• 대한약침학회지
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• 제7권3호
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• pp.77-88
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• 2004

Objectives ; This study was to investigate the anti-cancer effects of herbal acupuncture with distilled fresh ginseng. The herbal acupuncture was injected to Chung-wan($C.V_{12}$) and Wisu($BL_{21}$) of mice that were subjected to Sarcoma-180 adbominal cancer cell and A549 human epithelial lung cancer cells in vitro. Methods : Anti-cancer effects of distilled fresh ginseng herbal acupuncture were tested by measruing Cox, Bcl-2, and Bax by using RT-PCR in A549 human epithelial lung cancer cells in vitro. And four weeks old Balb/c line male mice weighing around $20\;{\pm}\;3g$ were used to measure survival rate and anti-cancer effect to outputs of interleukin-2 and interleukin-4 using flow cytometry, possibility of mRNA menifestation using RT-PCR, and Cox mRNA. The results are as follows. Results : 1. In measuring mRNA menifestation in Cox, Bcl-2, and Bax by using RT-PCR in A549 human epithelial lung cancer cells in vitro, the result showed that fresh ginseng decreased Cox-2 which is directly involved in Inflammation process. 2. Survival rate was measured in an anti-cancer effect experiment against Sarcoma-180 abdorminal cancer. Median survival time of controlled group was 27 days, of experiment group I was 21 days, and of experiment group II was 27 days. Therefore, experiment group I showed -22.2% increase in survival rate and experiment group II showed no difference compare to controlled group. 3. There was no difference between condition group and controlled and experiment group in measuring outputs of interleukin-2 and interleukin 4 by using flow cytometry 4. In measuring outputs of interleukin-2 by using ELISA, there was no significant difference between condition group and controlled group and there was decrease in experiment group II compared to conditioned and controlled group. 5. In measuring cytokine mRNA menifestation by using RT-PCR, experiment group I showed increase of mRNA menifestation in interleukin-2,4 and $interferon-{\gamma}$ and experiment group II showed no significant difference in $interferon-{\gamma}$. Conclusion : According to the results, fresh ginseng herbal-acupuncture took a little effects in cancer. In using distilled fresh ginseng herbal acupuncture has effect on Cox-2 decrease. However, the difference in concentration of fresh ginseng showed no effect on killing cancer cell. It is assumed that inaccurate concentration of herbal acupuncture and fresh ginseng component could be the reason for this result. Therefore, future consideration will be studies on herbal acupuncture concentration.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.76-76
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• 2001

The objective of this study was to optimize the vitrification method of in vitro produced bovine embryos. Thus, in vitro produced embryos at 8 cell, morula and blastocyst stages were vitrified on electron microscope grids (EM grids) or in open pulled straws (OPS) with EG5.5 (5.5 M ethylene glycol, 1.0 M sucrose and 10% FBS in m-DPBS medium) freezing solution and their survival rates after thawing were compared. The embryos on EM grids or in OPS were briefly exposed to EG5.5 freezing solution and plunged directly into liquid nitrogen within 30 to 35 sec. Post-thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-DPBS, each for 1 min, and then cultured in CRI aa medium supplemented with 10% FBS. Embryonic survival rate was assessed as re-expanded and hatched rates of those embryos after warming. The rates of re-expansion embryos did not significantly different between EM grid (8 cell: 42.10%, morula: 66.66% and blastocyst: 77.08%) and OPS (8 cell: 47.36%, morula: 61.90% and blastocyst: 83.33%) methods. In addition, the hatched rates in EM grid (8 cell: 31.57%, morula: 57.14% and blastocyst: 72.91%) were similar to those in OPS (8 cell. 34.21%, morula: 50.00% and Blastocyst: 77.08%). Interestingly, even at the same blastocyst stage, the in vitro survival of day 7 embryos (EM grid: 79.48 and OPS: 87.18%) was higher than those of day 8 embryos (EM grid: 72.10 and OPS: 82.06%). The total cell number of blastocyst developed in vitro after vitrification was examined with Hoechst 33342 staining to compare the embryo quality among different treatment groups. The total cell number of blastocyst was not significantly different between vitrified groups (EM grid: 162.4$\pm$8.0 and OPS: 158.4$\pm$7.1) and unvitrified control (168.0$\pm$5.6). These results indicate that both vitrification containers can provide the high rate of embryo survival. Moreover, the OPS container may not need a cap to protect the container from floating after immersion in L$N_2$. Therefore, this study suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification method using EM grid or OPS with EG5.5 freezing solution. In the future, the Pregnancy rate would be investigated after transfer of our vitrified embryos into the appropriated recipients.

