• Tuberculosis and Respiratory Diseases
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• v.63 no.3
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• pp.242-250
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• 2007

Background: Abnormal angiogenesis can induce hypoxia within a highly proliferating tumor mass, and these hypoxic conditions can in turn create clinical problems, such as resistance to chemotherapy. However, the mechanism by which hypoxia induces these changes has not yet been determined. Therefore, this study was conducted to determine how hypoxia induces changes in cell viability and extracellular microenvironments in an in vitro culture system using non-small cell lung cancer cells. Methods: The non-small cell lung cancer cell line, A549 was cultured in DMEM or RPMI-1640 media that contained fetal bovine serum. A decrease in the oxygen tension of the media that contained the culture was then induced in a hypoxia microchamber using a $CO_2-N_2$ gas mixture. A gas analysis and an MTT assay were then conducted. Results: (1) The decrease in oxygen tension was checked the anaerobic gas mixture for 30 min and then reoxygenation was induced by adding a 5% $CO_2-room$ air gas mixture to the chamber. (2) Purging with the anaerobic gas mixture was found to decrease the further oxygen tension of cell culture media. (3) The low oxygen tension resulted in a low pH, lactic acidosis and a decreased glucose concentration in the media. (4) The decrease in glucose concentration that was observed as a result of hypoxia was markedly different when different types of media were evaluated. (5) The decrease in oxygen tension inhibited proliferation of A549 cells. Conclusion: These data suggests that tumor hypoxia is associated with acidosis and hypoglycemia, which have been implicated in the development of resistance to chemotherapy and radiotherapy.

• Journal of Animal Science and Technology
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• v.54 no.2
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• pp.103-109
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• 2012

Present study was performed to investigate the effect of single and co-culture of adipocyte and muscle cell lines on cell differentiation. 3T3-L1 (adipocyte) and L6 (muscle) cell lines were single-cultured on the condition of 10% fetal bovine serum (FBS)/Dulbeco's modified eagle's medium (DMEM) for 48 h followed by culture within 5% FBS/DMEM as a growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without additives in single- or co-culture of the 3T3-L1 and the L6 cells to induce differentiation of both cell types. In co-culture system, the 3T3-L1 and the L6 cells were grown in separated places by being seeded on a $0.4{\mu}m$ insert membrane and on the bottom of 6 well plate, respectively. Cell differentiation was measured using morphological investigation and cytosolic analysis of glycerol-3-phosphate dehydrogenase (GPDH; for 3T3-L1) and creatine kinase (CK; for L6). Based on the GPDH results, the presence of L6 cells did not stimulate 3T3-L1 differentiation showing more differentiation of 3T3-L1 cells in the single-culture compared to the co-culture condition. In contrast, 3T3-L1 cells in the co-culture promoted differentiation of L6 cells. Enzymatic analysis supported this result showing that 3T3-L1 cells showed statistically (P<0.05) higher GPDH activity in the single-culture than the co-culture, whereas CK results of L6 cells were vice versa (P<0.05). Overall, present results may indicate that co-culture system is more reliable and precise technique compared to single-culture. Further studies on several co-culture trials including different media conditions, supplementation of differentiating substances, molecular biological analysis, etc. should be required to obtain practical and fundamental mass data.

• Journal of Periodontal and Implant Science
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• v.29 no.1
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• pp.15-30
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• 1999

The purpose of this study was to evaluate the effect of mixed extracts of aralia cortex and phellodendron cortex (P55A) on activities of human gingival fibroblasts and periodontal ligament cells in vitro. First experiment was done to evaluate the effect of P55A in normal condition. In control group, the cells($4.5{\times}10^4$ cells/ml) were cultured with Dulbecco's Modified Eagle's Medium contained with 10% fetal bovine serum. In experimental groups, P55A was added to the above culture condition at the final concentrations of 0.1 ${\mu}g/ml$(Test group 1), 1 ${\mu}g/ml$(Test group 2) and 10 ${\mu}g/ml$(Test group 3). Then each group was tested for the cell proliferation rate at $\frac{1}{2}$, 2, 5 days, protein levels at 2, 5 days, and alkaline phosphatase activity at 2, 5 days. Second experiment was done to evaluate the effect of P55A in high glucose condition. 200 mg/dl glucose was added to the same culture condition of all groups in first experiment. Then each group was tested for the cell proliferation rate at $\frac{1}{2}$ , 2, 5 days, protein levels at 2, 5 days, and alkaline phoaphatase activity at 2, 5 days. The results were as follows ; 1. First experiment 1) As P55A concentration increased, cell proliferation rate increased significantly in test group 2 at 2 days, and test group 2 and 3 at 5 days in human gingival fibroblasts and periodontal ligament cells(P<0.05). 2) In human gingival fibroblasts, all test groups showed significantly increased protein levels as compared to control group at 5 days. In periodontal ligament cells, test group 2 and 3 showed significantly increased protein levels as compared to control group at 2, 5 days(P<0.05). 3) Alkaline phosphatase activity of human periodontal ligament cells increased as P55A concentration increased. The test group 2 and 3 showed significant increase as compared to control group at 5 days(P<0.05). 2. Second experiment 1) As P55A concentration increased, cell proliferation rate increased significantly in test group 2 at 2 days, and test group 2 and 3 at 5 days in human gingival fibroblasts and periodontal ligament cells(P<0.05). 2) In human gingival fibroblasts, test group 3 showed significantly increased protein levels as compared to control group at 2 days, and all test groups at 5 days. In periodontal ligament cells, test group 2 and 3 showed significantly increased protein levels as compared to control group at 2, 5 days(P<0.05). 3) Alkaline phosphatase activity of human periodontal ligament cells increased as P55A concentration increased. The test group 2 and 3 showed significant increase as compared to control group at 2 days, and all test groups at 5 days(P<0.05). From the above results, mixed extracts of aralia cortex and phellodendron cortex appeared to enhance cellular activities including cell proliferation rate, protein levels and alkaline phosphatase activity of human gingival fibroblasts and periodontal ligament cells in normal and high glucose condition. This study suggests that mixed extracts of aralia cortex and phellodendron cortex seem to be able to subside the inflammation of periodontal tissue and regenerate the destructed periodontal tissue.

• Journal of Periodontal and Implant Science
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• v.29 no.3
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• pp.483-509
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• 1999

Biodegradable barrier membrane has been demonstrated to have guided bone regeneration capacity on the animal study. The purpose of this study is to evaluate the effects of cultured calvarial cell inoculated on the biodegradable barrier membrane for the regeneration of the artificial bone defect. In this experiment 35 Sprague-Dawley male rats(mean BW 150gm) were used. 30 rats were divided into 3 groups. In group I, defects were covered periosteum without membrane. In group II, defects were repaired using biodegradable barrier membrane. In group III, the defects were repaired using biodegradable barrier membrane seeded with cultured calvarial cell. Every surgical procedure were performed under the general anesthesia by using with intravenous injection of Pentobarbital sodium(30mg/Kg). After anesthesia, 5 rats were sacrificed by decapitation to obtain the calvaria for bone cell culture. Calvarial cells were cultured with Dulbecco's Modified Essential Medium contained with 10% Fetal Bovine Serum under the conventional conditions. The number of cell inoculated on the membrane were $1{\times}10^6$ Cells/ml. The membrane were inserted on the artificial bone defect after 3 days of culture. A single 3-mm diameter full-thickness artificial calvarial defect was made in each animal by using with bone trephine drill. After the every surgical intervention of animal, all of the animals were sacrificed at 1, 2, 3 weeks after surgery by using of perfusion technique. For obtaining histological section, tissues were fixed in 2.5% Glutaraldehyde (0.1M cacodylate buffer, pH 7.2) and Karnovsky's fixative solution, and decalcified with 0.1M disodium ethylene diaminetetraacetate for 3 weeks. Tissue embeding was performed in paraffin and cut parallel to the surface of calvaria. Section in 7${\mu}m$ thickness of tissue was done and stained with Hematoxylin-Eosin. All the specimens were observed under the light microscopy. The following results were obtained. 1 . During the whole period of experiment, fibrous connective tissue was revealed at 1week after surgery which meant rapid soft tissue recovery. The healing rate of defected area into new bone formation of the test group was observed more rapid tendency than other two groups. 2 . The sequence of healing rate of bone defected area was as follows ； test group, positive control, negative control group. 3 . During the experiment, an osteoclastic cell around preexisted bone was not found. New bone formation was originated from the periphery of the remaing bone wall, and gradually extended into central portion of the bone defect. 4 . The biodegradable barrier membrane was observed favorable biocompatibility during this experimental period without any other noticeable foreign body reaction. And mineralization in the newly formed osteoid tissue revealed relatively more rapid than other group since early stage of the healing process. Conclusively, the cultured bone cell inoculated onto the biodegradable barrier membrane may have an important role of regeneration of artificial bone defects of alveolar bone. This study thus demonstrates a tissue-engineering the approach to the repair of bone defects, which may have clinical applications in clinical fields of the dentistry including periodontics.

• Tuberculosis and Respiratory Diseases
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• v.43 no.4
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• pp.579-589
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• 1996

Background : Activation of neutrophil is critical for the clearance of microorganisms and toxic host mediators during sepsis. Unfortunately the activated neutrophil and its toxic byproducts can produce tissue injury and organ dysfunction. The leucocyte CD11/18 adhesion complex regulates neutrophil-endothelial cell adhesion, the first step in neutrophil migration to sites of injection and inflammation. To investigate the potential of neutrophil inhibition as a treatment strategy for sepsis, we evaluated the effects of monoclonal antibody against CD11b (MAb 1B6) in rats intrabronchial challenged with Escherichia coli. Methods : Animals were randomly assigned to receive monoclonal antibody against CD11b (1 mg/kg, sc) and bovine serum albumin(BSA, 1 mg/kg, sc) 6 hr before, at 0 and 6 hr after intrabronchial challenge of $20x10^9$ CFU/kg E. coli 0111. Animals were randomized to treat either 24, 60 or 90% oxygen after bacterial challenge and begining 4 hr after inoculation, all animals were received 100 mg/kg ceftriaxone qd for 3 days. Peripheral and alveolar neutrophil(by bronchoalveolar lavage) counts and lung injury parameters such as alveolar-arte rial $PO_2$ difference, wet to dry lung weight ratio and protein concentration of alveolar fluid were measured in survived rats at 12 hr and 96 hr. Results : Monoclonal antibody against CD11b decreased circulating and alveolar neutrophil especially more in 12 hr than in 96 hr The lung injury parameters of antibody-treated animals were not different from those of BSA-treated animals. but It was meaningless due to small number of survived animals. The early(6 hr) mortality rate was significantly increased in antibody-treated group(51%) compared to BSA-treated group(31%) (P=0.02) but late(from 12 hr to 72 hr) mortality rate was not different in antibody-treated group(44%) from BSA-treated group(36%) (P =0.089). Conclusion : Leucocyte CD11b/18 adhesion molecule is known to regulate neutrophil migration to the site of infection and inflammation. The monoclonal antibody against CD11b decreased alveolar neutrophil in rats with pulmonary sepsis and increased early mortality rate. Therefore, we can speculate that monoclonal antibody against CD11b blocks of alveolar recruitment of neutrophils, impairs host defense mechanism and increases early mortality rate of pulmonary sepsis in rat.

• Journal of fish pathology
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• v.24 no.1
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• pp.39-45
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• 2011

The specific antibody response of olive flounder, Paralichthys olivaceus to different water temperature were tested by enzyme-linked immunosorbent assay (ELISA). In the rearing temperature of $15^{\circ}C$, first anti-bovine serum albumin (BSA) antibody titer was appeared after 14 days of immunization, whereas 24~48 days post-immunization (PI) resulted maximum antibody titer in all 5 experimental fish with optical density (OD) values 1.94~3.04. At the end of the experiment (84 days), 0.03~1.28 OD values were observed. In the rearing temperature of $12{\sim}13^{\circ}C$, first antibody titer was found 28 days PI in 2 out of 5 fish. Three fish shown high OD titer (1.88~2.68) between 56 and 70 days and OD values of 0.49 to 2.35 were observed at 84 days. However, the anti-BSA antibodies of two fish showed less than 0.8 OD values until 84 days. In the rearing temperature of $10^{\circ}C$, specific antibody appeared at 56 days, maximum antibody titer was observed at 70 days in 2 out of 5 fish (OD values: 1.37~1.53) and 1.00 to 1.11 OD values were observed at 84 days. Rest 3 fish showed OD values of 0.12 to 0.68 much below to that of other 2 fish, throughout the experimental period. In conclusion, specific antibody response of olive flounder at high temperature was much faster, higher and longer than that at lower temperature.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.1-10
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• 2004

These studies were carried out to improve the reproductive efficiency through embryos transfer of Hanwoo IVM/IVF embryos. Following routine IVM/IVF procedure, oocytes and zygotes were cultured far 40 to 44 h in CRlaa medium with BSA. Then 2 to 8-cell embryos were removed the cumulus cell and were cultured in CRlaa medium containing 10% fatal bovine serum and 2.5 mM taurine in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. The fresh embryos of the morulae and blastocysts cultured for 6 to 9 days in vitro or the frozen-thawed embryos were transferred into recipients. The pregnancy rates of the blastocyst produced for 6, 7, 8, and 9 days in vitro culture were 59.4, 68.2, 66.0 and 100%, respectively. In the developmental stage, pregnacy rates of early blastocysts (61.1%), blastocysts(64.7%) and expanded blastocysts(69.5%) were higher than that of morulae stage(20.0%). The pregnancy rates according to the corpus luteum grades of A, B and C in recipients were 73.6, 62.9 and 50.0%, respectively. Effects of donor-recipients synchrony of after day 2, 1 and 0, before day 1 and 2 on the pregnancy rates were 35.7, 65.5, 72.6, 67.9 and 60.0%, respectively. Pregnancy rates of the body condition score of recipients $\leq$2(71.3%) were higher than those of $\geq$3.0 score(40.0%). The pregnancy rates according to the parity of recipients when embryo was transferred to cow(70.6%) was higher than in heifer(59.1%). The pregnancy rates according to hormone treatment before embryo transfer were 69.9% in hCG + GnRH administration group and 63.0% in control group. Fresh and frozen-thawed embryos on the pregnancy rates were 70.6 and 36.4%, respectively. Pregnancy rates in single and AI+single was 90.0% and 64.8%. Pregnancy rates in twin induction was better than in single. These results indicate that pregnancy rates after transfer were affected on the embryo ages, donor-recipient synchrony, body condition score of recipients, corpus luteum status, parity and hormone treatment to recipients.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.4
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.205-212
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• 1999

The present study was designed to examine the effects of selenium(Se), vitamin E(Vit. E) and recombinant Bovine Somatotropin(rBST) administration on the selenium and vitamin E concentrations of blood in Hanwoo sires Hanwoo sires were randomly assigned to five groups; 1. control, 2. rBST, 0.09mg/kg body weight (BW) 3. Vit E, 1,500IU/kg BW, 4. Se 0.lmg/kg BW, 5. Vit E, 1.500IU plus Se 0.1mg/kg BW. rBST, Vit. E and Se for each experimental group were given 6 times at 15 days interval by intramuscular injection. Blood samples were collected 10 times for experimental periods, separated the serum by centrifugation, and stored at －7$0^{\circ}C$. Se and Vito E concentrations in blood were measured by fluorophotometer and HPLC. Se concentrations of blood in control, rBST, Vito E, Se and Se plus Vito E groups were 64.55, 65.50, 68.15, 73.11 and 74.09 ppb/$m\ell$, respectively. Se concentration in Vit. E plus Se group was significantly higher than in control and rBST groups (P＜0.05), but Vito E group was not significantly different in control and rBST groups(P＞0.05). The Vit. E concentrations of blood in control, rBST, Vit. E, Se and Se plus Vit. E groups were 2.27, 2.32, 2.80, 2.58 and 2.75 ppm/$m\ell$, respectively. Vit. E and Vit. E plus Se groups were slightly higher than those of any other groups, but not significantly difference in 외 I experimental groups(P＜0.05). These results indicate that Se and Vit. E concentrations of blood were slightly increased with the injection of Se and Vit. E in Hanwoo sires.

• Journal of Yeungnam Medical Science
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• v.11 no.2
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• pp.293-302
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• 1994

The objective of this study was to establish a good methodology to isolate single smooth muscle cells that are alive and respond properly to pharmacological agents. Canine urinary bladders were employed as the source of single cells, and acetylcholine, atropine and imipramine were used as indicators of pharmacological responsiveness. Imipramine, an antidepressant drug exhibited the anticholinergic and calcium antagonizing properties on rat detrusor muscle. To establish a control value for a further experiment to elucidate the mechanism of action of imipramine on detrusor muscle, we measured the concentration-response of single cells to acetylcholine in the presesnce of imipramine by length of the cells and compared the result with the response in the presence of atropine. Tiny chops of smooth muscle taken from anesthetized canine urinary bladder were incubated in collagenase solution at $36^{\circ}C$ for 17-20 minutes. The collagenase solution included collagenase 1.2 mg/ml, soybean tryspin inhibitor 0.08 mg/ml, bovine serum albumin 2% in 10 ml Krebs-Henseleit buffer solution aerated with a consistent breeze of 95/5% $O_2/CO_2$, to maintain the pH at 7.4. After washing with plain K-H solution on 450 mesh, cells were dissociated from the digested tissue for 12-15 minutes. Cell suspension was transfered in 5 ml test tubes and acetylcholine was added for the final concentration to be $10^{-14}M{\sim}10^{-9}M$. To find the optimal time to fix the cells to determine the contractile responses, 1% acrolein was added 5, 10, 20, 30, 60 and 120 seconds after the administration of ACh. The length of cells fixed by acrolein were measured by microscaler via CCTV camera on phaes-contrast microscope. The average length of 50 cells from a slide glass was taken as the value of a sample at the very concentration point. Single cells were isolated from canine detrusor. The length of untreated cells varied from 82 ${\mu}m$ to 94 ${\mu}m$. The maximal response to actylcholine $10^{-9}M$ was accomplished within 5 seconds of exposure, and the shortening was $19{\pm}3$%. Atropine reduced the contraction of the cells concentration-dependently. Imipramine which exerts a cholinergic blocking action on some smooth muscles also reduced the contraction concentration-dependently and by a similar pattern as atropine. These findings document that imipramine may exerts a cholinergic blocking activity in the single smooth muscle cells isolated from canine urinary bladder.