• Journal of Periodontal and Implant Science
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• 제28권1호
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• pp.17-35
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• 1998

Chitosan, with a chemical structure similar to hyaluronic acid, has been implicated as a wound healing agent. The purpose of this research was to evaluate the effects of chitosan on the characteristics of periodontal ligament cells, calvaria cells and gingival fibroblasts and to define the effects of chitosan on bone formation in vitro. In control group, the cells were cultured alone with Dulbecco's Modified Eagle's Medium contained with 10% Fetal bovine serum, 100unit/ml penicillin, $100{\mu}g/ml$ streptomycin, $0.5{\mu}g/ml$ amphotericin-B. In experimental group, chitosan($40{\mu}g/ml$) is added into the above culture condition. And then each group was characterized by examining the cell proliferation at 1,3,5,7,9,12,15 day, the amount of total protein synthesis, alkaline phosphatase activity at 3, 7 day and the ability to produce mineralized nodules of rat calvaria cell at 11 day. The results were as follows : 1. At early time both periodontal ligament cells and calvaria cells in chitosan-treated group proliferated more rapidly than in non-treated control group, but chitosan-treated group of periodontal ligament cells at 9 days and calvaria cells at 12days showed lower growth rate than control group. Gingival fibroblast in chitosan-treated group had lower growth rate than in control group but the difference was not statistically significant (P< 0.01).2. Both periodontal ligament cells and calvaria cells in chitosan-treated group showed much protein synthesis than in control group at 3 days, but showed fewer than in control group at 7 days. Amount of total protein synthesis of gingival fibroblast didn't have statistically significant difference among the two groups(P< 0.01). 3. At 3 and 7 days, alkaline phosphatase activity of periodontal ligament cells and calvaria cells was increased in chitosan-treated group, but at 7 days there was not statistically significant difference among the two groups of calvaria cells (P< 0.01). Alkaline phosphatase activity of gingival fibroblast didn't have statistically significant difference among the two groups(P<0.01). 4. Mineralized nodules in chitosan-treated group of rat calvaria cells were more than in control group. In summery, chitosan had an effect on the proliferation, protein systhesis, alkaline phosphatase activity of periodontal ligament cells and calvaria cells, and facilitated the formation of bone. It is thought that these effects can be used clinically in periodontal regeneration therapy.

• 한국가축번식학회지
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• 제26권4호
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• pp.329-338
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• 2002

This study was carried out to investigate the developmental rates of embryo reconstructed with different cell type and to estimate correlation of transcriptional level of octamer-binding transcription factor 4 (Oct4) and fibroblast growth factor 4 (FCF4) gene on peri-implantation stage embryos. Donor cells were transferred into perivitelline space of enucleated oocytes. The karyoplast-cytoplast couplets were accom- plished by cell to cell fusion and activated with ionomycin and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CR 1 aa medium. There is no difference in blastocyst formation rate following nuclear transfer UT) with fetal fibroblast cell (16/50; 32.0%), cumulus cell (16/49; 32.6%) and ear cell (17/52; 32.6%). The expression level of Oct4 and FCF4 in peri-implantation bovine embryo derived from in vitro fertilization (IVF) and NT were determined by reverse-transcription polymerase chain reaction (RT-PCR) technique. In peri-implantation of IVF result in a transient increased of FCF4 paralleled by an increased expression of Oct4. However, Oct4 gene was highly expressed in hatching blastocysts derived from NT compared to IVF. Also, FGF4 expression level in hatching blastocysts and outgrowth stage derived from NT was lower than that of IVF. In conclusion, it is suggested that the different transcription patterns observed in nuclear transfer embryos may lead to a lower rate of embryo development, implantation and pregnancy.