• 한국자원식물학회지
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• 제27권3호
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• pp.256-262
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• 2014

Viscum album var. coloratum (mistletoe) is considered as one of the endangered plant species in Korea. Our objective is to restore its population and multiplication of plant by ex situ method. In this research we explored the maximum germination (in vitro) of freshly harvested and stored seeds of mistletoe collected in different time intervals. Ex vitro germination after artificial inoculation on the branches of Prunus mume in different physiological conditions was also monitored. The research revealed that the lately harvested seeds (Feb. and March 2014) were superior over early harvested seeds (Nov. 2013 and Jan. 2014) of mistletoe due to the higher percentage of germination (above 93%). According to the data, it is also revealed that the survival and germination rate of mistletoe seeds decreased with the increase in storage duration. In ex vitro germination, the fluctuated temperature of a glass house in natural condition enhanced (four fold) the rate of germination on the branches of Prunus mume than the constant temperature condition in the glass house.

• Journal of Plant Biotechnology
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• 제5권1호
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• pp.43-49
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• 2003

Resistant cell lines to azetidine-2-carboxylic acid (AZCA) were selected through rice embryo culture after mutagenic treatment of callus irradiated with 30,50,70,90 and 120 Gy. The optimum AZCA concentration for the selection of resistant cell lines was 3 or 4 mM AZCA considering $LD_{50}$ and the fresh weight of callus. Survival rate of the AZCA resistant callus showed remarkable increase in the callus irradiated with 50 and 70 Gy. Regeneration rate of the AZCA resistant callus was much lower on the whole. Ninety and 120 Gy increased the regeneration rate for calli selected from 3 and 4 mM AZCA, respectively. Based on fresh weight, survival rate and regeneration for selection of the AZCA resistant cell line, 50-90 Gy was considered as the optimum range of gamma irradiation. Irradiated calli selected from AZCA were more tolerant to NaCl than those from non-irradiated calli. It suggests that elevated resistance to osmotic stress resulted from mutagenic treatment. The level of free proline content in the AZCA resistant cell line was increased up to 3.5 times compared with that in the control. Proline content in the regenerant derived from the AZCA resistant cell line also increased to 1.7 times that from the control plants regenerated from callus grown in AZCA free medium. Selection of proline overproducing cell lines by in vitro mutagenesis was successful and seems to be useful for improvement of stress tolerance in this crop.

• 한국수정란이식학회지
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• 제29권1호
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• pp.73-81
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• 2014

돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin $K_1$(vit $K_1$) 첨가 농도, 시기 및 시간의 효과를 검토하였다. $1.0{\mu}M$, $3.0{\mu}M$ 및 $6.0{\mu}M$ vit $K_1$을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 $14.5{\pm}4.3$, 0.0 및 0.0%로써 대조군의 $35.5{\pm}3.2%$에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 $31.8{\pm}2.6%$로써 시험군의 $22.2{\pm}2.9$, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, $1.0{\mu}M$ 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 $26.5{\pm}2.9%$ 및 $47.2{\pm}2.8%$로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). $3.0{\mu}M$ 농도에서는 대조군의 배발달율이 $36.4{\pm}3.1%$로 가장 높았으나, 생존율은 3.0시간 첨가군이 $41.7{\pm}3.2%$로 대조군에 비하여 유의하게 높았다(p<0.05). $6.0{\mu}M$ 농도에서도 배발달율은 대조군($32.0{\pm}2.8%$), 생존율은 0.5시간 첨가군($42.9{\pm}1.8%$)이 가장 높았다. 각각의 vit $K_1$ 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, $1.0{\mu}M$ 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, $3.0{\mu}M$ 3.0시간 및 $6.0{\mu}M$ 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율($59.5{\pm}4.1%$ 및 $50.0{\pm}3.6%$)과 생존율($72.7{\pm}5.4%$ 및 $79.2{\pm}4.0%$)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit $K_1$ 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군($1.0{\mu}M$ 6시간 배양 2일째, $3.0{\mu}M$ 3.0시간 배양 4일째 및 $6.0{\mu}M$ 0.5시간 배양 6일째)이 $53.4{\pm}5.8$, $49.4{\pm}3.8$ 및 $51.5{\pm}4.5$개로써 대조군의 $40.2{\pm}2.3$개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 $3.2{\pm}0.9{\sim}3.7{\pm}2.1$개로써 대조군의 $4.2{\pm}1.2$개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, $6.0{\mu}M$ 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit $K_1$의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.

• 한국가축번식학회지
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• 제14권1호
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• pp.9-16
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• 1990

This study was carried out in order to investigate effects of cryoprotectant concentration and equilibration time on survival of ultrarapidly frozen 2-cell mouse embryos. Mouse 2-cell embryos, following dehydration by exposure to DMSO and sucrose, were directly immersed into liquid nitrogen and thawed in 37$^{\circ}C$ water. Viability was defined by development rate to the blastocyst stage after in vitro culture for 72 hours. The results are summarized as follows ; 1. When 0.25M of sucrose was added into the freezing medium at various concentrations of DMSO and dilution medium, higher development rate of embryo was obtained in 3.0M DMSO concentrations (82.6%). However, when sucrose concentraitons of 0.25 and 0.5 M were added to the freezing medium with 3.0 M DMSO and dilution medium, development rate of embryos were 81.7% and 24.1%, respectively. 2. In the equilibration time at room temperature, higher development rate was attained after short period of time (2.5min) in 3.0 M DMSO＋0.25 M sucrose (85.9%). 3. The development rate of embryos at in vitro 2-cell, in vitro 2-cell, solution control and untreated control was 84.6%, 90.9%, 89.9%,, and 89.7%, respectively.

• 한국가축번식학회지
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• 제22권3호
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• pp.207-211
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• 1998

본 연구는 소 미성숙 난자의 동결에 있어서 미성숙 난자의 1, 2, 4, 8 시간 성숙배양한 다음 동결 융해 후 체외배양하였을 때 체외수정율과 생존율을 조사하고저 수행하였다. 미성숙 난자들 1∼8시간 성숙배양후 동결융해 및 체외수정시켰을 때 수정율은 12.8∼30.9%로서 단시간의 체외성숙 배양이 높게 나타났으며(23.8∼28.8%), 또한 체외배양시 생존율은 11.8∼29.9%를 나타냈다.

• 한국가축번식학회지
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• 제16권2호
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• pp.125-131
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• 1992

This study was carried out to investigate the effects of concentration and equilibration time of cryoprotective agents on the survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rat to the morula and blastocyst stage after in vitro culture or by FDA test. The results are summarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5, or 4.0M glycerol was 65.3, 61.8, 64.3, 59.4 or 39.4%, respectively. Addition of 0.25M sucrose into the freezing medium containing 2.0M glycerol showed higher survival rate than those of 2.5~4.0M glycerol. 2. The survival rates of porcine embryos after ultraradpid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0, 2.5, 3.0, 3.5 or 4.0M DMSO was 65.6, 67.6, 68.6, 60.6 or 23.6%, respectively. However, addition of 0.25M sucrose into the freezing medium containing 3.0M DMSO showed higher survival rate than those of 2.0, 2.5, 3.5 or 4.0M DMSO. 3. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5 or 4.0M propanediol was 63.2, 60.3, 62.1, 52.3 or 24.3%, respectively. Addition of 0.25M sucroese into the freezing medium containing 2.0M propanediol showed higher survival rate than those of 2.5~4.0M glycerol. 4. The survival rates of porcine embryos after ultrarapid frozen-thawing the freezing medium of 2.0M glycerol added 0.10, 0.25, 0.50 or 0.75M sucrose was 61.8, 70.8, 67.6 or 52.2%, respectively. Addition of 2.0M glycerol into the freezing medium containing 0.25M sucreose showed higher survival rate than that those of 0.10, 0.50 or 0.75M sucrose. 5. The higher suvival rate of porcine embryos were attained at short period of equilibration time 92.5~5min.) in the freezing medium added 0.25M sucreose and 3.0M compared to those of 10 or 20min. equilibration time in the same condition.

