• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• 한국수정란이식학회지
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• 제26권2호
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• pp.123-128
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• 2011

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The matured oocytes (n = 248) were activated with 5 ${\mu}M$ ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was $3.5{\pm}0.5$. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was $42.1{\pm}4.7%$. Parthenogenetic activation, evidenced by formation of pronucleus, occurred in $37.2{\pm}15.8%$ of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

• 한국산학기술학회논문지
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• 제14권8호
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• pp.3843-3849
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• 2013

이배체 단위발생 돼지 난자를 체외 배양시 배반포 형성 단계에서 FBS (우태아 혈청), BSA (우혈청 알부민), EGF(상피세포 성장인자)를 배양액에 첨가하였을 때 이배체 단위발생 에서 총세포수, 세포사멸 및 세포사멸에 관여하는 유전자의 발현 효과를 조사하고자 본 연구를 수행하였다. 0.4% BSA를 배양액에 첨가 하였을 때 2 세포기 단계 단위발생의 발달은 배반포 까지는 강화 되었다 (p<0.01). FBS 처리 시는 배반포의 세포 수는 감소시켰으나 세포 사멸률은 증가하였다(p<0.01). 하지만 EGF가 존재할 때 BSA 처리는 총 세포수를 증가 시켰다. RT-PCR의 결과에 의하면 EGF는 0.4% BSA가 존재하는 배양액에서는 Bcl-xL mRNA 발현을 증가시키고 BSA와 EGF 가 단독으로 존재 할 때는 효과가 없었다. 하지만 FBS 처리시 Bcl-xL 유전자 발현은 감소하고 Bak 유전자의 발현은 증가시킨다. 이러한 결과 세포사멸에 관여하는 유전자의 발현은 배양액의 첨가물에 따라 유의적으로 영향을 받으며, 돼지 배아의 체외 배양시 세포사멸과 초기발달에 관여함을 시사한다.

• 대한수의학회지
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• 제59권2호
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• pp.81-88
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• 2019

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF ($113.0-114.8{\mu}m$) were significantly larger than that of no IVG-SAF ($111.8{\mu}m$). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.22-22
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• 2003

This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. Embryos were collected from 2-cell or late 4-cell diploid parthenotes that activated with electro pulse, and in vitro cultured in the NCSU 23 medium supplemented without or with 0.1% PVA, 10% FBS or 0.4% BSA for day 7. The morphological analysis of apoptosis in embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expressions of Bcl-xL, Bak and P53 in blastocyst stage parthenotes and in vivo-derived blastocysts were determined using semiquantitative RT-PCR. The addition of 0.4% BSA to the culture medium enhanced the development of 2- or late 4-cell stage parthenotes to the blastocysts stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced cell numbers of blastocysts developed from both 2- (P < 0.001) and late 4-cell (P < 0.05) embryos and increased percentage of apoptosis in the blastocysts (P < 0.001). The relative abundance of Bcl-xL mRNA in diploid parthenotes cultured from 2-cell stage in the presence of BSA is similar with that in in vivo derived embryos, but is significantly higher than in parthenotes cultured with FBS, PVA or none protein supplement control. Bak mRNA showed a significant increase at the blastocyst stage in FBS supplement medium. This result suggests that apoptosis related gene expression is significantly affected by protein supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

• Clinical and Experimental Pediatrics
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• 제46권2호
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• pp.167-172
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• 2003

목 적 : 소아 심장병의 주종을 이루고 있는 선천성 심장병 환아들에서 폐동맥 고혈압은 비교적 흔히 발생하지만 매우 치료하기 어려운 합병증이다. 폐동맥 고혈압의 원인과 치료 및 예방에 대해서는 아직 많이 알려지지 않은 실정이므로 이의 원인을 산소결핍이라는 전형을 이용하여 VEGF란 유전인자의 차원에서 규명하고, 나아가서는 폐동맥 고혈압의 치료 및 예방책을 마련하기 위하여 이 연구를 시행하였다. 방 법 : 폐동맥 평활근 세포는 생후 6주 Fischer rat의 주폐동맥을 적출하여 작은 조각으로 잘라 20% fetal bovine serum을 첨가한 DMEM 배지를 사용하여 5% 이산화탄소 배양기에서 배양하였다. 배양된 세포는 평활근 세포에만 선택적으로 염색되는 평활근 myosin과 ${\alpha}$-actin 항체를 이용하여 염색함으로써 순수 평활근 세포임을 확인하였다. 5% 이산화탄소 배양기에서 배양한 대조군 세포와 1 또는 3% $O_2$ tension에서 배양한 실험군 세포에서의 VEGF 발현 차이와 starvation한 군과 하지 않은 군에서의 VEGF 발현 차이를 RT-PCR과 northern blotting을 이용하여 비교하였다. 결 과 : 대조군과 저산소 조건에서 배양한 실험군에서 VEGF 발현 정도는 차이가 없었다. 결 론 : 아직 국내에서는 유전인자 차원에서의 폐동맥고혈압의 원인규명이나 이에 따른 치료에 대한 연구가 전혀 없는 상태이며, 이 연구에 이어 신생쥐와 성숙쥐와의 차이점 및 나아가서 사람과 쥐의 폐동맥 평활근 세포의 차이점 등을 규명할 예정이며, 이번 연구 결과를 바탕으로 폐동맥 고혈압의 원인기전 규명, 치료 및 예방방법 개발에 기여하고자 한다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• 폴리머
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• 제36권3호
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• pp.357-363
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• 2012

Poly(L-lactic acid)(PLLA) 고분자 필름 및 지지체의 세포 친화성을 향상시키기 위하여 산소 플라즈마 처리후 카복실기를 함유한 아크릴산(AA)을 $in$ $situ$ 그래프트시켰다. Stimulated body fluid(SBF) 용액에 15일간 담지시킨 후 hydroxyapatite(HA)를 형성시킨 시료와 phosphate-buffered saline(PBS), fetal bovine serum(FBS), 식염수 및 세포 배양용 배지에 담지시킨 다음 PLLA 시료 표면의 접촉각을 비교해 본 결과, HA 표면이 가장 낮은 접촉각을 나타내었다. 또한 연골세포와 조골세포는 HA 표면 위에서 높은 점착과 성장을 보였으며 연골세포가 HA에 많은 영향을 받는 것으로 확인되었다. 조골세포의 경우 HA 표면 이외에도 FBS나 세포 배양배지에 담지된 표면에서도 높은 세포 증식을 보였다. 더욱이 필름형태보다는 3차원 입체 구조의 다공성 지지체에서 연골세포와 조골세포의 점착과 세포 증식이 향상됨도 확인할 수 있었다. 이러한 표면개질된 PLLA는 조직공학적으로 연골이나 뼈 재생을 위한 유-무기 하이브리드 지지체로 응용될 수 있을 것으로 기대된다.

• The Journal of Advanced Prosthodontics
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• 제6권5호
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• pp.379-386
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• 2014

PURPOSE. These days, mesenchymal stem cells (MSCs) have received worldwide attention because of their potentiality in tissue engineering for implant dentistry. The purpose of this study was to evaluate various growth inducing factors in media for improvement of acquisition of bone marrow mesenchymal stem cells (BMMSCs) and colony forming unit-fibroblast (CFU-F). MATERIALS AND METHODS. The mouse BMMSCs were freshly obtained from female C3H mouse femur and tibia. The cells seeded at the density of $10^6$/dish in media supplemented with different density of fetal bovine serum (FBS), $1{\alpha}$, 25-dihydroxyvitamin (VD3) and recombinant human epidermal growth factor (rhEGF). After 14 days, CFU-F assay was conducted to analyze the cell attachment and proliferation, and moreover for VD3, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was additionally conducted. RESULTS. The cell proliferation was increased with the increase of FBS concentration (P<.05). The cell proliferation was highest at the density of 20 ng/mL rhEGF compared with 0 ng/mL and 200 ng/mL rhEGF (P<.05). For VD3, although the colony number was increased with the increase of its concentration, the difference was not statistically significant (P>.05). CONCLUTION. FBS played the main role in cell attachment and growth, and the growth factor like rhEGF played the additional effect. However, VD3 did not have much efficacy compare with the other two factors. Improvement of the conditions could be adopted to acquire more functional MSCs to apply into bony defect around implants easily.

• 생명과학회지
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• 제14권5호
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• pp.874-881
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• 2004

혈관 신생은 암의 성장 및 전이뿐만 아니라 염증, 관절염, 건성, 동맥경화 등의 병적인 진행에 주요한 역할을 하며, 혈관신생 억제를 통한 암의 치료를 시도하는 연구들이 활발하게 진행되고 있다. 혈관 신생 시 내피세포의 증식, 이동을 유도하는 활성화 과정이 필수적으로 일어나는 것으로 알려져 있다 본 연구에서는 in vitro에서 내피세포를 배양하여, 각종 growth factor가 풍부한 배지에서 활성화 시켰을 때, 그렇지 않는 세포들과의 유전자 발현 형태를 비교 조사하였다. HUVEC을 70∼80% cofluency로 배양시킨 후에 endothelial cell growth supplement (ECCS), 20% fetal bovine serum, heparin이 첨가된 Ml99 배지에서 13 시간 활성화시킨 세포(AHUVEC)와 대조군 세포(RHUVEC)로부터 분리한 total RNA로부터 CDNA를 제작하였고, 이것을 18,864 개의 유전자가 올려져있는 인간 올리고 칩과 hybridization 반응을 시켰다. 반응된 유전자를 이용하여 random clustering분석을 실시한 결과, 활성화 시켰던 HUVEC과 그렇지 않은 HUVEC으로 dendrogram 상에서 두개의 subgroup으로 나뉘어 지는 것을 확인할 수 있었다. 최소 2배 이상 발현 변화가 있는 유전자 122종이 활성화 시켰던 HUVEC으로부터 추출되었다. 이중에서 기능이 알려진 32 개의 유전자는 활성화시킨 HUVEC에서 발현이 증가하였고, 38 개의 유전자 발현은 감소하였다. 흥미롭게도 세포 증식과 이동, 염증, 면역반응에 관련한 유전자의 발현이 증가된 반면에 세포 흡착과 혈관 조직과 기능에 관련한 유전자의 발현이 감소된 것이 관찰되었다. 예상외로 규명이 잘된 혈관신생 인자와 관련한 유전자들의 발현에는 크기 차이를 보이지 않았으나, Eph-B4의 발현은 약 4 배 감소된 것으로 관찰되었다 또한, 2배 이상 발현에 차이를 보이고 기능이 알려져 있지 않은 유전자 52종이 발견되었다. 따라서, 이러한 연구 결과로부터 새로운 혈관 표적 물질 개발에 대한 기회가 제공될 수 있을 것이라 사료된다.