• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.41-49
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• 1996

본 연구는 체외수정에 의해 생산된 생쥐 배반포기배를 vitrification 방법으로 동결보존하였을때 높은 생존율을 얻기 위한 적정조건을 검토하고자 실시하였다. 배반포기배를 생산하기 위하여, B6CBA F1 (C57BL/6, (표현불가)${\times}CBA/N$, (표현불가)) 계통의 생쥐 미수정란에 $1{\times}10^6$ spermatozoa/ml 농도의 정자로서 수정을 유도하였으며, 이후 $37^{\circ}C$, 5% $CO_2$배양기내에서 96시간동안 체외배양하였다. 배양 4일째의 배반포기배는 발달상태에 따라 early, middle 그리고 hatching blastocysts로 구분하였다. 본 실험에 사용된 동결보존액은 30% Ficoll과 0.5mol의 sucrose가 첨가된 mDPBS 용액에 40%의 ethylene glycol를 첨가한 EFS 40 (Zhu et al., 1993) 이었고, 수정란은 $25^{\circ}C$의 상온에서 먼저 20% ethylene glycol에 노출된 후 EFS 40 용액으로 옮겨 액체질소에 침지하는 2단계 동결법에 의해 동결보존되었으며, 급속융해하여 다음과 같은 결과를 얻었다. 1. 체외수정율과 배양 4일째 배반포기까지의 배발달율은 각각 89.4%와 86.1%였다. 2. 20% ethylene glycol에서 5분간 평형된 후 EFS 40 용액에 냉동보존된 후 융해된 난자의 생존율은 20% ethylene glycol에 0, 1, 3분간 평형된 난자의 생존율에 비해 유의하게 높았다. 3. 배반포기배를 20% ethylene glycol에서 5분간, EFS 40 용액에 1분간 차례로 노출한 다음 체와배양하였던 바, 배양 24시간째 생존율은 $82.9%{\sim}88.4%$ 였다. 본 연구 결과, 체외수정, 배양된 생쥐 배반포기배는 20% ethylene glycol과 EFS40에 대한 노출만으로는 난자의 생존성에 나쁜 영향을 미치지 않는 것으로 미루어 보아 배반포기배의 초자화 동결이 가능함을 시사하였다. 따라서 동결 융해 후 높은 생존율은 상온에서 난자를 2단계 즉, 20% ethylene glycol에 5분간 평형시킨 후 EFS 40 용액에 노출하여 1분내에 LN2에 직접 침지하는 간편한 동결방법으로 얻을 수 있었다.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.1-9
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• 1998

This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.133-147
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• 2005

Objectives: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. Methods: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. Results: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46,XX karyotype. Conclusions: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

• Journal of Animal Environmental Science
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• v.13 no.1
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• pp.45-52
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• 2007

To evaluate the feed value of starfish, antimicrobial effects of its extract, nutrients contents, concentration of amino acids and its bioavailability were tested. Steaming and ether processes were applied to obtain the extract from starfish for antimicrobial effects examination. The starfish was dried at $60^{\circ}C$ for 3 days before grinding for processing and fermentation. Ground starfish(GS), extruded starfish(ES), fermented starfish(EFS) were added with enzyme and without enzyme(Non enzyme fermented starfish : NEFS). Then the nutrient composition and bioavailability of those were analyzed. The extract from starfish showed no inhibition of the growth of lactobacillus and pathogenic bacteria. Protein content showed significantly higher 62.86% and 52.82%, respectively in EFS and NEFS than GS and EGS(p<0.05). The Ca content of GS, EGS, EFS and NEFS was 17.26%, 18.26%, 5.37% and 8.55%, respectively. It was low in EFS and NEFS due to measure the Ca content after fermentation. Total amino acid was 17.17 mg/g, 20.28 mg/g, 36.30 mg/g and 29.96 mg/g in GS, EGS, EFS and NEFS, respectively. The ratio of total amino acid to protein tended to show the similar tendency as total amino acid. Both total amino acid and its ratio to protein were increased by the fermentation. Bioavailability of the protein and Ca showed more 80% in EFS and NEFS. The nutrients availability of EFS were significantly higher in laying hens than other treatments. The results of these experiments indicate that starfish would be applied as a feed ingredients if it was properly treated.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2967-2971
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• 2013

