• 대한의생명과학회지
• /
• 제2권1호
• /
• pp.121-126
• /
• 1996

Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할 수 있는 간편한 immunoassay체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA 방법으로 분석하였으며 이들 클론 중 bis-in-dole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids 와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids 와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05 nM정도의 dimeric Vinca alkoloids까지도 측정할 수 있었다.

• 한국식품과학회지
• /
• 제36권1호
• /
• pp.152-157
• /
• 2004

효소에 의한 단백질 분해로 우리 밀의 저 알레르겐화를 시도하기 위한 기초 연구로서 금강밀에서 gluten fraction을 추출하여 동물 실험을 통하여 경구 섭취된 gluten에 대한 항체 생성을 유도하였다. Gluten fraction은 UTH buffer로 추출하여 10-50kDa 사이의 분자량에서 여러 밴드가 확인된 분획을 얻었으며 약 25 kDa, 34 kDa 그리고 45 kDa 부근의 밴드가 주요하게 나타났다. 그리고, 경구 투여로 유도된 gluten에 대한 항체는 ELISA와 western blot의 항원-항체 반응을 실시하여 anti-gluten lgG, IgE를 확인하였다. 또한 경구 투여의 투여 시간 경과에 따라 항체가는 상승하였다. 추출된 gluten fraction은 bromelain, papain, ficin 그리고 b.p. protease 각 효소의 최적 pH와 온도에서 가수 분해하여 gluten fraction의 알레르겐 저감화를 시도하였다. 효소 분해 후 gluten fraction의 저분자화 정도는 anti-gluten 하체에 대한 ELISA를 실시하여 검토하였다. 그 결과 bromelain, papain, ficin의 경우보다 b.p. protease을 처리한 경우가 ELISA value를 크게 저하시켰다. 이것은 gluten fraction이 b.p protease 효소 가수분해에 의해 생성된 항체가 인식하는 amino acid 서열 (epitope)이 분해되었음을 나타낸 것이다. 마지막으로 b.p. protease의 분해능력을 1mg, 2mg, 3mg/10mL로 첨가량을 달리하여 검토한 결과 첨가량에 따른 변화는 거의 나타나지 않았다. 따라서 금강밀의 gluten fraction 10mL에 b.p protease 1mg으로 4시간 처리하여 단백질을 분해하여 우리밀의 항원성 저하 가능성이 탐색되었다.

• 한국식품과학회지
• /
• 제22권2호
• /
• pp.177-182
• /
• 1990

역류분배방법을 이용한 단백질의 분리정제 효율을 높이기 위하여, 4가지 모델단백질에 대하여 수용성액체 2상계에서의 분획계수를 KCl 농도와 pH변화에 따라 조사하였다. 또한, 역류분배방법을 이용한 다중분배 추출방식으로 모델단백질의 혼합물을 KCl 농도(0, 50, 250, 500mM)와 pH(4.5, 5.5, 6.5, 9.0, 12.0)를 달리한 20가지 조건에서 분획하였다. 그 결과 KCl 농도와 pH에 따라 단백질들의 분획계수(K)값이 다르게 나타나서, KCl을 첨가함으로서 역류분배방법을 이용한 단백질 분리효율을 향상시킬 수 있는 가능성을 보여 주었다. 단백질에 따라 분리가 잘 일어나는 KCl 농도와 pH는 다르게 나타나서 myoglobin의 경우 KCl 50mM, pH5.5조건의 14번 튜브 상층부와 KCl 250mM, pH6.5조건의 13번 튜브의 상층부에서 잘 분리가 되었으며, BSA의 경우는 KCl 250mM, pH4.5 조건의 14번 튜브에서 분리되었고, lysozyme의 경우는 KCl 50MM, pH4.5조건의 19번 튜브와 KCl 50mM, pH5.5의 16번 튜브 상층부에서 분리가 되었다.

• 분석과학
• /
• 제28권5호
• /
• pp.309-316
• /
• 2015

본 연구에서는 ICP-MS의 혈장 중 극미량의 납 분석법을 검증하기 위하여 Class 1,000 이내의 환경조건을 확보한 후 특이성, 일내 및 일간 정확도, 정밀도, 검정곡선 등을 확인하였다. 청정실의 부유 분진량을 측정한 결과, 0.3 µm 크기는 0.3~62개, 0.5 µm 크기는 0.0~28.3 개로 총 부유분진량이 최대 90.3개로 요구 환경에 부합하였다. 우태아 혈청 공시료로 조제한 시료의 MDL (Method detection limit)은 1.77 ng/L이고, LOQ (Limit of quantification)값은 5.55 ng/L 이었다. 검정곡선은 y=1.09×10⁻³ x+4.88×10⁻²이었고, 상관계수 r=0.9999이었다. 분석법 확립을 위한 특이성, 일내 및 일간 정확성, 정밀성, 그리고 검정곡선을 확인한 결과 50 ng/L 이상에서 분석법 확인 기준에 적합하였다. 이를 토대로 분석한 일반인의 혈장 중 납 농도 평균은 55.4 ng/L 이었으며, 현직 근로자는 440 ng/L 이었고, 퇴직 근로자는 132 ng/L 이었다.

