• 대한수의학회지
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• 제29권3호
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• pp.373-381
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• 1989

The aim of the present study was to test the efficiency of estrous induction in the premature, metestrous and anestrous bitches. The estrus was induced with prostaglandin $F_{2{\alpha}}$, estradiol-$17{\beta}$, pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin(HCG) in the treatment A, and with PMSG and HCG in the treatment B. Day 0 was the first day of estrone injection in the treatment A and the day of PMSG injection in the treatment B. Twenty three of the twenty six bitches were laparotomized under general anesthesia between 11 and 18 days after onset of behavioral estrus, whereas three bitches were not laparotomized and remained until parturition. Ovarian responses were evaluated with the total number of corpora lutea or ovulation sites. The uterine horns were flushed with phosphate-buffered saline added heat treated canine serum(10%), the flushing media was collected into watch glass and the ova were examined under stereomicroscope. The results obtained were as follows: 1. Standing estrus was observed on the day $17.7{\pm}1.5$ after injection of estrone in the treament A, but ovarian responses were not detectable. 2. Standing estrus was observed on the day $12.2{\pm}0.2$ after injection of PMSG in the treament Band 14 of 17 bitches showed ovarian responses. Ova were recovered in 9 of the 14 bitches. 3. Ovarian responses were observed in one of the three premature bitches. two of the three metestrous bitches and all of the 11 anestrous bitches. The average number of the ova collected from 9 bitchs were $12.2{\pm}1.4$. 4. Three bitches in the treament B exhibited behavioural estrus and all of them were mated with fertile male dog, resulting the pregnancy in only one bitch. The pregnant bitch gave the birth of two pups.

• 한국수정란이식학회지
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• 제10권1호
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• pp.65-72
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• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• 한국수정란이식학회지
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• 제10권1호
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• 한국수정란이식학회지
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• 제12권2호
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• pp.133-139
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• 1997

Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5$\mu$M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 $\mu$sec at 1.25kV/cm in $Ca^2$+, $Mg^2$+ - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 $\mu$sec at 1.25 kV/cm in 100$\mu$M $Ca^2$+, $Mg^2$+ 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.

• 한국가축번식학회지
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• 제11권3호
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• pp.161-169
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• 1987

This experiment was carried out to investigate the optimal cooling rate and the plunging temperature into liquid nitrogen of the 8-cell hamster embryos. The female hamsters were induced to superovulate by intraperitoneal injection of 30 i.u. PMSG. Embryos were flushed from oviduct and uterine horn. Embryos were frozen and incubated with a modified Dulbecco's phosphate buffered saline, and equilibrated with 1.5M-dimethyl sulfoxide by a 3-step procedure. The cooling rate of samples was 1$^{\circ}C$/min from room temperature to -5$^{\circ}C$ and the samples were seeded at -5$^{\circ}C$. The plunging temperatures into liquid nitrogen were -20, -25, -30, -35, -40, -45, -50 and -55$^{\circ}C$ at 0.3$^{\circ}C$/min, 0.5$^{\circ}C$/min and 1$^{\circ}C$/min cooling rates, respectively. This mean numbers of ovulation points and recovered embryos were 59.4 and 48.4 appearing 81.6% recovery rate. The percentage of 8-cell embryos in recovered embryos was 68.2. The survival rates of embryos plunged at -45$^{\circ}C$ were 73.5% at 0.3$^{\circ}C$/min, 44.8% at 0.5$^{\circ}C$/min and 30.3% at 1$^{\circ}C$/min cooling rates, respectively. This mean numbers of ovulation points and recovered embryos were 59.4 and 48.4 appearing 81.6% recovery rate. The percentage of 8-cell embryos in recovered embryos was 68.2. The survival rates of embryos plunged at -45$^{\circ}C$ were 73.5% at 0.3$^{\circ}C$/min, 44.8% at 0.5$^{\circ}C$/min and 30.3% at 1$^{\circ}C$/min cooling rates, respectively. The survival rates at 0.3$^{\circ}C$/min were significantly high. Under the condition of 0.3$^{\circ}C$/min cooling rate, the survival rates of embryos according to the plunging temperature were 70.0% and 73.5% at -40 and -45$^{\circ}C$, and those were higher than other plunging temperatures. Under the condition of 0.5$^{\circ}C$/min and 1$^{\circ}C$/min cooling rates, the survival rates according to the plunging temperatures were lower than the cooling rate of 0.3$^{\circ}C$/min, showing the similar tendency at all the plunging temperatures. In conclusion, 8-cell hamster embryos showed the best survival rates at 0.3$^{\circ}C$/min cooling rate and -45$^{\circ}C$ plunging temperature.

