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

• 한국응용곤충학회지
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• 제56권1호
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• pp.19-28
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• 2017

폴리드나바이러스(polydnavirus: PDV)는 일부 내부기생봉에 공생하는 이중나선형 DNA 바이러스 분류군이다. Cotesia plutellae bracovirus (CpBV)는 프루텔고치벌(C. plutellae)에 공생하는 일종의 PDV이다. 프루텔고치벌은 어린 배추좀나방(Plutella xylostella) 유충에 기생한다. 기생 초기에 발현하는 CpBV-ELP1 유전자는 혈구세포에 세포독성을 발휘하면서 기주의 세포성 면역을 억제하여 기생에 중요한 역할을 담당하고 있다. 본 연구는 이 유전자를 담배 식물에서 발현하여 해충에 대한 경구독성을 분석하는 데 목적을 두었다. 재조합 CpBV-ELP1 단백질이 배큘로바이러스 발현시스템을 통해 합성되어 세포배양액에 분비되었다. 수거된 세포배양액은 일련의 단백질 분리과정(ammonium sulfate 단백질 분획, size exclusion 크로마토그래피, 이온교환 크로마토그래피)을 통해 CpBV-ELP1 단백질을 분리하는 데 이용되었다. 분리된 rCpBV-ELP1 단백질은 파밤나방(Spodoptera exigua) 혈구에 대한 뚜렷한 세포독성을 보였다. CpBV-ELP1은 파밤나방 5령충에 대해서 혈강 주입하여 살충력을 나타냈고, 엽침지법을 이용하여 경구독성을 갖고 있는 것을 확인하였다. CpBV-ELP1 유전자를 CaMV 35S 유전자 프로모터와 opaline synthase 유전자 전사종결신호를 갖는 pBI121 벡터에 클로닝하여 Agrobacterium tumefaciens LBA4404 세균에 형질전환을 유도하였다. 형질전환된 세균은 담배(Nicotiana tabacum Xanthi)잎에 감염하여 캘러스를 유도하게 하였고 이후 차세대(T1)를 확보하였다. T1 세대 담배는 파밤나방에 대한 해충저항성을 갖고 있음을 확인하였다. 이러한 결과는 CpBV-ELP1 유전자가 형질전환작물을 통해 해충방제에 응용될 수 있다는 것을 제시하고 있다.

• 한국양식학회지
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• 제11권3호
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• pp.311-317
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• 1998

Estradiol-17${\beta}$($E_2$)에 의한 Vitellogenin (VTG) 함성에 미치는 Cu 및 Zn의 영향을 무지개송어의 배양 간세포를 이용하여 조사하였다. 간세포는 2일간 배양 한 후, E하(2) ($2{\times}10^6$ M)와 동시에 Cu ($2{\times}10^{-5}$~$10^{-4}$M) 또는 Zn ($10^{-5}$~$10^{-3}$M)을 배양액에 첨가하여 5일간 배양하였다. VTG 합성률은 총 단백질에 대한 VTG의 배율로 나타내었다. CU 및 Zn의 첨가는 배양 간세포의 생존율에는 영향을 미치지 않았다. 그러나, Cu의 첨가에 사용된 모든 농도에서, Zn의 첨가에는 농도 의존적으로 감소하여(10^{-3}$M에서 유의하게 감소하였다. 이러한 감소는, Zn의 제거시에는 회복되었으나, Cu에서는 회복되지 않았다. 그리고, Cu (10^{-4}$M의 첨가 시에 Ca (1.8 mM) 농도를 2.5 및 5.0 mM로 증가시켜도 Cu의 작용을 저해하지 못하였다. 이러한 결과로 보아, Cu 및 Zn은 간세포에서 합성되는 다른 단백질 보다 VTG 합성에 더 깊이 관여하는 것으로 추정된다.