• Clinical and Experimental Reproductive Medicine
• /
• 제28권2호
• /
• pp.121-129
• /
• 2001

Objective: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst.. Methods: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-fteezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of bias to cysts in experimental groups. Results: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower ($X^2$-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%,48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (${\pm}SD$) cell number of blastocyst in the exposure only ($89.2{\pm}11.5$), EM grids ($85.0{\pm}10.3$) and cryoloop ($89.0{\pm}11.0$) groups, except slow-freezing group ($79.0{\pm}10.0$), were not significantly different from that of control group ($93.1{\pm}13.9$) 24 h after thawing (Student's t-test). Conclusion: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.

• Asian-Australasian Journal of Animal Sciences
• /
• 제22권3호
• /
• pp.328-335
• /
• 2009

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature ($23.5{\pm}1.5^{\circ}C$), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes ($39^{\circ}C$) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44${\pm}$2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

• 대한한방종양학회지
• /
• 제5권1호
• /
• pp.47-60
• /
• 1999

Traditional oriental medicines have been used for treatment of various kinds of human cancers for long times and some of them proven to be effective clinically. However, the pharmacological actions and mechanisms related to cancer treatment are generally unknown. In an effort to clarify the action mechanisms of several oriental medicines used for cancer treatments, we planned this experimental procedures. We selected Cordyceps sinensis (冬蟲下草), Punellae Herba (夏枯草), Rehmanniae Radix (熟地黃), Paeoniae Radix (白芍藥), Oldenlandiae Herba (白花蛇舌草), Partulaceae Herba (馬齒? ), Scdopendra subspinipes mutilans (蜈蚣), Mylabris Phalerara (班蟄), Phellinus igniarius(桑黃), Ganodermae Lignum(靈芝) for evaluation, which have been used for patients of gastric cancers. The twenty grams of medicines were boiled in 100ml of water for 1 hour and filtered with $0.2\;{\mu}m$ pore-sized filter unit to remove insoluble particles. Initially we evaluated the effects oriental medicines on growth inhibition in stomach cancer cells. The gastric cancer cell line, AGS, was cultured in RPMI 1640 supplemented with l0% heat-inactivated fetal bovine serum and treated with $10{\mu}l$ oriental medicines per 1ml of medium up to 48 hours. The specimens were subjected to MTT assay for evaluation of growth inhibition. We found mat Mylabris phalerata (班蟄) markedly suppressed the growth of cancer cells as shown in results. Next, we checked the effects of oriental medicines on cancer on cell cycles and apptosis. Mylabrls phalerata (班蟄) inhibited cell cycle progression of cancer cells a compared with control cells and cells treated with other medicines. In addition, Mylabri phalerata (班蟄) induced apoptosis in 30-40% of stomch cancer cells within 24 hours. Other oriental medicines used for this experiments did not show apoptosis-inducing effects on cancer cells. Finally, we determined the expression levels of genes associated with cell cycle and apoptosis. The expressions of Bcl-2 and bcl-XL were downregulated by the treatment of Mylabris phalerata (班蟄). However, the expression levels of genes related to cell cycles were not altered significantly. In conclusion, we found that Mylabris phalerata (班蟄) has in vivo gowth-inhibiting and apptosis-inducing effects on stomach cancer cells. However, we think that at least animal experiments are necessay for evduations.

• Journal of Acupuncture Research
• /
• 제25권1호
• /
• pp.25-55
• /
• 2008

Objectives : The aim of the present study was to observe the effect of Astragali Radix Herbal-Acupuncture Solution(AR-HAS) at $ST_{36}$(jok-samni, $Z\acute{u}s\bar{a}n$ Li) on collagen- II -induced arthritis(CIA) in mice. Methods : DBA/1J mice were immunized with bovine type II collagen(CII) on days 0 and 21 to induce arthritis. The mice were divided into 5 groups : normal group(no CIA), control group(CIA+no treatment), needle prick group(CIA+single prick with an injection needle), saline group(CIA+saline injection) and ARHA group(CIA+ R-HA treatment). The needle prick, saline injection, and AR-HA groups were injected on the right $ST_{36}$(jok-samni) of mice for 9 weeks, 3 times a week, beginning 4 weeks after the booster immunization. Results : 1. The incidence of arthritis, AI(arthritis index), and joint edema decreased in the AR-HA group. 2. Weight gain, hypertrophy of the spleen, adhesion of the tissues, and transformation of the joint were restrained in the AR-HA group. 3. The concentrations of $IL-1{\beta}$, IL-6, $TNF-{\alpha}$, $IFN-{\gamma}$ in CIA mouse serum and $IFN-{\gamma}$, IL-10 in the CIA mouse spleen cell culture decreased significantly at $ST_{36}$ in the AR-HA group. 4. Total cell counts increased significantly, and the ratio of $CD3e^+$ to $CD45R^+$, $CD4^+$ to $CD8^+$, and $CD4^+$ to $CD25^+$ cells decreased significantly at $ST_{36}$ in the CIA mouse spleen cell culture of the AR-HA group. 5. Total cell counts decreased significantly, and $CD4^+/CD25^+$ and $CD45R^+/CD69^+$ decreased significantly at $ST_{36}$ in the CIA mouse lymph nodes of the AR-HA group. 6. $CD3e^+/CD69^+$ and $CD11b^+/Gr-1^+$ cells decreased significantly at $ST_{36}$ in the CIA mouse joints of the AR-HA group. 7. The histological examination showed that cartilage destruction and synoviocyte proliferation decreased in the CIA mouse joints of the AR-HA group, and collagen fiber was expressed similar to that seen in the normal group. Conclusions : Our experiments show that at $ST_{36}$, an anti-inflammatory mechanism of AR-HA controls synovial cell proliferation and protects against cartilage destruction in rheumatoid arthritis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제38권6호
• /
• pp.343-353
• /
• 2012

