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Cryopreservation of Bovine IVM/IVF/IVC Blastocysts by Vitrification (체외성숙, 체외수정 및 체외배양에서 생산된 소 배반포기배의 초자화 동결)

  • Nam, H.K.;Kim, E.Y.;Lee, K.S.;Yoon, S.H.;Park, S.P.;Lim, J.H.
    • Clinical and Experimental Reproductive Medicine
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    • v.26 no.2
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    • pp.231-238
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    • 1999
  • The objective of this study was to examine the effect of developmental stage and embryo age of in vitro produced bovine blastocysts after vitrification and thawing. In vitro cultured day 8 blastocysts after IVF were equilibrated 20% ethylene glycol (EG) for 3 min. and were vitrified using EFS40, which is consisted of 40% EG, 18% ficoll, 0.3M sucrose and 10% FBS added in mDPBS for 30 sec. before being plunged into $LN_2$. Also, survival in vitro was assessed by re-expansion and hatching or hatched at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) When the embryos were cultured for 8 day after IVF, 41.0% of the cleaved embryos developed to the blastocysts (early; 7.6%, expanded; 22.9%, hatching; 4.6% and hatched; 5.9%). 2) When the embryos were exposed or vitrified to the freezing solution, the re-expansion of vitrified embryos (73.3%) was significantly lower than that of control and exposed embryos (100, 97.0%) (p<0.05). But the formation rate of hatching or hatched blastocysts of vitrified embryos (66.7, 46.7%) at 48h after thawing was similar to that of exposed embryos (66.7, 39.4%) but not control (100, 100%) (p<0.01). However, in the total cell numbers of those developed hatched blastocysts, there were not significantly different among the treatment groups. 3) When the embryo survival rates by different developmental stage were examined, the re-expansion was not different among the groups $(64.5{\sim}75.6%)$. After warming 48 h, the hatching and hatched formation of early blastocysts (25.8, 9.7%) was significantly lower than those of expanded (69.7, 39.4%) and hatching blastocysts (53.3, 43.3%) (p<0.05). 4) In addition, when the expanded blastocysts at day 7, 8 and 9 were vitrified, the re-expansion of day 8 and 9 embryos was significantly lower than that of day 7 (day 7; 93.9%, day 8; 75.8% and day 9; 87.5%) (p<0.05). However, the rates of development to hatched blastocysts were no difference among the groups (day 7; 36.4%, day 8; 36.4% and day 9; 31.3%). These results suggested that in vitro produced expanded or hatching blastocysts can be efficiently cryopreserved by the two-step vitrification method using EFS40.

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A Preliminary Study on Depressive Symptoms and Glycemic Controls in Diabetic Patients (당뇨병 환자에서의 우울 및 관련증상에 관한 예비적 연구)

