• Korean Journal of Poultry Science
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• v.41 no.4
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• pp.249-259
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• 2014

Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps and freezing media on the rates of viability of cryopreserved chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos of Korean Ogye (KO) and Commercial breeds (C), using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). Gonads were harvested from stage 28 chick embryos and pooled in groups of 5, 10, 15, 20E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15% and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to slow freezing and vitrification, with different concentrations of the cryoprotectant solution, were examined. After vitrification and slow freezing, survival rates of the frozen-thawed PGCs from the 10% EG plus FBS treatment were 85.63%, and 66.14% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05) (85.63% vs 66.81%) by vitrification. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at a germplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.75-84
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• 1999

This study assessed development in vitro of pronuclear(PN) stage embryos cryopreserved by the method of either vitrification or slow freezing, by using of different cryoprotectants, and equilibration and cooling rate, in rabbit. Ethyleneglycol- ficoll- sucrose(EFS) or ethyleneglycol- polyvinylpyrrolidone - galactose- (EPG-I) for vitrification, and EPG- II for slow freezing as cryoprotectant were used. The pronuclear embryos were exposed to EFS for 0 to 5 min and diluted with D-PBS and/or pre-dilution with 0.5 M sucrose. To examine the viability of frozen-thawed embryos, PN embryos were co-cultured with bovine oviductal epitherial cell(BOEC) for 5 days to hatching blastocyst stage in 39 $^{\circ}C$ 5% $CO_2$incubator. The results obtained were as follows: The dilution with 0.5 M sucrose and D-PBS after the exposure to EFS for 1.0 min resulted in no significant(P＜0.05) decrease in the development of PN embryos to hatching blastocyst(72.0%), compared with controls. The development of PN embryos cryopreserved to hatching blastocyst was not significantly (P＜0.05) different between EFS for 1.0 min(72.0%), EPG-I for 1.0 min(72.0%) and EPG-II for 30 min(66. 7%). The post-thaw development of PN embryos to hatching blastocyst was similarly very low as 6.1% and 11.5% in vitrification with EFS and slow freezing with EPG-II, respectively. The incidence of post-thaw zona-crack in PN embryos cryopreserved by slow freezing with plunging to liquid nitrogen at －35$^{\circ}C$ was signicantly(P＜0.05) higher(25.0%), compared with －85$^{\circ}C$ (1.9%). These results indicated that the rabbit PN embryos could be cryopreserved with either vitrification or slow freezing procedure, and frozen PN embryos could be successfully developed in vitro to haching blastocyst. but the post-thaw development of cryopreserved PN embryos was still very low under the present conditions.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.133-145
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• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

• Journal of Chest Surgery
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• v.38 no.1 s.246
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• pp.38-43
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• 2005

Matrix metalloproteinase-2 (MMP-2) is a class of proteolytic enzymes that digest collagen type IV and other components of the basement membrane. It plays a key role in the local invasion and the formation of distant metastases by various malignant tumors. The aim of this study was to evaluate the activity of MMP-2 and its significance as a prognostic marker in resected stage I non-small cell lung cancer (NSCLC). Material and Method: In this study we obtained fresh-frozen samples of tumor and non-tumor tissues from 34 patients with stage I NSCLC who underwent resection without preoperative radiotherapy or chemotherapy. After the extraction of total protein from tissue samples, MMP-2 activities were assessed by gelatin-substrate-zymography. The activities were divided into the higher or lower groups. Result: The MMP-2 activities were higher in tumor tissues than in non-tumor tissues. The MMP-2 activity of non-tumor tissues in recurrent group was higher than in non-recurrent group (p<0.01). Also the patients with higher MMP-2 activity of non-tumor tissues showed poor 5 year survival (p<0.01). Conclusion: This result indicates that the higher level of MMP-2 activity in the non-tumor tissue is associated with the recurrence and survival after the resection of stage I NSCLC. Therefore, MMP-2 activity in the non-tumor tissue could be used as a potential prognostic marker for the resected stage I-NSCLC.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.369-375
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• 2011

The aim of this study was to evaluate the effect of ${\alpha}$-tocopherol on blastocysts development and subsequent cryosurvival of the vitrification. The ${\alpha}$-tocopherol(0, 100, 200, 400 ${\mu}M$) was added in to culture medium for the bovine embryos. The blasocysts from the ${\alpha}$-tocopherol and untreated control groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. There were no differences in the overall cleavage rate($56.14{\pm}4.66$, $58.18{\pm}4.70$, $62.97{\pm}6.86$ and $51.17{\pm}7.28$) among four treatment groups. However, in blastocyst development and total cell number were significantly higher in ${\alpha}$-tocopherol 200 ${\mu}M$ ($38.60{\pm}7.12$; $106.33{\pm}3.50$) to culture medium than other treatment groups($29.30{\pm}5.24$, $31.60{\pm}7.12$ and $26.37{\pm}4.18$; $101.36{\pm}5.12$, $97.27{\pm}2.87$, and $91.23{\pm}7.52$ respectively). Before and after vitrification, the total cell number and blastocyst development of embryo were significantly higher in July to August than September to October. In conclusion, addition of ${\alpha}$-tocopherol 200 ${\mu}M$ to in vitro bovine embryo culture medium was beneficial for improving embryo quality by decreasing the embryo damage blsstocysts cell number and improving the tolerance of the embryos to cryopreservation.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Tuberculosis and Respiratory Diseases
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• v.64 no.3
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• pp.200-205
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• 2008

