• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.639-645
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.

• Journal of Sericultural and Entomological Science
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• v.40 no.2
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• pp.111-116
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• 1998

To construct transformed Bm5 cells, Autographa californica nuclear polyhedrosis virus (AcNPV)IE1 gene, an immediate early viral gene was firstly used in this study. AcNPV IE1 gene, which shares on 95.3% uncleotide sequence homology with Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1 gene, was isolated and cloned into pBluescript. Neomycin gene from pKO-neo was inserted under the control of the IE1 promoter to yield pAcIE1-neo. The plasmid pAcIE1-neo was transfected into Bm5 or Sf9 cells, and neomycin-resistant cells were selected in TC100 medium containing 10% fetal bovine serum (FBS) and 1 mg/$m\ell$ G418 for two weeks. Individual clones were picked and each was amplified for further characterization. The genomic DNA from neomycin-resistnt cells was isolated and characterized by PCR using AcNPV IE1 gene-specific primers and by Southern blot analysis using neomycin gene probe. We concluded that AcNPV IE1 gene was functional in B. moridrived Bm5 cells as well as Spodaptera frugiperda-derived Sf9 cells to produce stably-transformed insect cells.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.379-384
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• 1996

This study was undertaken to investigate the sister chromatid exchange (SCE) frequency and embryonic development after exposure to cryoprotectants and vitrification of mouse zygotes. Mouse IVF zygotes were cryopreserved by vitrification using vitrification solution, EFS40 (40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). After mouse zygotes were exposed to EFS40 at $25^{\circ}C$ for 30 sec., they were immediately plunged into $LN_2$ or cultured for cryoprotectant toxicity test without freezing. The results obtained in these experiments were summarized as follows: After thawing, survival rates to the 2-cell stage of zygotes exposed to or vitrified in EFS 40 (98.5%, 95.2%) were not significantly difference compared with that of control (100%). However, the developmental rates upto blastocyst and hatching blastocyst in vitrified groups (66.7, 50.0%) were lower than those of control (93.9, 81.8%) or exposed group (94.0, 78.8%) (p<0.05). When the influence of vitrification and exposure to cryoprotectant on the in vitro development of mouse zygotes was assessed by the SCE frequency, the SCE frequency in exposed ($20.2{\pm}2.1$) to or vitrified embryos ($21.4{\pm}3.2$) was higher than that in control embryos ($16.8{\pm}1.5$). These results suggest that the frequency of SCE was increased after cryoprotectant exposure or Vitrification although developmental rates of zygotes upto blastocysts and /or hatching blastocysts were not afected by cryoprotectant.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.5
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• pp.385-391
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• 2011

Purpose: The periosteum is a well-known source of osteogenic precursor cells for tissue-engineered bone formation. However, cultured endothelial or endothelial-like cells derived from periosteum have not yet been investigated. This study focused on endothelial-like cell culture from the periosteum. Methods: Periosteal tissues were harvested from the mandible during surgical extraction of lower impacted third molars. The tissues were treated with 0.075% type I collagenase in phosphate-buffered saline (PBS) for 1 hr at $37^{\circ}C$ to release cellular fractions. The collagenase was inactivated with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant was centrifuged for 10 min at 2,400 rpm. The cellular pellet was filtered through a $100{\mu}m$ nylon cell strainer, and the filtered cells were centrifuged for 10 min at 2,400 rpm. The resuspended cells were plated into T25 flasks and cultured in endothelial cell basal medium (EBM)-2. Results: Among the hematopoietic markers, CD146 was more highly expressed than CD31 and CD34. The periosteal-derived cells also expressed CD90 and CD166, mesenchymal stem cell markers. Considering that the expression of CD146 was constant and that the expression of CD90 was lower at passage 5, respectively, the CD146 positive cells in passage 5 were isolated using the magnetic cell sorting (MACS) system. These CD146 sorted, periosteal-derived cells formed tube-like structures on Matrigel. The uptake of acetylated, low-density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL) was also examined in these cells. Conclusion: These results suggest that the CD146-sorted positive cells can be referred to as periosteal-derived CD146 positive endothelial-like cells. In particular, when a co-culture system with endothelial and osteoblastic cells in a three-dimensional scaffold is used, the use of periosteum as a single cell source would be strongly beneficial for bone tissue engineering.

