• 대한화장품학회지
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• 제25권3호
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• pp.47-66
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• 1999

Resazurin(alarmar Blue$^{TM}$)을 이용하여 in vitro 적으로 P. ovale의 최소 발육저지농도 (Minimum Inhibitory Concentration , MIC)를 측정함으로써 여러 가지 조성의 항비듬제의 항비듬 효과를 측정하고자 하였다. 최적의 실험 조건을 결정하기 위한 기초 실험 결과, 약 2.6$\times$$10^{5}$ cfu/$m\ell$ 이상의 농도로 P. ovate가 주입되는 경우 alarmar Blue$^{TM}$ 흡광도의 변화를 정 하게 관찰할 수 있었고 alannar Blue$^{TM}$ 자체의 희석 배율이 1:1-1:4이며, 16시간과 24 시간 배양을 하여야 비교적 정량적인 결과를 얻을 수 있었다. alarmar Blue$^{TM}$ 가 균체의 증식에 미치는 영향을 고찰하여 본 결과 1:1의 희석 용액에서도 alarmar Blue$^{TM}$가 균체의 증식에 영향을 미치지 못함을 알 수 있었다. Zinc pyrithione과 Climbazole의 혼합계에서 항비듬 효과를 관찰하여 본 결과 일정 혼합비에서 효과적인 항비듬 효과를 관찰할 수 있었고, 현미경 관찰에 의한 MIC 측정법과,SDDM(Skin-Disk Diffusion Method)법에 의한 항비듬 효과와 비교하여 본 결과 유사한 경향성을 관찰할 수 있었다. 이상의 결과를 종합해볼 때 alarmar Blue$^{TM}$를 사용한 in vitro 항비듬 효과 측정법은 시료나 배지의 탁도에 크게영향을 받지 않으며, Micro-plate reader를 사용함으로써 많은 종류의 시료에 대한 결과를 마르고 정확하게 얻을 수 있다는 장점을 가지고 있으므로, 향후 효율적인 in-vitro 항비듬 평가 방법으로 활용이 기대된다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제32권4호
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• pp.299-305
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• 2010

Purpose: Adipose tissue is located beneath the skin, around internal organs, and in the bone marrow in humans. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. Adipose tissue also has the ability to dynamically expand and shrink throughout the life of an adult. Recently, it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including, osteogenic, chondrogenic, endothelial cells, and myogenic lineages. Materials and Methods: This study focused on endothelial cell culture from the adipose tissue. Adipose tissues were harvested from buccal fat pad during bilateral sagittal split ramus osteotomy for surgical correction of mandibular prognathism. The tissues were treated with 0.075% type I collagenase. The samples were neutralized with DMEM/and centrifuged for 10 min at 2,400 rpm. The pellet was treated with 3 volume of RBC lysis buffer and filtered through a 100 ${\mu}m$ nylon cell strainer. The filtered cells were centrifuged for 10 min at 2,400 rpm. The cells were further cultured in the endothelial cell culture medium (EGM-2, Cambrex, Walkersville, Md., USA) supplemented with 10% fetal bovine serum, human EGF, human VEGF, human insulin-like growth factor-1, human FGF-$\beta$, heparin, ascorbic acid and hydrocortisone at a density of $1{\times}10^5$ cells/well in a 24-well plate. Low positivity of endothelial cell markers, such as CD31 and CD146, was observed during early passage of cells. Results: Increase of CD146 positivity was observed in passage 5 to 7 adipose tissue-derived cells. However, CD44, representative mesenchymal stem cell marker, was also strongly expressed. CD146 sorted adipose tissue-derived cells was cultured using immuno-magnetic beads. Magnetic labeling with 100 ${\mu}l$ microbeads per 108 cells was performed for 30 minutes at $4^{\circ}C$ a using CD146 direct cell isolation kit. Magnetic separation was carried out and a separator under a biological hood. Aliquous of CD146+ sorted cells were evaluated for purity by flow cytometry. Sorted cells were 96.04% positivity for CD146. And then tube formation was examined. These CD146 sorted adipose tissue-derived cells formed tube-like structures on Matrigel. Conclusion: These results suggest that adipose tissue-derived cells are endothelial cells. With the fabrication of the vascularized scaffold construct, novel approaches could be developed to enhance the engineered scaffold by the addition of adipose tissue-derived endothelial cells and periosteal-derived osteoblastic cells to promote bone growth.

• 구강회복응용과학지
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• 제19권2호
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• pp.87-96
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• 2003

The purposes of this study were to find out the optimum intensity of magnetic field where magnetism could promote the activity of osteoblast, and to discover the possibility of clinical application in the areas of dental implants and bone grafts by confirming the effect of clinically increasing bone formation. In this experiment, we used the Neodymium magnet, which had magnetic power six times as strong as the current ones and enabled the resistances against the demagnetization up to 20 to 50 times to be minimized with the size of 1mm in sight. In order to culture cells, a specially designed device was used. It was made to adjust the distance and accordingly to control the intensity of the magnetic field, by placing the cell culture plate in the center with a magnet of 1mm long and thick installed on the both ends. Using MC3T3-E1 cell, a kind of osteoblast-like cell, we cultured, for 24 hours, not only the test group which had been cultured under the magnetic fields with different intensity of 5, 10, 50, 100, 500, and 1000 Gauss, but also the control group excluding the influences of the magnetic field. After observing the cell's form and the density of the culture medium through an inverted microscope, we made a series of proceedings needed for the immunofluoroscence staining, such as fixation, normal serum reaction, primary antibody reaction, and secondary antibody reaction. And with a fluorescence microscope, we observed those-above and compared the frequency of expression of IFG-1 receptor. To make a Western immunoblotting analysis, the cells cultured under the same condition as the above had the procedure of the lysis buffer and the acrylamide gel electrophoresis was carried out. Protein transferred into the nitrocellulose membrane and tested on the primary and the secondary antibody reactions was observed and compared. The results were as follows: When observed through an inverted microscope, the nuclear divisions of the cells under the magnetic field of 10 Gauss were the most active, and the density of the cells could be observed the most enormously. As the result of an immunofluoroscence staining of IGF-1 receptor, the expression of IFG-1 was the most frequently observed under the magnetic field of 10 Gauss. On the other hand, few differences of consideration were made between the test group cultured under the magnetic fields of 5, 500, and 1000 Gauss and the control group. In respect of the expression of IFG-1 receptor, the test group cultured under the magnetic fields of 50 and 100 Gauss were higher than the control group, and lower than that cultured under the magnetic field of 10 Gauss.(p<0.05) According to the Western immunoblotting analysis, the band of IFG-1 receptor which had 85KDa of molecular weight was the darkest. Judging from the above-mentioned results, the growth factor receptor of an osteoblast cell which was an important criterion for the bone formation was increased in maximum under the magnetic field of 10 Gauss. Moreover it was observed that the optimum intensity of magnetic field in which magnetism made the activity of the osteoblast cell increase was about 10 Gauss.

• 생명과학회지
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• 제29권4호
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• pp.492-498
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• 2019

기존 연구를 통하여 토양에서 분리한 Acinetobacter schindleri DYL129로부터 3개의 lipase 유전자(lipAD1, lipAD2와 lipAD3)들과 1개의 chaperone (lipBD) 유전자를 보고하였다. 본 연구에서는 각 유전자들의 발현을 위해서 pET32a(+)와 pGEX-6P-1 벡터에 클로닝하여 각각을 pETLAD1-3와 pETLBD 또는 pGEXLAD1-3와 pGEXLB로 명명하였으며, 단백질의 발현량은 pET 시스템을 사용할 때 1.5 배 정도 향상됨을 확인하였다. LipAD1과 LipAD2는 inclusion body 형태로 발현이 되었으며, LipAD3과 LipBD는 soluble type으로도 발현되었다. Inclusion body 형태의 LipAD1과 LipAD2는 고농도의 우레아를 처리하여 refolding 시켰다. LipAD1은 C4와 C2를, LipAD2는 C2와 C14를 그리고 lipAD3은 C2, C4와 C14를 기질로 잘 이용하는 것을 확인하였다. 그리고 모든 효소들은 $50^{\circ}C$에서 최적 활성을 나타내었다.