• Tuberculosis and Respiratory Diseases
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• 제60권2호
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• pp.187-193
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• 2006

배 경 : 결핵균의 성장 속도가 늦은 이유가 mammalian cell이나 대장균에 비해 결핵균 AK의 매우 낮은 활성도에 의한 것이라는 추측으로부터 AK1과 유사한 촉매능을 나타내는 AKmt 돌연변이 유전자와 human muscle-type AK 합성 유전자(AK1)를 각각 Mycobacterium/E.coli 발현벡터에 재조합(pMVAKmtDM, pEMAK1)하여 성장 속도가 매우 느린 BCG에 형질전환함으로써 이들 단백질들의 촉매능에 의한 성장 속도 변화가 일어나는지를 확인하고자 하였다. 방 법 : Human AK1의 촉매 활성도와 유사하도록 결핵균 AK (AKmt)유전자의 ATPbd와 LID domain을 돌연변이하여 제조한 유전자(AKmtDM)와 human muscle-type adenylate kinase 합성 유전자(AK1)를 Mycobacterium/E.coli 발현벡터에 클로닝하여 재조합 BCG를 제조하였고, 이들 재조합 BCG와 BCG Pasteur $1173P_2$ (wild-type)를 7H9 액체배지에 접종하여 2-3 일 간격으로 $A_{600}$ 값을 측정하였다. 결 과 : 재조합 BCG의 성장 속도는 Wild-type BCG의 성장 속도에 비해 변화가 없었다. 결 론 : 결핵균 adenylate kinase의 정확한 기능은 알 수 없으나, adenylate kinase의 촉매 활성도의 증가는 BCG의 성장 속도에는 영향을 주지 않는 것으로 판단된다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제31권6호
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• pp.453-460
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• 2009

Background and Purpose: Vascular endothelial growth factor (VEGF)-C, VEGF-D and their tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. Oral mucosal squamous cell carcinoma (OMSCC) easily metastasizes to cervical lymph nodes, so we determined the expression levels of VEGF-C, VEGF-D and VEGFR-3 in oral squamous cell carcinoma. Materials and Methods: We performed Western blot analyses with 4 OMSCC cultured tumor cell lines (SCC9, KB, YD-10B, YD-38), and with 7 surgical specimens of OMSCC for the detection of VEGF-C, VEGF-D and VEGFR-3 proteins. Expression of VEGF-C mRNA as well as mRNA for VEGFR-3 in 4 OMSCC cell lines (KB, SCC-4, SCC-9, YD-10B) was investigated by RT-PCR. We also measured VEGFC/VEGF-D protein concentrations in the media and protein concentration of VEGFR-3 in cell lysates of 4 OMSCC cell lines (SCC9, KB, YD-10B, YD-38) using commerical ELISA kits. Finally, we performed immunoprecipitation for the detection of VEGF-C in cell lysates of 4 OMSCC cells (KB, SCC-4, SCC-9, YD-10B) and real-time RT-PCR for the quantification of VEGF-C mRNA. Results: In the result of Western blotting with cell lysates of 4 OMSCC cells, we could not detect the protein expression of VEGF-C, VEGF-D, and VEGFR-3. But, all tumor tissues demonstrated VEGF-C and VEGFR-3. VEGF-C mRNA was detected at various levels in 4 OMSCC cell lines. Moreover, OMSCC cells secreted VEGF-C, not VEGF-D and VEGFR-3 was also detected in cell lysates of OMSCC by ELISA. Immunoprecipitation and real-time RT-PCR revealed VEGF-C was also expressed in 4 OMSCC cell lines. Conclusion: Taken together, tumor cells of OMSCC secrete VEGF-C, not VEGF-D. And VEGFR-3 is expressed tumor cells as well as OMSCC tumor tissues, needs further study.

• 한국응용곤충학회지
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• 제57권1호
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• pp.25-32
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• 2018

곤충의 면역반응에 대한 연구는 곤충 체내 침입한 미생물들과 직접 반응하는 기작들을 중심으로 연구되었다. 그러나 미생물들이 곤충 체내에 침입 한 후 발생되는 다양한 미생물 분비물질에 의한 곤충 면역반응의 시작여부 등에 대한 연구는 거의 없는 실정이다. 이를 위하여 흰점박이 꽃무지(Protaetia brevitarsis seulensis) 유충의 장내에 존재하는 공생균과 체외 병원균을 동일한 조건에서 배양 하고 다양한 분비물질들이 존재 할 거라 예상되는 배양액만을 분리, 유충에 주사하여 면역반응 여부를 조사하였다. 공생균 배양액을 주입한 유충들은 비교적 건강하고 면역반응도 발생하지 않았으나 병원균 배양액을 주입한 유충의 경우 150시간 후 60% 이상 사망하였고 주사된 자리도 짙은 갈색의 멜라닌화가 관찰 되었다. 이러한 면역반응은 과립혈구세포의 리소좀(Lysosomes) 활성화 여부로 재확인 하였다. 병원균 배양액이 주입된 유충들의 경우 12시간 후 리소좀이 ~50% 이상 활성화 되었으나 공생균 배양액이 주입된 유충들의 경우 ~5% 미만으로 활성화 되는 것으로 나타났다. 따라서 공생균 배양액내에는 기주면역반응을 유도하는 물질들이 없거나 량이 매우 적게 존재하는 것을 추측 할 수 있었다.