Background: Tumor angiogenesis plays an important role in tumor growth, maintenance and metastatic potential. Tumor tissue produces many types of angiogenic growth factors. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have both been implicated to have roles in tumor angiogenesis. In this study, the expression of tissue VEGF and bFGF from non-small cell lung cancer (NSCLC) patients were analyzed. Methods: We retrospectively investigated 35 patients with a histologically confirmed adenocarcinoma or squamous cell carcinoma of the lung, where the primary curative approach was surgery. An ELISA was employed to determine the expression of VEGF and bFGF in extracts prepared from 35 frozen tissue samples taken from the cancer patients. Results: VEGF and bFGF concentrations were significantly increased in lung cancer tissue as compared with control (non-cancerous) tissue. The VEGF concentration was significantly increased in T2 and T3 cancers as compared with T1 cancer. Expression of VEGF was increased in node-positive lung cancer tissue as compared with node-negative lung cancer tissue (p=0.06). VEGF and bFGF expression were not directly related to the stage of lung cancer and patient survival. Conclusion: Expression of VEGF and bFGF were increased in lung cancer tissue, and the expression of VEGF concentration in lung cancer tissue was more likely related with tumor size and the presence of a lymph node metastasis than the expression of bFGF. However, in this study, expression of both VEGF and bFGF in tissue were not associated with patient prognosis.

• Journal of Sasang Constitutional Medicine
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• v.27 no.2
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• pp.270-287
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• 2015

Objectives To evaluate the neuroprotective effects of modified Yuldahanso-tang (MYH) in a Parkinson's disease mouse model. Methods 1) Four groups (each of 8 rats per group) were used in this study. 2) The neuroprotective effect of MYH was examined in a Parkinson's disease mouse model. C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day), intraperitoneal (i.p.) for 5 days. 3) The brains of 2 mice per group were removed and frozen at $-20^{\circ}C$, and the striatum-substantia nigra part was seperated. The protein volume was measured by Bradford method following Bio-Rad protein analyzing kit. Using mouse/Rat Dopamine ELISA Assay Kit. 4) The brains of 2 mice per group were separated and removed. TH-immunohistochemical was examined in the MPTP-induced Parkinson's disease mice to evaluate the neuroprotective effects of MYH on ST and SNpc. 5) Two mice out of each group were anesthetized and skulls were opened from occipital to frontal direction to take out the brains. The brains added TTC solution for 20 minutes for staining. 6) The water tank used for morris water maze test was filled with $28^{\circ}C$ water, and a round platform of 10cm in diameter was installed for mice to step on. The study was carried out once a day within 30 seconds, keep exercising to step on the platform in the pool. 7) The brains of two mice out of each group were fixed in 10% formaldehyde solution and paraphillin substance was infiltrated. They were fragmented by microtome, and observed under an optical microscope after Hematoxylin & Eosin staining. 8) A round acrylic cylinder with its upper side open was filled with clean water and depressive mouse models were forced to swim for 15 minutes. After 24 hours the animals were put in the same equipment for 5 minutes and were forced to swim. 9) The convenient, simple, and accurate high-performance liquid chromatography (HPLC) method was established for simultaneous determination of Neurotransmitters in MPTP-MYH group. Results 1) MYH possess Dopamine cell protective effect on MPTP-induced injury in striatum and substantia nigra pars compacta. 2) MYH inhibits the loss of tyrosine hydroxylase-immunoreacitive (TH-IR) cells in the striatum and substantia nigra pars compacta on MPTP-induced injury in C57BL/6 mice. 3) MYH possesses improvement effect on MPTP-induced memory deterioration in C57BL/6 mice through the reduction of prolongated Sort of lost time by MPTP injection using the Morris water maze test. 4) MYH possesses hippocampal neuron protective effect on MPTP-induced injury in C57BL/6 mice. 5) MYH possesses improvement effect on MPTP-induced motor behaviour deficits and depression in C57BL/6 mice through the reduction of prolongated losing motion by MPTP injection using the Forced swimming test. 6) MYH increases serotonin product amount on MPTP-induced injury in C57BL/6 mice. Conclusions This experiment suggests that the neuroprotective effect of MYH is mediated by the increase in Dopamin, TH-ir cell, Hippocampus and Serotonin. Furthermore, MYH essential oil may serve as a potential preventive or therapeutic agent regarding Parkinson's disease.

• Journal of Korean Medicine Rehabilitation
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• v.19 no.1
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• pp.23-38
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• 2009

Objectives : The purpose of this morphological studies was to investigate the relations between Sanyinjiao(Sp6), Yinlingquan(Sp9) and pancreas of rats using peudorabies virus(PRV). Methods : We observed labeled neurons following the injection of PRV, Bartha strain, into the Sanyinjiao(Sp6), Yinlingquan(Sp9) and pancreas of rats. After survival times of 4 days following the injection of PRV, the rats were perfused, and their spinal ganglia, spinal cord and brain stem were frozen sectioned($35{\mu}m$). These sections were strained by PRV immunohistchemical staining methods and observed with light microscope. Results : The results were as follows. 1. In the spinal ganglia, the overlap areas of PRV labeled neurons projecting to Sanyinjiao(Sp6), Yinlingquan(Sp9) and pancreas were observed in T10-13 dorsal root ganglia. 2. In the spinal cord, the overlap areas of PRV labeled neurons projecting to Sanyinjiao(Sp6), Yinlingquan(Sp9) and pancreas were lamina I, IV, V, VII, IX, X, intermediolateral nucleus(IML), intermediomedial nucleus(IMM) in thoracic segments. In lumbar segments, the overlap areas of PRV labeled neuron were lamina I, IV, V, VI, IX, X and IMM. In sacral segments, the overlap areas of PRV labeled neuron were lamina I, IV, V, VI, VII, IX, X. 3. In the brain, the overlap areas of PRV labeled neurons projecting to Sanyinjiao(Sp6), Yinlingquan(Sp9) and pancreas were area postrema, nucleus tractus solitarius, caudoventrolateral reticular nu., medullary reticular nu., lateral paragigantocellular nu., C3 adrenalin cells, gigantocellular nu., raphe pallidus nu., raphe obscurus nu., ambiguus nu., raphe magnus nu., pontine reticular formation, A5 cell group, subcoeruleus nu., locus coeruleus, Barringnton's nu., $K{\ddot{o}}lliker$-Fuse nu., dorsal raphe nu., Edinger-Westphal nu., central gray matter, perifornical nu., dorsomedial hypothalamic nu., arcuate nu., lateral hypothalamic nu., paraventricular hypothalamic nu., hindlimb area. Conclusions : In conclusion, these results suggest that the interrelationship of meridian(spleen meridian), acupoints(Sp6 and Sp9) and viscera(pancreas) may be related the central autonomic centers.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.15 no.1
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• pp.63-79
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• 1993

To repair bony defects with tansplanted bone in the body, fresh autogenous bone is undoubtly, the most effective bone graft for clinical applications. But the demineralized bone has the matrix-induced bone formation which was suggested by Urist in 1965. Many authors assisted that demineralized bone powder induces phenotypic conversion of mesenchymal cells into osteoblasts, with high-density bone formation. The process of inducing differentiated cells becomes osteogenic properties. The purpose of this study was to evaluate the osteoinductive capacity of allogenic freeze-dried demineralized bone block (FDD, $7{\times}7mm$) and to compare FDD with the same sue of deep-frozen allogenic bone(DF), fresh autogenous bone (A) after implantation. The histological and ultrastructural features of tissue responses were examined after 1, 2, 4, 6, 8 weeks implantation of each experimental groups in the operative site of the New Zealand white rabbits. The results were as follows : 1. Inflammatory cell infiltration generally has appeared at 1 week, but reduced at 4 weeks in each group, but most severe in DF group. 2. Osteoblastic activity has increased for 4 weeks, but decreased at 6 weeks in each group and there was no significant difference among experimental groups. 3. New bone formation has begun at 1week, least activations in A groups, and showed the revesal line of bone formation among each group at 6 to 8 weeks. 4. Bone resorption has appeared at 1 week, but disappeared at 4 weeks in both A and DF groups, but more severe in DF than A groups. 5. In ultrastructural changs, the DF group have showed the most remarkable osteoclastic activities among experimental groups. 6. Osteoid or tangled collagen fibrils near the implanted sites were replaced by more mature, lamellated bony trabeculae during bone remodeling. There was little difference among each experimental groups. 7. During the convertion osteoblasts to osteocytes which embedded within the bone matrix, there was organ-less-poor cytoplasm, increased nuclear chromatin, abundant rough endothelial reticulum (RER) in each groups. From the above the findings, the DF group shored more bone resorption and foreign body reaction than FDD and A groups, and FDD group showed more new bone formation or osteoblastic activity than DF and A groups in early stage. There was no significant difference of cellular activities among the FDD DF, and A groups according to the time.