Objectives: This aim of this study was to effectively isolate mesenchymal stem cells (hSMSCs) from human submandibular skin tissues (termed hSMSCs) and evaluate their characteristics. These hSMSCs were then chemically induced to the neuronal lineage and analyzed for their neurogenic characteristics in vitro. Materials and Methods: Submandibular skin tissues were harvested from four adult patients and cultured in stem cell media. Isolated hSMSCs were evaluated for their multipotency and other stem cell characteristics. These cells were differentiated into neuronal cells with a chemical induction protocol. During the neuronal induction of hSMSCs, morphological changes and the expression of neuron-specific proteins (by fluorescence-activated cell sorting [FACS]) were evaluated. Results: The hSMSCs showed plate-adherence, fibroblast-like growth, expression of the stem-cell transcription factors Oct 4 and Nanog, and positive staining for mesenchymal stem cell (MSC) marker proteins (CD29, CD44, CD90, CD105, and vimentin) and a neural precursor marker (nestin). Moreover, the hSMSCs in this study were successfully differentiated into multiple mesenchymal lineages, including osteocytes, adipocytes, and chondrocytes. Neuron-like cell morphology and various neural markers were highly visible six hours after the neuronal induction of hSMSCs, but their neuron-like characteristics disappeared over time (24-48 hrs). Interestingly, when the chemical induction medium was changed to Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS), the differentiated cells returned to their hSMSC morphology, and their cell number increased. These results indicate that chemically induced neuron-like cells should not be considered true nerve cells. Conclusion: Isolated hSMSCs have MSC characteristics and express a neural precursor marker, suggesting that human skin is a source of stem cells. However, the in vitro chemical neuronal induction of hSMSC does not produce long-lasting nerve cells and more studies are required before their use in nerve-tissue transplants.

• Journal of Periodontal and Implant Science
• /
• 제31권1호
• /
• pp.163-180
• /
• 2001

The aim of this study was to evaluate the effects of chitosan coating on the attachment, proliferation, functional and morphological change of periodontal ligament cells. Primary human periodontal ligament cells were cultured in dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. In experimental group, cells of 4th to 7th passage were inoculated in the multiwell plates coated with chitosan in concentration of 0.22, 0.2, and $2mg/m{\ell}$. Cell counting and MTT assay were done after 0.5, 1.5, 3, 6 and 24 hours of incubation to evaluate the cell attachment, and then after 2 and 7 days of culture to evaluate the cell proliferation. The alkaline phosphatase activity was measured after 4 and 7 days of culture and the ability to produce mineralized modules was evaluated after 21 days of culture. The results were as follows : 1. The morphology of periodontal ligament cells on the chitosan coating was round or spheric. Round cells were aggregated after 6 hours of culture. Aggregated cells on the chitosan coated surface showed nodule-like appearance after 24 hours of culture and not achieved confluency at 7 days. 2. During early period of culture, the attachment of periodontal ligament cells were inhibited by chitosan coating. Inhibition of cell attachment tended to increase with the concentration of chitosan. 3. At the chitosan concentration of 0.02 and $0.2mg/m{\ell}$, periodontal ligament cells were more rapidly proliferated at 7 days, compared to the control group. At the concentration of $2mg/m{\ell}$, the proliferation of periodontal ligament cells was inhibitied(p<0.01). 4. Alkaline phosphatase activity of periodontal ligament cells was increased in chitosan coated group, especially at the concentration of $0.02mg/m{\ell}$after 4 days of culture.5. Periodontal ligament cells produced mineralized nodules on chitosan coated wells without the addition of mineralized nodule forming materials (ascorbic acid, ${\beta}-glycerophosphat$, dexamethasone). With the addition of mineralized nodule forming materials, periodontal ligament cells produced more mineralized nodules at the concentration of $0.02mg/m{\ell}$, compared to the control. In summary, the attachment, proliferation, cell activity, and alkaline phosphatase activity of periodontal ligament cells depended on the concentration of coated chitosan. Chitosan stimulated mineralized nodule formation by periodontal ligament cells. At the appropriate concentration($0.02mg/m{\ell}$), chitosan could increase alkaline phosphatase activity and stimulate the formation of mineralized nodule by periodontal ligament cells. These results suggest that chitosan can be used as an adjunct for bone graft material, and the matrix of tissue engineering for periodontal regeneration, especially bone regeneration.