• Journal of Periodontal and Implant Science
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• 제33권2호
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• pp.277-288
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• 2003

It has been suggested that increased number and activity of phagocytes in periodontitis lesion results in a high degree of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, nitric oxide and peroxynitrite. There are few reports on the relationship between ROS and MMPs expressions in gingival fibroblast. We studied to elucidate whether and how ROS, especially nitric oxide affects the MMP expression. Human gingival fibroblasts and HTl080 cells (human fibrosarcoma sell line as reference) were grown in DMEM supplemented with 10 mM HEPES, 50 mg/L gentamicin, and 10% heat inactivated fetal bovine serum with addition of various reactive oxygen species (ROS). Culture media conditioned by cells were examined by gelatin zymography. HT1080 cells expressed proMMP-2 and proMMP-9, but human gingival fibroblasts (HGF) produced only proMMP-2. Hydrogen peroxide upregulated MMP-9 expression in HT1080 cells, whereas in human gingival fibroblast SNP treatment showed marked increase in MMP-2 level compared to other ROS. These results suggest that the effects of ROS on MMPs expressions are cell-type specific. RT-PCR for MMP-2 and TIMP-2 m-RNA were performed using total RNA from cultured cells under the influence various kinase inhibitors. In HT1080 cells, treatment with FPTI III (Ras processing inhibitor) and LY294002 (PI3-kinase inhibitor) resulted in inhibition of MMP-2 and MMP-9 expressions, suggesting that Ras/P13-kinase pathway is important for MMPs expression in HT1080 cells. In gingival fibroblasts, treatment with FPTI III and PDTC (NF-kB inhibitor) showed marked decrease in MMP-2 regardless of the of SNP , suggesting that Ras/NF-kB could be the key pathway for NO-induced MMP-2 expression in gingival fibroblasts. This study showed that ROS, especially nitric oxide, could be the critical mediator of periodontal disease progression through control of MMP-2 expression in gingival fibroblasts possibly via Ras/NF-kB pathway.

• Journal of Periodontal and Implant Science
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• 제26권4호
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• pp.1013-1021
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• 1996

There are many important factors in periodontal inflammation. $IL-1{\beta}$, $PGE_2$ and collagenase are predorminantly key factors. These inflammatory mediators induce gingival tissue and alveolar bone destruction. For the prevention and treatment of periodontal disease, it is necessary to inhibit $IL-1{\beta}$, $PGE_2$ production and collagenase activity. Ursodeoxycholic acid(UDCA) has immunomodulatory properties, and there is evidence that some natural extracts show antiinflammatory activity to some degree. The purpose of this study was to assess the inhibitory effect of UDCA and its mixture with natural extracts on $IL-1{\beta}$, $PGE_2$ production and collagenase activity. Accordingly we assessed the effect of UDCA and its mixture combined with some natural extracts on inhibition of $IL-1{\beta}$, $PGE_2$ production and collagenase activity. For the $IL-l{\beta}$ inhibition study, cultured cells were exposed to $25{\mu}g/ml$ LPS. $IL-1{\beta}$ activity was measured by $IL-1{\beta}$ enzyme immunoassay system. Human gingival fibroblasts were prepared and cells (l05/well) were seeded into culture plates. $rhIL-1{\beta}$ was added to induce $PGE_2$. The amount of $PGE_2$ in sample media was measured using enzyme immunoassay system. Crude collagenase was prepared from Porphyromonas gingivalis and collagenolytic activity was determined using a Collageno kit CLN-100. The test inhibitor was added to the assay mixture consisting of 0.1ml of 50mM Tris buffer(pH 7.5) and 0.2ml of substrate solution. UDCA and UDCA combined with natural extracts generally inhibited $IL-1{\beta}$ production. groups above 0.01% UDCA strongly inhibited $IL-l{\beta}$ synthesis. Both groups inhibited $IL-1{\beta}-induced$ synthesis of $PGE_2$. In low concentration, the degree of inhibition was as same as prednisolone. In high concentration, each group was superior to prednisolone. UDCA group and UDCA mixture group exerted a moderate inhibition of collagenolytic enzyme. The present study suggested that UDCA and its mixture with natural extracts could be further investigated as antiinflammatory drug for periodontal disease.