  • Ko, Seung-Hyun;Jeong, Jong-Hyun;Hong, Seung-Chul;Han, Jin-Hee;Lee, Seung-Pil;Ahn, Yoo-Bae;Song, Ki-Ho
    • Korean Journal of Psychosomatic Medicine
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    • v.12 no.2
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    • pp.165-173
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    • 2004
  • Objectives: Diabetes mellitus is a heterogeneous, chronic, progressive disease characterized by hyperglycemia and abnormality in protein, carbohydrate, fat metabolism. Recent studies have reorted two times prevalence of depression in individuals with diabetes compared to individuals without diabetics. This study was designed to investigate glycemic controls, anxiety, alexithymia, stress responses between depressed diabetic patients and non-depressed diabetic patients. Methods The subjects were 60 diabetic patients(mean age : $50.3{\pm}9.7$ years, 31 men and 29 women) who were confirmed to have diabetes depending on the laboratory findings as welt as clinical symptoms at the St. Vincent Hospital Diabetes Clinic, from Mar. 2004 to Sep. 2004. Laboratory test including, blood chemistry. glycated hemoglobin, urinalysis for proteinuria and Korean version of Beck Depression Inventory(BDI), State and Trait Anxiety Inventory(STAI), Toronto Alexithymia Scale(TAS) and Stress Response Inventory(SRI) were used for assessment. Based on BDI scores, all diabetics were divided into 13 depressed-diabetics group(above 20 point) and 47 non-depressed group(below 20 point). We compared demographic data. glycemic controls, STAI, TAS and SRI scores between two groups by independent t-test. Results : 1) Depressed diabetic groups were 13(mean age : $55.4{\pm}7.2$ years, 7 men and 6 women) and non depressed groups were 47(mean age $48.9{\pm}9.8$ years, 24 men and 23 women). In depressed diabetics, compared with non-depressed group, manifested aged(p=0.031), but other demographic data showed no difference between two groups. 2) No significant differences were noted in FBS, PP2h, Hb A1C, total cholesterol, HDL-cholesterol, SGOT/SGPT, BUN levels between depressed and non-depressed groups. But, blood creatine levels of depressed group were significantly increased than non-depressed group(p=0.026). 3) No significant differences were found in the score of STAI, STAI-S, STAI-T, TAS between depressed and non-depressed groups. 4) The SRI scores of depressed groups were significantly higher than non-depressed groups$(59.7{\pm}24.9\;vs.\;31.5{\pm}22.0)(p=0.000)$. Conclusion : The above results suggest that depressed diabetic patients are have more stress responses and higher blood creatine levels. However, there were no differences in laboratory data related to glycemic controls, and anxiety. alexithymia levels between two groups. We suggest that physicians should consider integrated approaches for psychiatric problems in the management of diabetes.

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Transformation of Adult Mesenchymal Stem Cells into Cardiomyocytes with 5-azacytidine: Isolated from the Adipose Tissues of Rat (성체 백서의 지방조직에서 추출한 중간엽 줄기세포의 5-azacytidine을 이용한 심근세포 분화 유도)

  • Choe Ju-Won;Kim Yong-In;Oh Tae-Yun;Cho Dai-Yoon;Sohn Dong-Suep;Lee Tae-Jin
    • Journal of Chest Surgery
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    • v.39 no.7 s.264
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    • pp.511-519
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    • 2006
  • Background: Loss of cardiomyocytes in the myocardial infarction leads to regional contractile dysfunction, and necrotized cardiomyocytes in infracted ventricular tissues are progressively replaced by fibroblasts forming scar tissue. Although cardiomyoplasty, or implantation of ventricular assist device or artificial heart was tried in refractory heart failure, the cardiac transplantation was the only therapeutic modality because these other therapeutic strategies were not permanent. Cell transplantation is tried instead of cardiac transplantation, especially bone marrow is the most popular donated organ. But because bone marrow aspiration procedure is invasive and painful, and it had the fewer amounts of cellular population, the adipose tissue is recommended for harvesting of mesenchymal stem cells. Material and Method: After adipose tissues were extracted from abdominal subcutaneous adipose tissue and intra-abdominal adipose tissue individually, the cellular components were obtained by same method. These cellular components were tried to transformation with the various titers of 5-azacytidine to descript the appropriate concentration of 5-azacytidine and possibility of transformation ability of adipose tissue. Group 1 is abdominal subcutaneous adipose tissue and Group 2 is intra-abdominal adipose tissue-retroperitoneal adipose tissue and omentum. Cellular components were extracted by collagenase and $NH_4Cl$ et al, and these components were cultured by non-induction media - DMEM media containing 10% FBS and inducted by none, $3{\mu}mol/L,\;6{\mu}mol/L,\;and\;9{\mu}mol/L$ 5-azacytidine after the 1st and 2nd subculture. After 4 weeks incubation, tile cell blocks were made, immunostaining was done with the antibodies of CD34, heavy myosin chain, troponin T, and SMA. Result: Immunostaining of the transformed cells for troponin T was positive in the $6{\mu}mol/L\;&\;9{\mu}mol/L$ 5-azacytidine of Group 1 & 2, but CD34 and heavy myosin chain antibodies were negative and SMA antibody was positive in the $3{\mu}mol/L\;&\;6{\mu}mol/L$ 5-azacytidne of Group 2. Conclusion: These observations confirm that adult mesenchymal stem cells isolated from the abdominal subcutaneous adipose tissues and intra-abdominal adipose tissues can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.

The Usefulness of Visceral Fat Thickness Measured by Ultrasonography as an Abdominal Obesity Index (초음파로 측정된 내장지방두께의 복부 비만지표로서의 유용성)

  • Kim, Yong-Kyun;Han, Man-Seok
    • Journal of radiological science and technology
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    • v.31 no.3
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    • pp.249-258
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    • 2008
  • Purpose : Abdominal obesity with visceral fat accumulation have been known to be intimately associated with the development of metabolic syndrome. Therefore, it is important to estimate the precise amount of visceral fat. Ultrasonography has been reported that it is a simple and noninvasive method for visceral fat evaluation. Purpose of this study is to evaluate the association of ultrasonographic visceral fat thickness, anthropometric indexes, and risk factor of metabolic syndrome, and to investigate the cut-off value of abdominal visceral fat thickness leading to increased risk of metabolic syndrome. Materials and methods : The subject included 200 men and 200 women who visited D healthcare center in Daejeon from January to April 2008. The subcutaneous fat thickness and visceral fat thickness were measured by ultrasonograph. As anthropometric index, we measured body mass index, waist circumference and waist/height ratio. As for the risk factor of metabolic syndrome, we measured blood pressure, high density lipoprotein cholesterol, triglyceride and fasting serum glucose. Results : VFT was significantly correlated with waist circumference, (r=0.683/M, r=0.604/F), waist to height ratio (r=0.633/M, r=0.593/F) and BMI (r=0.621/M, r=0.534/F) in both men and women. In addition it was significantly correlated with Systolic blood pressure (r=0.229/M, r=0.232/F), Diastolic blood pressure ((r=0.285/M, r=0.254/F), high density cholesterol (r=-0.254/M, r=-0.254/F), Triglyceride (r=0.475/M, r=0.411/F), and Fasting blood sugar (r=0.158/M, r=0.234/F) in both men and women. The cut-off value of visceral fat thickness leading to the increased risk of metabolic syndrome was 4.58cm (sensitivity89.2%, specificity 71.2%) in men and 3.50cm (sensitivity61.2% specificity 80.8%) in women respectively. The odds ratio of the risk of metabolic syndrome was dramatically increased with the abdominal visceral fat thickness level over 6cm in men and 5cm in women. Conclusion : The visceral fat thickness using ultrasonography was significantly correlated with anthropometric indexes and risk factors of metabolic syndrome in both men and women. The cut-off value of visceral fat thickness leading to the increased risk of metabolic syndrome was 4.58cm in men and 3.50cm in women.

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The Risk Factors of the Pre-hypertension and Hypertension of Rural Inhabitants in Chungnam-do (충남 농촌 지역 주민의 고혈압 전단계와 고혈압의 위험요인)

  • Eom, Ji-Sook;Lee, Tae-Ryong;Park, Seon-Joo;Ahn, Youn-Jin;Chung, Young-Jin
    • Journal of Nutrition and Health
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    • v.41 no.8
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    • pp.742-753
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    • 2008
  • The purpose of this study is to investigate risk factors of pre-hypertension and hypertension in rural residents. Nine hundred and ninety four subjects aged 40-70 yrs in Chungnam-do participated in this study. The subjects (n = 824) were classified into three groups of hypertensive, pre-hypertensive, and normotensive according to the Joint National Committee (JNC)-7 criteria. The weight, body mass index (BMI), waist-hip ratio (WHR), and serum total protein, albumin, BUN, and triglyceride (TG) were positively correlated with SBP and DBP. After adjusted by age, sex and BMI, the total protein, albumin and TG were significantly correlated with SBP and DBP (p < 0.01). There was no significant difference in eating habits according to the level of blood pressure. The serum albumin, creatinine, Glu-FBS, Glu-PP l20, and triglyceride were higher in both prehypertensive and hypertensive group than in the normotensive group. However, mean serum cholesterol was not different among three blood pressure groups. In this study, the common risk factors of pre-hypertension and hyper-tension were male, age of fifties, lower education level, ex-smoking, higher drinking frequency, higher BMI, body fat %, waist circumference, WHR, serum albumin and diabetes, even though the degree of risks in these variables were higher in the hypertensive group. The higher BUN was a risk factor of prehypertension, while the family history, prediabetes, serum total protein, Glu-PP l20 and higher alcohol drinking amount were the risk factors of hypertension. This result suggests that maintaining good health habit and normal range of blood parameters as well as controlling body weight have to be paid attention in order to prevent hypertention, and further reseasch on the relationship of blood pressure and BUN are needed.

THE EFFECTS OF VARIOUS TETRACYCLINE HCL CONCENTRATION TREATED ROOW SURFACES ON PROLIFERATION AND SPREADING OF PERIODONTAL LIGAMENT CELLS (다양한 농도의 테트라사이클린로 처리된 치근면이 치주인대세포의 증식과 전개에 미치는 영향)

  • Jung, Oh-Chul;Sun, Jo-Young
    • Journal of Periodontal and Implant Science
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    • v.24 no.3
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    • pp.581-596
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    • 1994
  • This in vitro study was undertaken to obtain optimal tetracycline concentration that aids proliferation and spreading of human periodontal ligament cells, for clinical application in root surfaces of periodontally diseased teeth. Periodontal ligament cells used in this study were obtained from explants of periodontal ligament of 1st premolar teeth which were extracted for the purpose of orthodontic treatment. The cells were cultured in Dulbecco's Modified Eagle Medium(DMEM) supplemented with 100 U/ml penicillin, $100\;{\mu}g/ml$ streptomycin and 10% FBS at $37^{\circ}C$, 100% humidity, 5% $CO_2-95%$ air. Cells were used between the third to 4th passage. After root planing of periodontally extracted teeth, the root slabs were cut with carborundum disk. In the cell proliferation experiment, experimental groups were root planing only group, immersed groups in 25, 50, 75, 100, 150mg/ml aqueous solution of Tetracycline HCl followed by a vigorous rinse in PBS. Human PDL cells at concentration of $1{\times}10^5\;cells/ml$ were seeded in each culture well which contained root slabs and incubated for 6 hours. Then, all of the root slabs were moved into new 24 culture well and incubated 24, 48 and 72 hours. The cell counting was done by inverted phase contrast microscope after trypsinization. The following results were obtained. The cell number was increased in order root planing only group, 25, 150, 50, 75, 100mg/ml of Tetracycline HCl treated group in 24, 48 and 72 hours. The maximal cell number was obtained when the root slabs were immersed in solution with 100mg/ml of Tetracycline HCl. There were statistically significant between the root planing only group and 75, 100 mg/ml of Tetracycline HCl treated group in 24 hours, between the root planing only group and 100mg/ml of Tetracycline HCl treated group in 48 hours, between the root planing only group and 50, 75, 100mg/ml of Tetracycline HCl treated group, between 25 and 100mg/ml of Tetracycline HCl treated group in 72 hours(p<0.05). In the cell spreading experiment, after 30 minutes of incubated, in the root planing only group, the cells were generally round in shape. The cell surface was mostly covered with blebs. The cells started to attach to root surface by cytoplasmic extension in 50, 100mg/ml of Tetracycline HCl treated groups, more numerous cells attached to root surface than root planing only group. Many orifices of dentinal tubule were exposed, cells showed radially spreaded cytoplasm and unspreaded central region of the cell was covered with blebs. After 6 hours of incubation, in the root planing only group, cells showed radially spreaded cytoplasm and were attached flat appearance. In 50, 100mg/ml of Tetracycline HCl treated groups, cellular margin was concaved and cytoplasm showed elongated appearance with polarity. After 24 hours of incubation, in the root planing group, cells showed characteristic polarity. In 50, 100mg/ml of Tetracycline HCl treated groups, cells showed more elongated and spindle - like appearance.

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