

Bone remodeling results from the combined process of bone resorption and new bone formation which is regulated in part by some of the polypeptide growth factors such as platelet derived growth factor(PDGF), which has been known to be an important local regulator of bone cell activity and participate in normal bone remodeling. This process includes strictly regulated gene expression of several bone matrix proteins such as type I collagen and osteopontin, a 44 kDa phosphorylated glycoprotein, which has important roles in bone formation. The purpose of this study is to evaluate the effecs of PDGF-BB on the mRNA expression of bone matrix protein, type I collagen and osteopontin, in MC3T3- E1 cell culture. Cells were seeded at $5{\times}10^5$ cells in 10 ml of minimum essential medium alpha(${\alpha}-MEM$) containig 10% fetal bovine serum, 10 mM beta glycerophosphate. 0.1, 1, 10 ng/ml PDGF-BB were added to the cells for the day 3, 7, 14, 21, 28 and cultured for 24 hours. Type I collagen cDNA, Hf677, and osteopontin cDNA were used as probes for northern blot analysis. Total cellular RNA was purified at indicated day and northern blot analysis was performed. The results were as follows : Type I collagen mRNA expressions were higher at the day 3 and 7, and lower in the day 14, 21 in the control groups. In the experimental groups, mRNA expressions were increased when 0.1 ng/ml PDGF-BB were added on the day 3, 7, 21, and decreased in dose-dependent manner on the day 14, decreased at all added dose on the day 28. Osteopontin mRNA expressions were highest in the day 21 groups and lowest in the day 14 groups in the control groups. Interesting results were shown in the day 14 and 21 groups. We found that osteopontin mRNA level was increased in dose dependent manner in the day 14 groups, and decreased dose dependent manner in the day 21 groups. In conclusion, PDGF-BB may have various control effects on type I mRNA expression in the growth and differentiation process of MC3T3-E1 cells and may have contrary regulatory effects on osteopontin mRNA expression. For examples, when the baseline level of osteopontin mRNA was low, as in the day 14, PDGF-BB up-regulated osteopontin mRNA expression in dose dependent manner, and when the baseline level was high as in the day 21, PDGF-BB down-regulated dose dependent manner. Thus, it may be useful for clinical application in periodontal regeneration procedure if further study were performed.

• 한국가축번식학회지
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• 제23권1호
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• pp.53-61
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• 1999

본 연구는 생쥐 preantral follicle 의 체외성장 및 성숙에 있어서 gonadotrophins 인 FSH 와 LH 의 효과를 조사하기 위하여 실시하였으며, 그 결과는 다음과 같다. 1. FSH 첨가군들은 대조군에 비하여 유의하게 높은 생존율과 성숙율을 나타냈으며, 100$m\ell$D/$m\ell$ 의 FSH 농도가 preantral follicle 의 체외배양에 적정한 농도인 것으로 나타났다. 2. HMG 첨가군은 FSH 첨가군보다, 통계적 유의차는 인정되지 않았지만, 높은 생존율과 성숙율을 나타냈다. 3. FSH 와 LH 의 첨가비율이 100$m\ell$U/$m\ell$ 대 10$m\ell$D/$m\ell$(10:1) 에서 가장 높은 생존율과 성숙율을 나타냈다. 4. FSH 혹은 HMG 첨가시, 정상적인 oestradiol 과 progesterone 분비양상을 나타냈으며, HMG 첨가군에서 유의하게 높은 농도의 oestradiol 과 progesterone 을 분비하였다. 이상의 결과를 종합해 볼 때, gonadotrophins 은 preantral follicle 의 체외성장 및 성숙뿐만 아니라 steroidogenesis 에서 중요한 역할을 수행한다는 것을 확인할 수 있었다.

• 한국식품위생안전성학회지
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• 제28권2호
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• pp.89-94
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• 2013

마늘 숙성 중 생성되는 S-allylmercaptocysteine의 콜레스테롤 생합성 억제 효과에 대하여 Hepatocytes를 이용하여 조사하였다. HepG2 cells을 Dulbecco's modified Eagle's medium (DMEM)에 배양하여 S-allylmercaptocysteine의 농도를 20, 40, 60, 80 및 100 mL 씩 각각 첨가하여 cell viability를 살펴본 결과 20~40 ${\mu}g/mL$에서는 높았으며, 60 ${\mu}g/mL$ 농도에서 약 50%가 유지되었다. S-allylmercaptocysteine을 5, 10, 15 및 20 ${\mu}g/mL$ 농도로 [$^{14}C$]-acetatecholesterol에서 처리하였을 경우 15 ${\mu}g/mL$ 농도에서 cholesterol 생합성이 79%로 억제되었다. Fatty acid synthase의 활성은 0.95 nmol에서 19%의 억제효과를 나타내었으나, Glucose 6-phosphate dehydrogenase (G6PDH)의 활성에는 거의 영향을 미치지 않았다. S-allylmercaptocysteine의 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase의 활성은 용량 의존형으로 감소하였다. 이상의 결과로 보아 마늘 숙성 과정에서 생성되는 주요 성분인 S-allylmercaptocysteine은 간 세포에서 cholesterol의 생합성을 억제하는데 기여하는 것으로 나타났다.