• 대한세포병리학회지
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• 제6권2호
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• pp.106-115
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• 1995

Reactive humsn mesothelial cells were examined by immunocytochemical stain with intermediate filaments (cytokeratin [CK1, CK7, CK8, CK18, CD19), vimentin, desmin, actin), epithelial membrane antigen, carcinoembryonic antigen (CEA), MHC class II antigen (HLA-DR), LeuM-1 (CD15), $\alpha1-antitrypsin$(ACT), $\alpha1-antichymotrypsin$ (ACHT), CD68(KP-1) and FcyRIII(CD16). The mesothelial cells were isolated from patients with liver cirrhosis and pleural effusion, and short-term cultured in RPMI 1640 media containing 10% heat inactivated fetal calf serum and 1% identical supernatant fluid of the patients' transudates. The results obtained are as follows 1. The cultured-reactive mesothelial cells were positive for the protein of cytoskeleton such as cytokeratin and vimentin, but negative for desmin and actin. The resting mesothelial cells showed positive reactions for cylokeratin, but negative for vimentin, desmin and actin. 2. The primary antibodies to the cytokeratin were strongly reactive for CK1, CK8 and CK18 but negative for CK7 and CK19 in both reactive and resting mesothelial cells. 3. Resting mesothelial cells showed negative reactions for CEA, but strong positive reactions in cultured-reactive mesothelial cells. 4. The markers for the monocytes/histiocytes(CD11b, CD14, CD16, CD68, Iysozyme and $\alpha1-antitrypsin$ and $\alpha1-antichymotrypsin$) were nonreactive in resting mesothelial cells, but lysozyme and $\alpha1-antitrypsin$ were weakly reactive in reactive and proliferative mesothelial cells. 5. MHC Class II molecule(HLA-DR antigen) was negative in both resting and reactive mesothelial cells. These results suggest that the short-term cultured, reactive mesothelial cells show a newly aberrant expression of the vimentin and calcine-embryonic antigen. The reason of the aberrant expression of the intermediate filament and oncofetal antigen in reactive and proliferative mesothelial cells should be further evaluated.

• 한국가축번식학회지
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• 제20권2호
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• pp.191-196
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• 1996

The objective of this study were to investigate the effects of culture media and additives on the development of bovine in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. In experiment 1, bovine oocytes were cultured in droplets of TC 199 supplemented with 10% fetal bovine serum(FBS) with or without hormones (5$\mu\textrm{g}$/ml FSH, 5$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml E2). Cleavage rates of embryos cultured for 40~44hrs after IVF were higher when embryos were cultured in TC 199 supplemented hormones (68.1%, 921/35) than without hormones (52.7%, 77/146), but the percentages of development beyond morulae stage were not difference (20.7%, 19.4%). In experiment 2, the effects of various media such as TC 199, synthetic oviduct fluid(SOF), CR1aa with different energy source (fatal bovine serum, FBS; bovine serum albumin, BSA) on developmental capacity of IVM/IVF bovine embryos were investigated. The developmental rates into morulae and blastocysts were 27.1, 10.7, 6.3 and 0%, respecitvely, in CR1aa plus 3mg/ml BSA, SOF plus 10% FBS, TC 199 plus 10% FBS, SOF plus 3mg/ml BSA. In experiment 3, the comparisons of bovine embryos developed to morulae and blastocysts in different culture media (TC 199, SOF, CR1aa, Menezo's B2) were investigated. The developmental capacity beyond morulae stage were 32.9, 26.6, 11.1 and 7.1%, respectively, in Menezo's B2 plus BSA, CR1aa plus BSA, SOF plus BSA, TC 199 plus FBS medium. The cell numbers of the blastocyst were not different in different cultrue media. In experiment 4, bovine embryos were co-cultured with vobine oviduct epithelial cells(BOEC) in TC 199 plus FBS, SOF plus BSA, CR1aa plus BSA, Menezo's B2 plus BSA. The morula and blastocyst rates were 44.7, 32.9, 26.0 and 23.3%, respectively, in CR1aa TC 199, SOF, and Menezo's B2 medium. The cell numbers of the blastocyst were similar to those of blastocyst developed in different culture media.

• Animal Bioscience
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• 제36권11호
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• pp.1757-1768
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• 2023