Background: Extraskeletal Ewing sarcoma (EES)/primitive neuroectodermal tumours (PNET) are rare soft tissue sarcomas. Prognostic factors and optimal therapy are still unconfirmed. Materials and Methods: We performed a retrospective analysis on patients to explore the clinic characteristics and prognostic factors of this rare disease. A total of 37 patients older than 15 years referred to our institute from Jan., 2002 to Jan., 2012 were reviewed. The characteristics, treatment and outcome were collected and analyzed. Results: The median age was 28 years (range 15-65); the median size of primary tumours was 8.2 cm (range 2-19). Sixteen patients (43%) had metastatic disease at the initial presentation. Wide surgical margins were achieved in 14 cases (38%). Anthracycline or platinum-based chemotherapy was performed on 29 patients (74%). Radiotherapy was delivered in 13 (35%). At a median follow-up visit of 24 months (range 2-81), the media event-free survival (EFS) and overall survival (OS) were 15.8 and 30.2 months, respectively. The 3-year EFS and OS rates were 24% and 43%, respectively. Metastases at presentation and wide surgical margins were significantly associated with OS and EFS. Tumour size was significantly associated with OS but not EFS. There were no significant differences between anthracycline and platinum based chemotherapy regarding EFS and OS. Conclusions: EES/PNET is a malignant tumour with high recurrence and frequent distant metastasis. Multimodality therapy featuring wide surgical margins, aggressive chemotherapy and adjuvant local radiotherapy is necessary for this rare disease. Platinum-based chemotherapy can be used as an adjuvant therapy.

• Resources Recycling
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• v.21 no.1
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• pp.30-40
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• 2012

Electric arc furnace slag is made in ironworks during steel refining, it is been increasing chemical and physical resistibility using ageing method of unstable state of melting steel slag for using concrete's fine aggregates. Which is been changing stable molecular structure of aggregates, it restrains moving of ion and molecule. In Korea, KS F 4571 has been prepared for using the electric arc furnace oxidizing slag to concrete aggregates(EFS). In this study, Electric arc furnace oxidizing slag is used in the PRCC(Polymer Rapid setting Cement Concrete) which is applied a bridge pavement of rehabilitation, largely. The results showed that the increment of compressive strength development by 10- 20%. The flexural strength of EFS-Con increased greatly as the electric arc furnace oxidizing slag changed. The compressive strength and flexural strength developed enough for opening the overlayed EFS-Con to the traffic after 4 hours of EFS-Con placement. The permeability of EFS-Con was evaluated as negligible due to its very low charge passed. Thus, EFS-Con could be used at repairing or overlaying the concrete at fast-track job sites.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.47-52
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• 1997

The objective of this study was to investigate the in vitro / in vivo embryonic development after vitrification of mouse zygotes and the chrom osomal normality of delivered live young after embryo transfer. Mouse IVF zygotes were cryopreserved by vitrification using vitrification solution, EFS40 (40% ethylene glyc이, 30% Ficoll a and 0.3 M sucrose in phosphate buffer saline c containing 10% FBS ) . After mouse zygotes were exposed to EFS40 at 25"C for 30 sec., they were immediately plunged into LN$_2$. Vitrified thawed mouse zygotes were cultured upto bIastocysts in M16 for 4 days. The rates of in vitro development were 71.5% under this condition. Cultured blastocysts were transferred to recipients (3 day of pseudopregnant) on one or both uterus horns (6-8 embryos per a uterus horn). And all recipients were allowed to produce litters. The results obtained in these experiments were summarized as follows: The pregnancy rates and in vivo survival rates, live fetus rates, for vitrified zygotes (80.0, 39.6%) were not significantly difference in those of control zygotes (77.8%, 50.0%). Also, all of live-born young mice were chromosomally normal (n=40). This results suggested that proposed rapid vitrification procedures can be effectively use in 1-cell mouse zygotes cryopreservation.

• The Korean Journal of Physiology
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• v.30 no.2
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• pp.219-229
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• 1996