• 한국수정란이식학회지
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• 제16권2호
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• pp.91-98
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• 2001

본 연구는 phosphatidylinositol 대사의 기질 및 억제물질이 돼지 난모세포의 체외 성숙·수정에 미치는 영향에 대하여 실험을 하였다. 난모세를 inositol (250 mM) 첨가 또는 무첨가한 mTLP-PVA 배양액에서 46시간 체외배양하였다. 성숙이 완료된 난모세포는 신선 돼지정액을 이용하여 6시간 동안 mTALP-PVA 배양액에서 체외수정을 시켰다. 수정 후 6시간에 이들 난모세포를 10% 혈청을 첨가한 TCM-199 배양액에서 추가로 12시간 배양하였다. 제 2성숙분열중기로 성숙한 남모세포의 비율은 대조구 (67.3%)에 비하여 inositol (81.4%)을 첨가하여 46시간 체외성숙시킨 난모세포에서 더 높았다 (P<0.05). 체외수정 후 18시간에 웅성전핵 형성율은 대조구의 27.3%보다 inositol (42.0%)을 첨가한 배지에서 성숙시킨 난모세포에서 더 높게 나타났다 (P<0.05). 난모세포의 성숙에 대한 inositol의 작용을 조사하기 위하여 LiCl (10 mM) 또는 dbcAMP (0.5 mM)를 첨가한 배지에서 난모세포를 배양하였을 때, 이들 두 약제는 난모세포의 성숙을 억제하였다 (P<0.05). 그러나 이 두 약제의 억제작용은 inositol 첨가에 의해 해제되어 대조구 수준까지 성숙율이 개선되었다. DbcAMP와 verapamil은 상승작용을 하여 난모세포의 성숙을 억제하였다. Verapamil을 단독으로 첨가하였을 때, 난모세포의 성숙분열 개시에는 영향이 없었으나 제 2성숙분열중기로의 성숙은 억제되었다. 이상의 결과로부터, inositol 첨가에 의해 PI-Cycle이 활성화되어 간모세포의 핵 및 세포질 성숙과 막의 변화가 유도되어 난모세포의 성숙이 inositol 첨가에 의해 개선되었다는 것이 시사되었다.

• Journal of Pharmaceutical Investigation
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• 제31권4호
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• pp.241-256
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• 2001

Alginate microspheres, containing fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) or green fluorescent protein (GFP) were prepared and used as a model drug to develop the oral vaccine delivery system. The alginate microspheres were coated with poly-L-lysine or chitosan. Two methods, w/o-emulsion and spray, were used to prepare alginate microspheres. To optimize preparation conditions, effects of several factors on the particle size and particle morphology of microsphere, and loading efficiency of model antigen were investigated. In both preparation methods, the particle size and the loading efficiency were enhanced when the concentration of sodium alginate increased. In the w/o-emulsion preparation method, as the concentration of Span 80 was increased from 0.5% to 2%, the particle size was decreased, but the loading efficiency was increased. The higher the emulsification speed was, the smaller the particle size and loading efficiency were. The concentration of calcium chloride did not show any effect on the particle size and loading efficiency. In the spray preparation method, the particle size was increased as the nozzle pressure $(from\;1\;kgf/m^2\;to\;3\;kgf/m^2)$ and spray rate was raised. Increasing calcium chloride concentration (<7%) decreased the particle size, in contrast to no effect of calcium chloride concentration on the w/o-emulsion preparation method. Alginate microspheres prepared by two methods were different in the particle size and loading efficiency, the particle size of microspheres prepared by the spray method was about $2-6\;{\mu}m$, larger than that prepared by the w/o emulsion method $(about\;2{\mu}m)$, and the loading efficiency was also higher with spray method. Furthermore, drying process for the microspheres prepared by the spray was simpler and easier, compared with the w/o emulsion preparation. Therefore, the spray method was chosen to prepare alginate microspheres for further experiments. Release pattern of FITC-BSA in alginate microspheres was evaluated in simulated intestinal fluid and PBS (phosphate buffered saline). Dissolution rate of FITC-BSA from alginate/chitosan microsphere was lower than that from alginate microsphere and alginate/poly-L-lysine microsphere. By confocal laser scanning microscope, it was revealed that alginate/FITC-poly-L-lysine microspheres were present in close apposition epithelium of the Peyer's patches of rabbits following inoculation into lumen of intestine, which proved that microspheres could be taken up by Peyer's patch. In conclusion, it is suggested that alginate microsphere prepared by spray method, showing a particle size of & $10\;{\mu}m$ and a high loading efficiency, can be used as a model drug for the development of oral vaccine delivery system.

