• Journal of Nutrition and Health
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• 제29권1호
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• pp.97-103
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• 1996

This study was performed to investigate the effects of lipid on cellular senescence, lipid peroxide production, and morphological changes. For this study we used primary skin fibroblasts from neonate rats grown in media various lipid contents. Fibroblasts were cultured until they lost their proliferation potential either in control medium (Dulbecco's modified Eagle's medium supplement with 10% fetal bovine serum) or in media supplemented with various concentrations of lipid-cholesterol rice component from bovine serum. Cumulative population doublings(CPD, as an index of cellular life span), and cellular thiobarbituric acid reactive substances (TBARS, as an index of lipid peroxide) concentrations were measured and morphological changes were observed. CPD were shortened with increasing lipid concentration in media ; 28.12 for cells grown in control medium and 13.42, 11.42, and 6.19 for those grown in 0.1%, 1% and 5% lipid rich components containing media, respectively. Cellular proliferation ratios were those grown in 5% lipid rich components containing media were delayed and they were degenerated soon. TBARS concentrations were increased with increasing concentration of lipid in media. Morphological changes were observed in cells grown in control medium by cellular senescence. Especially lipid droplets were observed in cells grown in 5% lipid rich components containing media. Therefore it seems that lipid contents in media had an effect on cellular proliferation and cellular life span, possibly via lipid peroxide production.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

• 한국가축번식학회지
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• 제24권1호
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• pp.59-67
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• 2000

본 연구는 핵이 제거된 소난자 내에 소의 태아 섬유아세포를 이식한 후 난자의 발달능력을 조사하였다. 소 태아 섬유아세포는 45일된 응성 태아로부터 분리한후 MitoTracker로 염색하고 세포 주기를 동기화시키지 않은 세포를 핵이 제거된 소난자의 위란강 내에 이식하였다. 섬유아세포와 소난자의 복합체는 전기 자극을 주어 융합시키는 방법을 이용하였으며, 융합된 난자는 Calcium ionophore와 6-DMAP를 이용하여 난활성을 유도시킨 다음 CR1aa 에서 배양하였다. 핵치환 된 난자의 핵은 핵질 응축과정, 팽대과정, 전핵 형성과정이 일어났으며, 이러한 과정에서 재구성된 난자는 분열 과정을 거쳐 18∼26시간 사이에는 2-세포기로 발달하였다. 섬유아세포의 미토콘드리아는 핵치환시 난자 내로 이전되었는데 이것들은 난자 내에서 빠르게 사라지는 것으로 관찰되었으며, 핵융합 후 8시간째에는 섬유아세포의 미토콘드리아가 전혀 관찰되지 않았다. 2-세포기로 분열된 난자의 21% 가 이식 가능한 단계인 배반포 단계까지 발달하였다. 이러한 결과는 소의 태아 섬유아세포가 탈핵된 소난자 내에서 성공적으로 재분화과정을 거치며, 이렇게 재조합 된 수정란은 배반포 단계까지 발달이 가능하다는 것을 보여주고 있다.

• 한국수정란이식학회지
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• 제14권2호
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• pp.93-97
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• 1999

The development potential of bovine somatic cells was evaluated using nuclear transfer. A single donor cell derived from fetus of HanWoo(Korean Native Cattle) was selected and deposited into perivitelline space of each enucleated oocyte before electrical fusion and activation. Nuclei of donor cells starved for 7 days (37%) tended to support the development of reconstitute embryo the blastocyst stage better than those of donor cells starved 3, 14 and 30 days. The cleavage rate was significantly lower(P<0.05) in reconstitute embryos derived from large size donor cells(51.2%), than those from small medium size donor cells(76.6 and 73.5, respectively). The developmental rate to blastocyst of reconstructed embryos from medium size donor cells was higher than those from small and medium size donor cells. This study demonstrates that an appropriate culture period for induction into quiescent stage and the size of donor cells effect on the efficiency of nuclear transfer using cultured bovine cells.