• 한국가축번식학회지
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• 제11권3호
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• pp.155-160
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• 1987

This study was carried out to determine the effects of cryoprotectants, freezing and thawing rates on the survival of 8-cell mouse embryos. The female ICR mice were induced to superovulated by intraperitoneal injections of 5 i.u. PMSG and 5 i.u. HCG given 48h apart and then were paired with males of the same strain. They were killed and embryos were flushed from the oviducts and uteri on 3 days after injection of HCG. Embryos were flushed with modified Dulbecco's phosphate buffered saline and equilibrated with 1.5M-dimethyl sulfoxide (DMSO) or 1.5M-glycerol by 3-step procedure. The freezing rates of the embryos were 1$^{\circ}C$/min from room temperature to -5$^{\circ}C$ and the embryos were seeded at -5$^{\circ}C$. After being held for 3 min at the seeding temperature, the rates were 0.3$^{\circ}C$/min from -5$^{\circ}C$ to -35$^{\circ}C$. From -35$^{\circ}C$ to -7$0^{\circ}C$, the rates were divided into 0.1$^{\circ}C$/min, 1$^{\circ}C$/min and 1$0^{\circ}C$/min, respectively. After being held for 5 min at -7$0^{\circ}C$, the embryos were plunged directly into liquid nitrogen. The embryos were thawed at 4$^{\circ}C$/min and 12$^{\circ}C$/min from -196$^{\circ}C$ to 37$^{\circ}C$, and for 2 min in 37$^{\circ}C$ water bath, respectively. The average number of ovulation points and embryos recovered were 42.7 and 34 appearing 79.5% recovery rate. Eight cell embryos in the embryos recovered were 26.3. The survival rates of embryos according to the freezing rates in the presence of 1.5M-DMSO were 73.5~80.6% at 0.1$^{\circ}C$/min, 75.0~79.5% at 1$^{\circ}C$/min and 52.8~54.7% at 1$0^{\circ}C$/min, but in the presence of 1.5M-glycerol were 62.9~67.6% at 0.1$^{\circ}C$/min, 61.4~68.3% at 1$^{\circ}C$/min and 25.5~30.2% at 1$0^{\circ}C$/min. The survival rates of embryos were not affected by the thawing rates.

• 한국산학기술학회논문지
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• 제15권9호
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• pp.5644-5651
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• 2014

본 연구는 칸디다 알비칸스(C. albicans)에 대한 프로폴리스의 억제효과를 확인하고자 수행되었다. 프로폴리스는 양봉 꿀벌에서 채취하여 C. albicans는 $37^{\circ}C$에서 액체 배지에서 2시간 배양 하였다. 항균 활성검삼를 위해서 생리식염수 (PBS), 3% 차아염소산나트륨 (NaOCl에), 0.1% 클로로헥시딘 (CHX), 프로 폴리스 추출물 ($5{\mu}L/m{\ell}$, $10{\mu}L/m{\ell}$)에서 평가하였다. C. albicans는 3%의 NaOCl, 0.1% CHX, 프로 폴리스($5{\mu}L/m{\ell}$, $10{\mu}{\ell}/m{\ell}$)에서 15, 14.5, 16, 17mm의 억제구간을 확인하였다. 항균활성을 확인하기 위하여 집락형성을 분석한 결과, 3%의 NaOCl, 0.1 % CHX, $5{\mu}L/m{\ell}$와 $10{\mu}L/m{\ell}$의 프로 폴리스가 7, 7, 5, 7 로그지수가 감소를 보였다. C. albicans서는 $10{\mu}L/m{\ell}$의 프로폴리스에서 유의하게 억제되는 결과를 보여주었다. 이러한 결과를 통하여 프로폴리스는 구강 점막 질환에서 새로운 향균 약제가 될수 있는 것으로 생각된다.

• 한국안광학회지
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• 제15권4호
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• pp.347-353
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• 2010