• Journal of Preventive Medicine and Public Health
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• v.31 no.1 s.60
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• pp.59-71
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• 1998

To examine the association of hypertension with cluster of obesity, abnormal glucose and dyslipidemia in Korean urban population, we conducted this cross-sectional study among 3027 men and 2127 women age 20-85 years who visited a prevention center between May 1991 and June 1995 for a multiphasic health check at St. Mary's Hospital, Seoul. By the self-administered questionnaire, the informations of educational attainments, monthly income, alcohol consumption, cigarette smoking, and physical excercise level were obtained. Height, weight, and blood pressure were measured by a trained nurse. The fasting blood sugar (FBS), total cholesterol, high density lipoprotein (HDL) cholesterol and triglyceride were tested by enzyme method. Low density lipoprotein (LDL) cholesterol was calculated by 'total cholesterol - HDL cholesterol - triglyceride/5'. For testing the differences of cardiovascular risk factors between hypertension and normotension group, 1-test and $\chi^2$-test were performed. For the age adjusted odds ratios of hypertension in persons with obesity, abnormal glucose, and dyslipidemia compared with normal, logistic regression was performed by using SAS pakageprograme. The results obtained were as follows: 1. Age, weight, body mass index(BMI), blood glucose, total cholesterol, LDL cholesterol, and triglyceride of hypertension group in men and women were significantly higher than normotension group, but height and HDL cholesterol of hypertension group only in women significantly lower than normotension group. The frequency of obesity $(BMI\geq25kg/m^2)$, abnormal glucose $(\geq\;120mg/dl)$, hypercholesterolemia $(\geq\;240mg/dl)$, lower HDL cholesterol (<45 mg/dl in women only), higher LDL cholesterol $(\geq\;160mg/dl)$, and hyper hypertriglyceridemia $(\geq\;250mg/dl)$ in hypertension group of men and women were significantly higher than normotension group. 2. Systolic and diastolic blood pressure were negatively correlated with hight, but positively with age, weight, BMI, total cholesterol, LDL cholesterol, and triglyceride in men and women. BMI was positively correlated with fasting blood sugar, total cholesterol, LDL cholesterol and triglyceride but negatively with HDL cholesterol. 3. The age adjusted odds ratios of hypertension were as follows in men and women : among persons who were obese compared with those nonobese, 2.53 (95% Confidence Intervals [C.I.] 2.08-3.07) and 2.22 (95%C.I. 1.71-2.87): among persons who were abnormal glucose compared with those normoglycemic, 1.43 (95%C.I 1.13-1.82) and 2.01 (95%C.I 1.36-2.94): and among persons who were dyslipidemia (hypercholesterolemia or lower HDL cholesterol or higher LDL cholesterol or hypertriglyceridemia) compared with those normal lipid, 1.59 (95%C.I 1.30-1.95) and 1.51 (95%C.I 1.16-1.96). After combined more than one risk factor, the odds ratios were increased. Among persons with cluster of obesity, abnormal glucose, and dyslipidemia, the odds ratio of hypertension was 2.25 (95%C.I 1.47-3.37) in men and 3.02 (95%C.I 1.71-5.30) in women. In conclusion, it was suggested that hypertension was associated with cluster of obesity, abnormal glucose, dyslipidemia in this Korean urban population.

• Proceedings of the Ginseng society Conference
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• 1993.09a
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• pp.102-109
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• 1993

Backgrounds Diabetes mellitus is associated with accelerated atherosc lerosis and predispose to specific microvascular problems. This study was performed to evaluate the usefulness of red ginseng as adjunctive therapeutic agent of NIDDM especially in preventing chronic diabetic complications. Materials and Methods We treated 50 patients with NIDDM for 5 month with 2 regimens: 1)oralhypoglycemic drug therapy only(the control group), 2)oral hypoglycemic group). The patients were recruited at Korea university hospital from June, 1992 to October, 1992 and the following inclusion criteria were used: l)age above 35 years 2)initial body weight within or above ideal body weight 3)fasting blood glucose level greater than 140mg/dl 4)no previous history of diabetes mellitus or no history of blood glucose control for recent 3 months of more. The patients were seen every 2 weeks for remaining 3 months. At every visit FBS and PP2hr blood glucose were measured with blood pressure and body weight. Lipid profiles were checked every 4 weeks and platelet function test was perfomed with aggregometer after administration of ADP, epineprine and collagen every 4 weeks. Free fatty acid were also analyzed every 8 weeks and glycosylated hemoglobin was measured every 12 weeks. Results The results were as follows: 1. The mean values for fasting and PP2hr blood glucose decreased significantly in the control group than in the ginseng group. 2. The weight gain was less in the ginseng group than in the control group. The levels of systolic blood pressure decreased' significantly in the ginseng group than in the control group. 3. There was no significant differences of lipid profiles in both groups. 4. The platelet hyperaggregation was improved more significantly in the ginseng group than in the control group. Conclusions In patients with NIDDM who were recieving oral hypoglycemic drug therapy, the addition of red ginseng improved platelet function and blood pressure, but induced less weight gain. The data suggests that red ginseng may be useful as a therapeutic adjunct especially in preventing chronic complications of NIDDM.