• Journal of Periodontal and Implant Science
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• 제37권1호
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• pp.45-51
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• 2007

This study evaluated the possibility of the 3-dimensional attachment of human periodontal ligament fibroblasts to a periodntally involved root surface after an EDTA treatment in vitro. The human PDL fibroblasts were isolated from the middle third of the root of periodontally healthy teeth extracted for orthodontic reasons. The cells were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $37^{\circ}C$ in humidified air containing 5% $CO_2$. Eight single-rooted teeth were obtained from patients diagnosed with periodotitis. After scaling and root planing, four teeth were etched with 24% ethylenediaminetetracetic acid (EDTA) for two minutes (Experimental group). The other four teeth were not treated with EDTA and were used as the control group. The human PDL fibroblasts were placed in the total root surface and cultured for 4 weeks. The teeth were fixed in 2.5% glutaraldehyde in PBS before preparation for the scanning electron microscopy (SEM) examination. The human PDL fibroblasts showed a healthy morphology on the root surfaces treated with EDTA (Experimental group) and a relatively unhealthy appearance on the treated root surfaces (Control group). This suggests that EDTA favorably affects the 3-dimensional attachment of human PDL fibroblasts cultured on the root surfaces. which may play an important role in periodontal healing and regeneration.

• Journal of Periodontal and Implant Science
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• 제26권1호
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• pp.188-201
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• 1996

One of the initial events required for periodontal regeneration is the attachment, spreading and proliferation of fibroblasts at the healing sites. These have been reported that minocycline stimulates the attachment of gingival fibroblasts and periodontal ligament cells and $TGF-{\beta}1$ enhances the proliferation of periodontal ligament cells. The purpose of this study was to evaluate and confirm the effect of minocycline and $TGF-{\beta}1$ on human gingival fibroblasts and periodontal ligament cells. That gingival fibroblasts and periodontal ligament cells used in this study were obtained from the explants of healthy periodontal ligaments and gingival tissues of extracted 3rd molars or premolar teeth extracted from the patients with orthodontic treatment. The cells were cultured in ${\alpha}-MEM$(minimal essential medium) supplemented with antibiotics and FBS(fetal bovine serum) at $37^{\circ}C$ in a humidified atmosphere of 5% carbon dioxide-95% air. Cells were used between the 5th to 8th passage in this study. The attachment and activity of both cells were evaluated by MTT assay. The results were as follows: 1. Maximum gingival fibroblast attachment was seen at a $50{\mu}g/ml$ dose of minocycline, while maximum periodontal ligament cell attachment was seen at a $100{\mu}g/ml$, and exposure of both cells to minocycline above maximal attachment dose results in a decline from maximum attachment. 2. The activity values of both cells tested minocycline were below to the control activity values at all concentrations. 3. The attachment values of both cells tested $TGF-{\beta}1$ were below or similar to control attachment values. On the above the findings, minocycline stimulated the cell attachment of gingival fibroblasts and periodontal ligament cells and $TGF-{\beta}1$ enhances the cell activity of periodontal ligament cells.

• BMB Reports
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• 제45권2호
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• pp.102-107
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• 2012

The aim of this study was to investigate protein profiles related to the induction of adipogenesis within the bovine longissimus dorsi muscle (BLDM) by proteomic analysis. We analyzed BLDM proteins at different growth stages to clarify the physiological mechanisms of marbled muscle development in 20 head of Korean native cattle (11 month: 10 head, 17 month: 10 head). BLDM proteins were analyzed by two-dimensional electrophoresis and image analysis. Villin 2 was specifically identified by mass spectrometry and a protein search engine. Villin 2 protein expression in BLDM decreased during the fat development stage in test steers. In a Western blot cell culture study of spontaneously immortal bovine muscle fibroblasts, the abundance of Villin 2 was shown to be down-regulated during differentiation into muscle. In 3T3-L1 mouse embryonic fibroblasts, Villin 2 was decreased during differentiation into adipocytes. The results suggest that Villin 2 may be related to the induction of transdifferentiation and adipogenesis in bovine longissimus dorsi muscle.

• Asian-Australasian Journal of Animal Sciences
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• 제10권2호
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• pp.233-239
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• 1997

A mammary epithelial cell line (MAC-T) established as a model for lactation was utilized to identify and characterize effects of various hormones upon insulin-like growth factor binding protein secretion. Ligand and immunoblot analyses of conditioned media indicated that insulin-like growth factor binding protein-2 was secreted by MAC-T cells. Insulin-like growth factor-I stimulated insulin-like growth factor binding protein-2 secretion in a dose-dependent manner, but prolactin and bovine somatotropin did not alter insulin-like growth factor binding protein-2 secretion. Insulin increased and cortisol decreased insulin-like growth factor binding protein-2 secretion. Effects of insulin-like growth factor-I on insulin-like growth factor binding protein-2 secretion support previous studies using primary cultures of bovine mammary cells and bovine fibroblasts. Effects of cortisol and insulin on insulin-like growth factor binding protein-2 secretion may be explained by changes in protein synthesis. In addition, supraphysiological doses of insulin can cross-react with the insulin-like growth factor-I receptor and stimulate insulin-like growth factor binding protein-2 secretion. MAC-T cells provide a model system to study mechanisms that regulate local insulin-like growth factor-I bioactivity.

• Journal of Nutrition and Health
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• 제29권2호
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• pp.159-165
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• 1996

This study was performed to investigate the effects of concentration and degree of unsaturation of fatty acids on cellular proliferation and lipid peroxide production, using primary skin fibroblasts from neonate rats Fibroblasts (CPD : 2.8-5.4). Cells were cultured either in control medium (Dulbecco's modified Eagle's medium supplement with 10% fetal bovine serum) or in media supplemented with various kinds (stearic, oleic, linoleic, arachidonic, linolenic, eicosapentaenoic acid) and amounts (5, 10, 25, 50, 100, 150uM)of fatty acids. Cellular proliferation ratio and lipid peroxice production were measured and morphological changes were observed. Cellular proliferation was inhibited and morphological changes were observed. Cellular proliferation was inhibited and morphological changes were observed in cells grown in stearic containing media. Oleic, arachidonic, and eicosapentaenoic aicd tend to stimulate cellualar proliferation, and linolenic acid had no effects. Lipid peroxide concentrations in fibroblasts increased in proportion to the contents and unsaturation of fatty acids in media. Especially supplementation of arachidonic acid accelerated cellualr lipid peroxidation. Free radicals may cause severs damage to biological molecules, so lipid peroxidation probably contributes cellular membrane damages. However there were little relationship between lipid peroxide production and cellular proliferation in this study. (Korean J Nutrition 29(2) : 159~165, 1996)

• 한국가축번식학회지
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• 제26권3호
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• pp.235-243
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• 2002

본 연구는 몇 몇 포유류로부터 얻은 공여세포핵의 proliferation을 돕기 위한 수핵난자로 소 난자의 세포질을 사용하였으며, 공통 세포질로써의 능력을 시험하였다. 소 난자와 소, 사람, 돼지, 생쥐의 체세포와의 결합에서 유래한 각 종별 핵이식란의 융합율, 분할율, 배발달률 그리고 염색체 수를 조사하였다. 1. 소, 돼지, 생쥐, 사람 각각의 체세포를 이용한 핵이식란의 융합율은 70.2% 70.2% 72.4%와 63.0 %로 유의차를 보이지 않았다. 2. 소, 돼지, 생쥐, 사람 각각의 체세포를 이용한 핵이식란의 체외분할 ($\geq$2cell cleavage)율 또한 60.6%, 63.7%, 54.1%와 62.7%로 유의차가 없었다. 3. 각 종별로 체외분할이 일어난 시기를 조사한 결과 종에 관계없이 대체로 활성화 처리 후 24시간째에 대부분 일어나는 것을 볼 수 있었다. 그러나 점차적으로 분할이 계속 이루어지면서 발달시기가 종 특이적인 특성을 나타내는 것을 볼 수 있었다. 4. 이종간 핵이식의 후기 배발달 (상실배와 배반포) 률을 살펴보면 소와 사람의 체세포를 사용했을 경우 17.5%, 4.3%의 결과를 나타내었으며, 돼지와 생쥐의 체세포를 사용한 경우 16세 포기 이후 단계에 발달중지 현상을 볼 수 있었다. 5. 4~8 세포기 정도의 각 종별 핵이식란 할구를 분석에 사용하였을 때, 공여핵으로 사용된 종의 염색체 수와 일치하는 결과를 볼 수 있었다. 이상의 결과를 종합해 볼 때 성숙한 소 난자의 세포질은 소뿐만 아니라 돼지, 생쥐, 사람과 같은 다양한 포유류의 핵을 사용하여 핵이식을 하였을 때에도 발달이 가능하다는 것을 보여주고 있다.