• Journal of Animal Science and Technology
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• 제52권3호
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• pp.175-186
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• 2010

The aim of this study was to analyze the proteome expressions of proliferating stromal vascular cells from Hanwoo omental, subcutaneous and intramuscular depots subjected to hormone deprivation and IGF-1, Estradiol-$17{\beta}$ addition. For hormone deprivation or addition studies, the cells were either grown in 10% charcoal-dextran stripped fetal bovine serum (CD-FBS) or in 10% FBS supplemented medium. Further, to analyze the effect of insulin like growth factor (IGF-1) and $17\beta$-Estradiol (E2), cells were grown in 10% CD-FBS containing IGF-1 (10 ng/ml) or E2 (10 nM). The results showed that hormone deprivation had a negative impact on proliferation among the cells from all depots without any growth difference. On comparison of proliferation levels, higher levels were observed in cells that were grown in 10% FBS than in 10% CD-FBS alone or with IGF-1/E2. Proteome expression from preadipocytes grown in hormone deprivation conditions were compared by 2D-DIGE and MALDIToF/ToF. A total of twelve different proteins were found to be differentially expressed under hormone deprivation conditions. Further, our proteomic analysis with DIGE under IGF-1 and E2 addition revealed four proteins with differential expression levels. Moreover, the results highlighted in this study offer a role for each differentially expressed protein with respect to their effect in positive or negative regulation on proliferation.

• 한국축산식품학회지
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• 제20권2호
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• pp.101-106
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• 2000

유지방구로부터 분리 정제한 lipase의 활성과 안정성에 미치는 pH의 영향을 야자유와 균질유를 기질로 하여 조사하였다. 효소원으로 buttermilk를 사용하였을 경우는 반응온도 $37^{\circ}C$에서 야자유, 균질유 모두 pH 9.5에서 최대 활성을 나타내었으며, 전형적인 종형의 pH의 존곡선을 나타내었다. 그러나 반응온도 $0^{\circ}C$에서는 pH 10.0까지 활성이 증가하였다. 정제 lipase를 사용한 경우는 반응응도 $37^{\circ}C$에서 균질유를 기질로 사용하였을 때 종 모양의 곡선을 나타내었으며, 최대 활성은 pH 9.0에서 나타났다. 반응온도 $0^{\circ}C$에서는 pH 10.0까지 활성이 계속 증가하였다. BSA를 첨가한 야자유를 기질로 사용한 경우는 반응온도 $37^{\circ}C$, $0^{\circ}C$ 모두 최대 활성이 pH 9.5에 나타났으며, pH 10.0에서는 활성이 현저하게 저하하였다. 정제 lipase의 안정성에 미치는 pH의 증가에 따라 현저하게 저하하였다. pH 10.0에서 $37^{\circ}C$로 20분간 효소를 유지하였을 경우 lipase의 활성은 pH 8.5 때의 활성에 비하여 13%로 감소하였다. 그러나, $4^{\circ}C$에서 60분간 효소를 유지하였을 경우는 lipase 활성이 pH 7.5∼10.0의 범위에서 안정하였다.

• 한국수정란이식학회지
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• 제24권1호
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• pp.21-27
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• 2009

This study was performed to elucidate the effects of addition of ${\beta}-lactoglobulin$ and bovine serum albumin (BSA) in vitro maturation (IVM) and in vitro culture (IVC) medium on porcine embryo production. The development rate to the 2 cell ($71.4{\sim}75.6%$) and blastocyst stages ($6.8{\sim}13.3%$) with different BSA concentrations in IVM medium were similar among treatment groups. Blastocyst hatching rate was significantly higher in the control group (0.0mg/ml) than in the group of 1.0mg/ml supplement (20.0% vs. 0.0%; p<0.05). The development rate to the 2 cell ($62.0{\sim}70.6%$) and blastocyst stages ($15.4{\sim}38.5%$) with different ${\beta}-lactoglobulin$ concentrations in IVM medium was similar among treatment groups. The development rate to the blastocyst was significantly higher in the group of 1.0mg/ml(15.3%) than in the group of 0.5mg/ml supplement (7.6%, p<0.05). The development rate to the 2 cell and blastocyst stages following the first addition of ${\beta}-lactoglobulin$ in IVM medium was significantly higher in the control group (77.0% and 18.9%) and was $0{\sim}44\;hr$(77.2% and 16.9%) greater than that observed in other treatment groups (p<0.05). The development rate to the 2 cell stage ($68.1{\sim}74.8%$) and blastocyst stages ($9.2{\sim}12.7%$) with different BSA concentrations in IVC medium was similar among treatment groups. However, blastocyst hatching rate was significantly higher in the group of 3.0mg/ml supplement (30.0%) than in the control group (0.0%; p<0.05). The development rate to the 2 cell stage ($72.9{\sim}78.0%$), blastocyst ($7.1{\sim}14.2%$) and hatching stages ($33.3{\sim}38.1%$) were not different. The development rate to the 2 cell stage ($63.6{\sim}72.5%$), blastocyst ($8.4{\sim}16.1%$) and hatching stages ($18.2{\sim}37.5%$) at the different culture periods were similar among treatment groups. This study suggested that if the addition level and periods of ${\beta}-lactoglobulin$ addition are adjusted, it is possible to replace BSA in the in vitro porcine embryo production.

• 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.