• International Journal of Human Ecology
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• v.4 no.2
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• pp.27-37
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• 2003

Although the idea that obese people consume higher calorie diets is widely accepted, many dietary surveys have shown that obese people do NOT consume larger amounts of energy. We had an opportunity to study the relationship between calorie intake and obesity in Korea from the data contained in the '98 National Health and Nutrition Examination Survey of Korea. The survey was executed nationwide for two months - from Nov. 1 to Dec.30 in 1998. The survey included 10,876 (aged >10 years) subjects of whom 9,771 underwent health examinations. Surveyors visited each household and checked health status, measured anthropometry and blood pressures, collected blood and urine samples, and interviewed from the health questionnaires. Well-trained dietitians evaluated the food consumption of 11,525 subjects over the age of 1 year with the 24-hour recall method. The number of subjects from whom a complete health examination and food consumption information was obtained was 8,004. Subjects were classified by BMI (< 20, 20-22, 22-24, 24-26, 26-28, 28 $\leq$) and into newly diagnosed patients with DM (FBS $\geq$ 126 mg/㎗), hypertension (SBP $\geq$ 140 mmHg or DBP $\geq$ 90 mmHg) and hyperlipidemia (Total cholesterol $\geq$ 220 mg/㎗ or TG $\geq$ 200 mg/㎗). Our main results were as following：1) their average energy intake was 2,029.6 $\pm$ 908.5 ㎉ and BMI is 22.6 $\pm$ 3.4 kg/$m^2$；2) a comparison of nutrient intakes by BMI level did not show a significant difference of energy intake even though BMI increased (BMI, < 20： 1,999 ㎉ ∼ 28 $\leq$： 2,028 ㎉)；and 3) Even in newly diagnosed patients with diabetes, hypertension or hyperlipidemia, their energy consumption was not significantly increased as BMI increased (from BMI 20). There are several possible explanations for these results：1) Reduced physical activity caused the weight of obese people to increase even with the same energy intake；2) people underreported their energy consumption；or, people intentionally reduced their energy consumption due to self-image regarding their obesity. We might also hypothesize that there is a metabolic problem conceiving obese people, because calorie intake was not higher in obese people than in non-obese people in Korea. Further research is necessary for re-evaluating these current conclusions.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.137-146
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• 2006

The objective of this study was to examine the effect of donor cell types, the source of recipient oocytes and estrous synchronization on pregnancy and delivery rates of somatic cell nuclear transfer (SCNT) embryos in Korean native goats. Recipient oocytes were surgically collected after superovulation. Ear cells and fetal fibroblasts were collected and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. The zonae pellucidae of in vivo- and in vitro-matured oocytes were partially drilled using a laser system. Single somatic cell was transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3 M mannitol. After the fusion, embryos were activated by Ionomycin+6-DMAP. NT embryos were cultured in mSOF medium supplemented with 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 12 to 20 hr. One hundred and two SCNT embryos were transferred into 20 recipients and pregnancy rate at days 30 was 20.0%. Of them, one developed to term and delivered 1 kid. Ear cells showed significantly higher fusion (63.8 vs. 26.5%) and pregnancy rates (20.0 vs. 0.0%) than those of fetal fibroblast (p<0.05). The recipients synchronized by CIDR showed significantly lower pregnancy rates compared to that of recipient in natural estrus ($0.0{\sim}25.0%$ vs. 100%) (p<0.05). Cloned kid was born from the recipient in natural estrus. For the synchronization of estrus between recipient and donor, there was no difference between treatments (${\pm}0$ vs. +12 hr) in pregnancy rate. The first healthy cloned kid (Jinsoonny) was produced by transfer of SCNT embryos derived from in vivo oocytes and ear cells into a recipient goat whose estrus was synchronized with the donor. These results imply that donor cells for nuclear transfer may affect the success rate, and the estrus synchronization between donor and recipient animals can also be important.