• 한국수정란이식학회지
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• 제13권2호
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• pp.147-157
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• 1998

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.347-353
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• 1997

본 실험은 초자화 동결된 생쥐 배반포기배의 융해 후 배양조건 및 이식방법이 난자의 생존에 미치는 효과를 조사하고자 실시하였다. 체외수정후, M16배양액에서 4일동안 배양하여 얻어진 생쥐 배반포기배는 EFS40 (40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 함유된 PBS)으로 초자화동결하였다. 실험 I에서는 융해 후 배양조건에 따른 난자들의 체외/체내 생존율을 조사하였다. 융해된 난자가 M16과 4 mg/ml 소혈청알부민과 20 가지 아미노산이 함유된 m-CR1 (2% BME 아미노산 용액, 1% MEM 아미노산 용액) 및 단층배양이 유도된 난구세포 (10% FBS가 함유된 m-CR1배양액)에서 각각 배양되었을 때, 융해 후 24시간째 체외 생존율은 배양조건에 따라 차이가 없었다(75.6, 83.1, 82.4%). 그러나 체내 발달율에 있어서 임신 15일째 생존 산자율은 39.0, 49.0, 38.1%로서 유사한 성적을 나타냈으나, 전체 착상율에 있어서는 m-CR1 (80.4%)에 배양되었을 때, M16 (51.2%), 난구세포와 공배양시 (57.1%)보다 유의하게 높은 생존율을 보였다(p<0.05). 실험 II에서는 수정란 이식 방법에 따른 체내 발달율을 조사하였다. 배반포기배를 융해 후 체외배양없이 곧바로 가임신 2, 3일째 대리모에 이식을 실시하였을 때, 가임신 2일째 대리모에서는 임신징후를 얻지 못하였고, 가임신 3일째 대리모에서는 50.0%의 착상율과 15.4%의 정상산자율을 얻었다. 그러나, 이러한 결과는 융해 후, 16시간 배양하여 가임신 3일째 대리모에 이식 (73.5, 57.1%)하는 경우보다 유의하게 낮은 결과였다(p<0.05). 실험 III에서는 초자화 동결된 배반포기배의 융해 후 배양시 발달이 늦어진 수정란의 이용효율을 극대화시키기 위해 융해한 4일째 초기, 5일째 초기, 5일째 팽창 배반포기배의 체외/체내 생존율을 조사하였다. 가장 높은 체외 생존율은 5일째 팽창 배반포기배 (78.3%)에서 얻었으나, 체내 발달율 (산자율, 착상율)에 있어서는 4일째 초기 배반포기배 (33.3, 66.7%)의 경우가, 5일째 팽창 배반포기배(29.0, 38.7%)의 경우보다 높았다(p<0.05). 따라서 본 연구의 결과는 배양조건과 수정란 이식방법에 따라 초자화 동결된 배아의 체외/체내 발달율을 높일 수 있으며, 발달이 늦은 배반포기배의 체내 발달율은 체외 배양시간이 길어질수록 낮아짐으로, 5일째 팽창 배반포기배보다 4일째 초기 배반포기배를 동결하는 것이 더 유용하다는 것을 알 수 있었다.