Objective: The number of bovine mammary epithelial cells (BMECs) is closely associated with the quantity of milk production in dairy cows; however, the optimal levels and the combined effects of hormones and essential amino acids (EAAs) on cell proliferation are not completely understood. Thus, the purpose of this study was to determine the optimal combination of individual hormones and EAAs for cell proliferation and related signaling pathways in BMECs. Methods: Immortalized BMECs (MAC-T) were treated with six hormones (insulin, cortisol, progesterone, estrone, 17β-estradiol, and epidermal growth factor) and ten EAAs (arginine, histidine, leucine, isoleucine, threonine, tryptophan, lysine, methionine, phenylalanine, and valine) for 24 h. Results: Cells were cultured in a medium containing 10% fetal bovine serum (FBS) as FBS supplemented at a concentration of 10% to 50% showed a comparable increase in cell proliferation rate. The optimized combination of four hormones (insulin, cortisol, progesterone, and 17β-estradiol) and 20% of a mixture of ten EAAs led to the highest cell proliferation rate, which led to a significant increase in cell cycle progression at the S and G2/M phases, in the protein levels of proliferating cell nuclear antigen and cyclin B1, cell nucleus staining, and in cell numbers. Conclusion: The optimal combination of hormones and EAAs increased BMEC proliferation by enhancing cell cycle progression in the S and G/2M phases. Our findings indicate that optimizing hormone and amino acid levels has the potential to enhance milk production, both in cell culture settings by promoting increased cell numbers, and in dairy cows by regulating feed intake.

• 한국가축번식학회지
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• 제23권4호
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• pp.323-331
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• 1999

본 연구는 돼지난자의 체외성숙동안 insulin-like growth factor-I (IGF-I)과 난구세포의 영향을 검토하기 위하여 실시하였다. 미성숙난자를 0(60%), 1(61%), 5(62%) 및 l0ng/$m\ell$(72%)의 서로 다른 IGF-I의 농도로 첨가하여 배양했을 때 체외성숙율은 큰 차이를 나타내지 않았다. 또한 10ng/$m\ell$의 IGF-I이 첨가된 배양액내에서 난자를 배양한 경우, 난구세포부착시 (68%) 제거 (52%)된 난자에 비해 높은성숙율을 나타냈지만 유의적인 차이는 인정되지 않았다. 그러나 IGF-I이 첨가되지 않은 경우에는 난구세포 부착 (63%) 난자가 제거 (32%)된 난자에 비해 유의적 (P＜0.05)으로 높은 성숙율을 나타냈으며, IGF-I를 성숙 배양기간중 전반기 24시간 또는 후반기 24시간 동안만 첨가했을 때의 난구세포 부착시 (61과 49%) 제거된 (45 와 49%) 난자에 비해 높은 성숙융올 나타냈다. 한편, IGF-I의 존재 여부에 관계없이 FCS와 돼지난포액 (PFF)이 무첨가된 배양액에서 48시간동안 또는 배양전반기 24시간동안 난구세포를 제거한 경우 (16과 18%)로 난구세포 부착시 (46과 63%)에 비해 유의적 (P＜0.01)으로 낮은 성숙율을 나타냈다. 이와 같은 결과는 난구세포가 IGF-I의 존재 유무에 관계없이 난자의 체외성숙에 필수적이며, FCS와 PFF 첨가시 난구세포가 제거된 난자의 체외성숙을 촉진하는 것으로 밝혀졌다.

• Asian-Australasian Journal of Animal Sciences
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• 제28권3호
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• pp.411-419
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• 2015

We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained $10{\mu}g/mL$ insulin and $10{\mu}g/mL$ pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with $40{\mu}M$ palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta ($CPT1{\beta}$) gene expression, but the increase in $CPT1{\beta}$ gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17-fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased $AMPK{\alpha}$ gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

• 한국가축번식학회지
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• 제14권3호
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• pp.183-190
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• 1990

These studies were carried out ot investigate the effects of estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3-5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48 hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18$^{\circ}C$20 hrs. with motile capacitated sperm in the TCF(Tyrode calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes harvested, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 33.3%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5%~20% ECS and FCS were 74.0%~80.6, 26.2%~30.0% and 71.7%~76.9%, 51.9%~58.0%, and those values were higher the supplement of ECS than FCS. 3. The maturation rate(68.0%~64.6%) and fertilization rate(59.6%~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 4. The maturation rate(76.5%) and fertilization rate(61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$106/ml cumulus cells were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$104~5/ml and 1$\times$108/ml cumulus cells.lus cells.