• 대한한의학방제학회지
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• 제14권2호
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• pp.76-85
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• 2006

Objectives : We Investigated the effect of Dioscoreae Rhizoma(山藥) on the change of the corticosterone and the rectal temperature(直腸溫渡) of the mice induced by starvation stress(創戰 스트레스). Methods : After administration of Dioscoreae Rhizoma (0.25g/kg, 0.5g, 1.0g/kg, 3g/kg) three times, mice were starved. The corticosterone and rectal temperature were measured after 36.5 hours starvation stress. Results : The plasma cortiosterone levels in the S-2, S-3 and S-4 group were decreased significantly comparing with the control group (P<0.01) after 36.5 hours starvation stress. and rectal temperature was decreased in the control goup comparing with the normal group, but there is no significant change in the Dioscoreae Rhizoma treated group. Conclusion : it might be recognized that Dioscoreae Rhizoma has preventive-effect against starvation stress and also it might be needed further study in various viewpoints. Objectives : This study was disegned to elucidate the short term effect of Rossa rugosae Radix on proliferation. differentiation and maturation of 3T3-L1 Preadipocyte. Methods: 3T3-L1 preadipocytes obtained from Korean Cell Line Bank were cultured in a D ulbecco’ s modified eagle medium(MEM) culture solution containing 10% fetal bovine serum(FBS) and various concentrations of aqueous extract of Rossa rugosae Radix.. The short term effect of the extract of Rossa rugosae Radix on proliferation. differentiation and maturation of 3T3-L1 preadipocytes were investigate after treatment for 24 hours by measuring MTT. Oil Red 0 and latate dehydrogenase activity.. Results: The Rossa rugosae Radix extract inhibited significantly the proliferation of 3T3-L1 preadipocytes and tended to increase latate dehydrogenase activity in the media of differentiated 3T3-L1 preadipocytes & matured 3T3-L1 preadipocytes. the extract also inhibit the lipid accumulation of differentiated 3T3-L1 preadipocytes & matuered 3T3-L1 preadipocytes. Conclusions: These results demonstrated that the Rossa rugosae Radjx extract inhibited the proliferation. differentiation and maturation of 3T3-L1 preadipocytes. suggesting that Rossa rugosae Radix has anti-obesity effect: however further in vivo study is needed to demonstrate its pharmacological effects.

• Journal of Plant Biotechnology
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• 제38권4호
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• pp.251-257
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• 2011

벼의 염기서열 분석이 완료됨에 따라 유전자의 세포내 기능을 밝히기 위한 기능유전체 연구가 활발히 진행되고 있다. 이를 위해 효율적으로 아그로박테리움을 이용해 원하는 유전자를 식물체 내로 형질전환을 하기 위한 노력은 지금도 계속 진행되고 있다. 본 실험에서는 캘러스를 유기한 후 아그로박테리움을 이용해 접종하는 기존의 방법과 달리, 성숙 종자를 소독한 후 2,4-D가 포함된 액체배지에 24시간 침종하여 배 부분이 발아하기 시작하는 종자를 이용해 바로 아그로박테리움을 접종하여 체세포변이의 발생을 최소화하고 유전자를 포함하고 있는 아그로박테리움이 식물 조직내로 침투할 수 있는 효율을 증가시키며, 그 후 캘러스를 유기하여 재분화 시킴으로써 형질전환 식물체를 얻는 방법을 새롭게 수립하였다. 배양과정 중 공동배양 배지에 아그로박테리움 성장억제물질인 silver nitrate와 항산화 물질인 DTT를 첨가하여 공동 배양 기간을 7일 이상으로 늘림으로써 벼 형질전환효율을 증가시킬 수 있었고, PCR 분석을 통해 원하는 목표 유전자가 형질전환체에 안정적으로 도입이 되는 것도 확인할 수 있었다. 또한, 이 방법은 형질전환 효율이 낮은 일품벼와 같은 품종에도 적용할 수 있을 것으로 판단된다. 이러한 결과를 종합해 볼 때, 본 실험을 통해 얻어진 새로운 공동배양 방법은 우수한 농업적 형질을 가진 벼 육종 소재 및 품종 개발시 효율적으로 이용할 수 있을 것으로 생각된다.