In order to investigate intra-pancreatic cholinergic roles on insulin action in exocrine secretion, the pancreas was isolated from rats and continuously perfused with modified Krebs-Henseleit solution. Intra-arterial infusion of insulin (100 nM) or cholecystokinin (CCK, 14 pM) alone resulted in stimulation of the volume flow and amylase output. Also insulin potentiated the action of CCK in the exocrine secretion. Tetrodotoxin and atropine completely abolished the potentiating action of insulin and CCK as well as the action of insulin alone, but did not change the action of CCK alone. In order to see an effect of intra-pancreatic neural activation on the insulin action, electrical field stimulation (EFS) with parameters of 20 V, 2 msec and 8 Hz was applied to the isolated pancreas for 10 min under 2.5 or 18 mM glucose background. The EFS voltage-dependently elevated the flow rate and amylase output, and potentiated exocrine secretion in 18 mM glucose infusion compared with 2.5 mM glucose. The potentiating effects of EFS and 18 mM glucose were not observed in the streptozotocin-treated pancreas although it was perfused with 18 mM glucose. However, it was restored when the diabetic pancreas was perfused with porcine insulin(100 nM). Tetrodotoxin and atropine inhibited the pancreatic secretion induced by EFS with the background of 18 mM glucose. The results of present investigation indicate that the intra-pancreatic cholinergic tone exerts a stimulatory influence on the action of insulin in pancreatic exocrine secretion of rats.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.85-92
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• 1999

This study was designed to determine effect of cryoprotectant kinds and cell stages on OPP vitrification method in mouse embryos. The freezing speed, cryoprotectants and cell stage could affect of embryo viability following various vitrification methods. The vitrification solution used were consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose solution in holding medium (D-PBS supplemented with 5% FCS: HM) (EFS) or 16.5% ethylene glycol , 16.5% dimethyl sulfoxide, 0.5 M sucrose in HM (EDS). The embryos were collected from oviduct at 18 h after hCG injection and then washed and cultured in mHTF medium until use. In experiment 1, the blastocysts were vitrified by OPP straw to determine the optimal vitrification solution of EFS or EDS. The post-thaw survival rates at re-expanded stage rates were significantly different between EFS and EDS (95.0 vs 100%), but at hatching stage was not different between EFS and EDS (90.0 vs 95.0%). respectively. In experiment 2, zygotes, 2-, 4-cell, morula and blastocysts were vitrified by OPP method to determine the acceptable of early stage embryos. The development rates to expanded blastocyst in zygote (70.0%) were significantly lower rather than those in 2-, 4- 8-cell, compacted morula or blastocyst (89.7, 90.0, 92.8, 97.6 or 97.5%), respectively. However, the cell number of post-thaw developed to expanded blastocyst in blastocyst and control blastocyst stage (39.6$\pm$2.81, 35.7$\pm$2.98) were significanty higher than those in zygote, 2-, 4-, 8-cell, compacted morula (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 or 30.8$\pm$2.93). In experiment 3, the zygotes were exposed in VSl for 1, 2, and 3 min to the optimal exposed time. The cleavage rates (91.6, 88.5, 88.9%) and develop mental rates to blastocyst (83.3, 74.3 and 69.4%) depends on the exposed time in VSl were not significantly different among 1, 2, or 3 min, respectively. The cell number also were not significantly different among exposed time in VS1. respectively. These results indicate that OPP method could be useful for vitrification either EFS or EDS vitrification solution. The post-thaw survival rates at zygote were significantly lower than those at 2-, 4-, 8-cell, morula or blastocyst, respectively. The zygote stage were more sensitive rather than late stage embryos. The exposing time in VS1 for 1 min was better than that for 2 or 3 min, even it was not significantly different. The OPP vitrification method could be useful of mouse embryos either with EFS or EDS vitrification solution.

• YAKHAK HOEJI
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• v.41 no.4
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• pp.399-406
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• 1997

The effects of different $K^+$ channel blockers were investigated on the non-adrenergic non-cholinergic (NANC) relaxations in the circular muscle of the rabbit proximal stomach. Non-selective blockers of $K^+$ channels, 4-aminopyridine (4-AP, 3~30${\mu}M$) and tetraethylammonium (TEA, 100~1000${\mu}M$) significantly enhanced the NANC relaxations in a concentration-dependent manner. The enhancement was more prominent for the NANC relaxations induced by the electric field stimulation (EFS) with lower frequencies. Blockers of large conductance $Ca^{2+}$-activated $K^+$ channels, charybdotoxin and iberiotoxin, a blocker of small conduntance $Ca^{2+}$-activated $K^+$ channels, apamin and a blocker of ATP-sensitive $K^+$ channels, glibenclamide had no effect on the NANC relaxations, respectively. Exogeneous administration of nitric oxide (NO, 1~30${\mu}M$) caused concentration-dependent relaxations which showed a similarity to those obtained with EFS. None of the $K^+$ channel blockers had an effect on the concentration-dependent relaxation in response to NO. These results suggest that prejunctional $K^+$ channels regulate the release of NO from the NANC nerve in the rabbit proximal stomach as the inhibition of prejunctional $K^+$ channels increases the NANC relaxation induced by the EFS.