• 한국발생생물학회지:발생과생식
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• 제11권3호
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• pp.235-243
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• 2007

인간 제대혈 세포는 조혈모세포, 중간엽 줄기세포와내피전구세포를 풍부하게 포함하고 있다. 인간 제대혈 속의 중간엽 줄기세포는 조혈모세포와는 달리 다능성 줄기세포이며 신경세포로 분화할 수 있는 잠재성을 가지고 있다. 본 연구에서는 세포배양을 통해 제대혈의 중간엽 줄기세포를 신경세포와 콜린성 신경세포로 분화를 유도하였다. 중간엽 줄기세포를 신경세포로 분화시키기 위해 배양액에 dimethyl sulphoxide(DMSO)와 butylated hydroxyanisole(BHA)를 첨가하여 유도하였으며 basic fibroblast growth factor(bFGF), retinoic acid(RA), sonic hedgehog(Shh)를 처리하여 콜린성 신경세포로 분화시켰다. DMSO와 BHA에 처리된 중간엽 줄기세포가 빠르게 신경세포 모양으로 분화하는 것을 관찰하였으며, 이것은 면역조직학적 염색에서 신경세포 특이 표지인 $\beta$-tubulin III, 별아교세포에 대한 특이 표지인 GFAP, 희돌기아교세포에 대한 특이 표지인 Gal-C에 대해 양성반응을 나타내었고, 그 비율은 각각 $32.3{\pm}2.9%$, $11.0{\pm}0.9%,\;9.4{\pm}1.0%$였다. RT-PCR 분석에서 배양 단계에 따라 신경세포에 특이적인 표지 인자가 발현됨을 통해, 중간엽 줄기세포가 신경세포로 분화됨을 확인하였다. 또한, 중간엽 줄기세포에 bFGF, RA, Shh를 처리하여 콜린성 신경세포로 분화시켰을 때, 전체 중간엽 세포 중 $31.3{\pm}3.2%$가 신경세포 특이 표지인 $\beta$-tubulin III에 양성반응을 보였으며 이들 세포 중 $70.0{\pm}7.8%$가 콜린성 신경 특이 표지인 ChAT에 양성반응을 보였고, 이것은 Woodbury 방법에 의한 신경분화의 경우보다 3배 가량 높은 비율로 콜린성 신경의 분화를 유도한 것이다. 이러한 실험 결과들은 인간 제대혈의 중간엽 줄기세포가 콜린성 신경세포로 분화가 가능하고 이러한 잠재성을 가진 제대혈 중간엽 줄기세포는 퇴행성 신경질환에 대한 세포 치료제로서 가능성을 제시한다.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.119-124
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• 2006

The present study compared the developmental potential of cloned porcine embryos with mesenchymal stem cells (MSCs), fetal fibroblasts (FFs) and cumulus cells (CCs) by assessing the cleavage and blastocyst rate, total cell number, inner cell mass (ICM) ratio and apoptosis. MSCs were isolated by ficoll gradients from femur of -6 month old female pig, and maintained for primary cultures. FFs from a female fetus at ${\sim}30$ day of gestation were established, and CCs were obtained from cumulus oocyte complexes (COCs) aspirated from $3{\sim}6$ mm follicles in diameter. Donor cells at $3{\sim}4$ passage were employed for nuclear transfer (NT). COCs were matured and fertilized in vitro(IVF) as control. Cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs, FFs and CCs ($82.7{\pm}8.9%\;vs\;70.6{\pm}5.4,\;68.7{\pm}5.1\;and\;63.4{\pm}5.6%$, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs ($24.5{\pm}2.8\;and\;20.4{\pm}8.3%$) did not differ, but were significantly (P<0.05) higher than NT derived from FFs and CCs ($10.6{\pm}2.7\;and\;9.8{\pm}2.1%$). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs ($35.4{\pm}5.2\;and\;0.40{\pm}0.09%$, respectively) were significantly (P<0.05) higher than those from FFs and CCs ($24.9{\pm}6.2%\;vs\;0.19{\pm}0.16,\;23.6{\pm}5.5\;and\;0.17{\pm}0.16%$, respectively). Proportions of TUNEL positive cells in NT embryos from FFs and CCs ($6.9{\pm}1.5\;and\;7.4{\pm}1.7%$, respectively) were significantly (P<0.05) higher than in MSCs ($4.8{\pm}1.4%$) and IVF ($2.3{\pm}0.9%$). The results demonstrate that MSCs have a greater potential as donor cells than FFs and CCs in achieving enhanced production of cloned porcine embryos.