목적: 소프트콘택트렌즈 관리용액에 의한 balafilcon A 재질의 실리콘 하이드로겔렌즈에 부착된 지방 침전물 제거효과를 비교하였다. 방법: Balafilcon A 재질의 실리콘 하이드로겔렌즈를 실험실 조건에서 oleic acid, oleic acid methyl ester, cholesterol이 포함된 식염수에 담가 24시간 동안 오염시켰다. 오염된 콘택트렌즈는 식염수로 헹구어준 후 계면활성세척액, 알콜 성분이 포함된 세척액 및 다목적용액으로 각각 세척하였고, 반복적인 오염과 세척효과를 관찰하기 위해서는 오염과 세척과정을 14회 반복하였다. 초음파의 지방침전물 세척 효과를 관찰하기 위하여 오염된 렌즈에 초음파처리를 한 후 처리하지 않은 렌즈와 비교하였다. 지방침전물은 methanol:chloroform (1:1, v/v) 용액으로 추출하고 고성능액체크로마토그래피로 분석하여 정량하였다. 결과: 실리콘 하이드로겔렌즈의 지방세척효과는 알콜성분을 함유한 세척액이 계면활성세척액 및 다목적용액보다 높았으며, 초음파를 함께 처리해준 경우에는 세척 효과가 상승하였다. 콘택트렌즈 관리용액으로 세척한 후에도 지방침전물은 완전히 제거되지 않아 오염과 세척을 반복한 경우 지방 침전물의 양은 지속적으로 증가하였다. 결론: 실리콘 하이드로겔렌즈에 부착되는 지방 침전물에 대한 소프트 콘택트렌즈용 관리용액의 세척효과는 만족할 수준에 미치지 못하기 때문에 실리콘 하이드로겔렌즈의 지방 침전물 제거에 적합한 세척액의 개발이 필요할 것으로 사료된다.

• 한방안이비인후피부과학회지
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• 제19권2호
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• pp.1-18
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• 2006

Objective : Although management of asthma has become increasingly effective, its cure remains elusive, necessitating a new modality to prevent or eliminate causes triggering clinical progress. Based in the clinical experiences, a novel decoration Cheongsangbiyeum (CSB), has been developed to treat asthma, which consists of Polyporus, Semen Myristicae, Pericarpium citri Reticulatae, Rhizoma Cimicifugae, Cortex Albizziae, Fructus Rubi, Rhizoma Zedoariae, and Rhizoma Rhei. In the current study, its anti-asthmatic efficacy was evaluated using a mouse model of asthma. Methods : Experimental allergic asthma was induced by repeated intraperitioneal sensitization and intranasal challenge of ovalbumin (OVA). Water extract of CSB (1 mg/mouse/day) was administrated orally whereas control mice on given with identical volume of phosphate-buffered saline (PBS) for 5 days during the course of antigen challenge. When airway hyperreactivity(AHR) measured by ${\bata}-methacoline-induced$ airflow obstruction was compared, AHR of CSB-treated mice was significantly lower than those of control mice, indicating that CM extract can attenuate an asthmatic symptom. Airway recruitment of leukocytes and eosinophils was also markedly reduced by CSB treatment suggesting that oral treatment of CSB can alleviate the airway inflammation. For a better understanding of possible mechanisms underlying anti-asthmatic effet of CSB, cytokine (IL-4, IL-5, IL-13 and $IFN{\gamma}$ levels in bronchoalveola lavage fluid (BALF) and lung tissues were determined. Results : The results showed that cytokine levels were significantly lowered by CSB treatment. Additionally, number of draining lymph node cells was significantly lower than those of control mice. These data indicate that CSB suppress in vivo allergen-specific response. However, notably, levels of type 2 cytokines such as IL-5 and IL-13 were more profoundly influenced. Moreover, in vitro OVA-specific proliferative response and type 2 cytokine (IL-4, IL-5 and IL-13) production lymph node cells was markedly decreased in CSB-treated mice, whereas their $IFN{\gamma}$ production was not significantly altered Thrse data clearly showed a preferential inhibition of type 2 T cell (Th2) response by CSB treatment. This finding was also supported by serum antibody data showing that levels of OVA-specific type 2 antibodies, IgE and IgG1, in CSB-treated mice were significantly lower than in control mice, while type 1 antibody, IgG2a level m rather higher than controls, although the difference was in significant. Conclusions : In conclusion, oral administration of CSB attenuates asthmatic manifestations including AHR ad airway recruitment of eosinophils in a mouse model which possibly results from selective inhibition of Th2 cell response to allergen. Our data suggest a potential clinical application of CSB for control of allergic asthma.

• Tuberculosis and Respiratory Diseases
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• 제78권3호
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• pp.218-226
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• 2015

Background: Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury. Methods: Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure). Results: EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group ($4.30{\pm}2.93$ vs. $11.45{\pm}1.20$, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: $11.33{\times}10^4{\pm}8.84{\times}10^4$ vs. IgG+LPS: $208.0{\times}10^4{\pm}122.6{\times}10^4$; p=0.018) and total protein concentrations (EphA2 mAb+LPS: $0.52{\pm}0.41mg/mL$ vs. IgG+LPS: $1.38{\pm}1.08mg/mL$; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase $110{\gamma}$, phospho-Akt, nuclear factor ${\kappa}B$, and proinflammatory cytokines. Conclusion: This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation.