E. fetida를 방사선과 수은에 각각 노출시킨 후, 체강세포를 추출하고 단세포 겔 전기영동 기법을 이용하여 DNA의 손상정도와 시간의 경과에 따른 수복 양상을 평가해 보았다. 그 결과, 방사선 조사 후의 시간이 경과할수록 대체로 DNA 손상정도가 감소했으며, 12시간 내에 모든 실험군의 DNA가 완전히 수복되었다. 정확한 수복 완료 시간을 알아보기 위해 OTM 값을 대조군과 비교해 보면 2.5와 5Gy는 방사선 조사 후 약 2시간, 10과 20 Gy

This study was performed to investigate the in vitro effect of a corn water extract on immune function. Splenocyte proliferation was determined by the MTT(3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl terazolium bromide) assay after preparing asingle cell suspension. Production of macrophage-secreted interleukin(IL)-1β, IL-6, and interferon(IFN)-γ, was detected by ELISA using a cytokine assay kit. After a 48-hr incubation with mitogens(ConA or lipopolysaccharide), mice splenocyte proliferation increased with the addition of a corn water extract supplement at 10, 50, 100, 250, 500, or 1, 000㎍/㎖. Production of IL-1β, IL-6, and IFN-γ increased in treatments supplemented with the corn water extract. In an in vitro study, splenocyte proliferation increased when 50~1, 000㎕/㎖ corn water extract was added. In an ex vivo experiment, the highest production of cytokines by activated peritoneal macrophages was observed in mice orally administered 500㎎/㎏ body weight/day.

NAC는 GSH의 전구물질로, thiol기를 포함하는 항산화제 중 하나로 잘 알려져 있으며, 방사선 조사 시 발생하는 생체 내 영향을 감소시켜 생체 손상의 방호 및 회복에 도움을 주는 방사선 방어제로 이용된다. S. cerevisiae에서 항산화제 NAC를 전처리 함에 따라 이온화 방사선 조사에 따른 효모의 세포사멸 방어효과 및 superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx)와 같은

In this study, the Comet assay (evaluation of DNA damage) used the fish hepatocellular carinoma cell, PLHC-1, was tried to the sediment extract obtained from freshwater to understand its applicability as a tool for monitoring sediment toxicity. In paralle

본 실험에서는 대장암 세포 HT-29의 증식을 억제하고 세포 사멸을 유도하는 천연소재 발굴을 목적으로, 오미자(Schizandra chinensis Baillon) 열수 추출물을 이용하여 인체 대장암 세포 HT-29의증식에 미치는 영향을 확인하였다. 오미자 열수 추출물이 HT-29 대장암 세포의 apoptosis 유도 효과 및 기전에 미치는 영향을 분자생물학적 방법으로 실험하여 다음과 같은 결론을 얻었다. MTT assay를 통해 인체 대장암세포 HT-29는 오미자 시료농도 0, 1.0, 2.0, 4.0 mg/mL에서 암세포 사멸농도가 각각 0%, 10%, 70%, 88%를 나타내었다. 대장암세포에 오미자 추출물을 처리하고 cell cycle 분석 결과, 시료농도 의존적으로 sub-G1기가 증가하였고, G0/G1기는 감소되는 것을 통해, apoptosis가 일어나 세포 증식을 저해하는 것으로 확인되었다. 대장암세포 핵의 형태학적 변화를 보면, 오미자 추출물 처리 시 농도 의존적으로 세포수가 감소되는 것이 뚜렷이 관찰되었고 cell shrinking, chromatin condensation 등 apototic body 등과 같은 형태학적 변화들이 뚜렷하게 관찰되었다. RT- PCR을 통한 유전자 발현은, 오미자 추출물 농도 의존적으로 p53 유전자 발현이 증가되는 것을 통해 대장암세포의 증식억제를 확인할 수 있었다. In vitro 실험에서 오미자 열수추출물이 대장암세포의 성장을 저해하는 효과가 있음을 확인하였으며, 오미자 추출물에 함유된 활성 본체 규명 및 apoptosis를 유도하는 작용기작에 관한 연구가 수행중이다.

Diabetic patients tend to exhibit delayed bone formation and osteoblast differentiation, which results in osteopenia. Recently, numerous clinical reports suggest that 635-nm light irradiation improves bone regeneration and wound healing, and reduces pain in patients suffering from diabetes. The purpose of the present study was to test the hypothesis that 635-nm irradiation can influence bone formation by MC3T3-E1 osteoblasts cultured on high concentrations of glucose(25mmol/L D-glucose) in the presence or absence of phorbol 12-myristate 13-acetate(PMA), and to establish an in vitro pathological model of bone formation. The effect of 635-nm irradiation on bone formation was investigated using Alizarin Red S staining, and alkaline phosphatase enzyme activ ity and calcium deposition assays. In addition, gene expression of the o steogenic markers BMP-2, osterix and osteocalcin were assayed by RT-PCR. Calcium deposition by MC3T3-E1 cells was reduced in the presence of high concentrations of glucose or by PMA supplementation. However, 635-nm irradiation led to an increase in calcium deposition by MC3T3 cells, followed by increased bone mineralization. mRNA expression of BMP-2 and osterix at an early stage and of osteocalcin at a late stage was significantly upregulated by 635-nm irradiation in MC3T3-E1 cells supplemented with high concentrations of glucose. Irradiation at 635 nm increases bone mineralization in MC3T3-E1 cells cultured in vitro on high concentrations of glucose and alters osteogenic gene expression, which accelerates bone formation in hyperglycemic conditions.

Cyclosporin A(CsA) as immunosuppressive drug is used to prevent immune reactions after organ transplant. And also It is reported that the effect of CsA on osteoblast differentiation has been controversial according to dosage. The purpose of this study was to examine the effect of various CsA concentrations on osteoblast differentiation. According to different concentration o f CsA, growth curve, apoptosis index MTT assay, ALP activation and osteocalcin secretion, in cultured NHost were analyzed. Treating osteoblasts with low concentrations of CsA increased growth rate, MTT assay activity, ALP activation and osteocalcin protein levels in a dose-dependent manner, while high concentration showed opposite results. Therefore, these results showed that low concentrations of CsA increased osteoblast differentiation, while high concentrations elicited an opposite response, showing inhibition of CsA on osteoblast differentiation. It suggested that different CsA concentrations might play in regulating NHost differentiation, and its specific activation of lower concentration will represent a viable anabolic therapy for bone resorption disease in future.

Malignant tumor cells outgrow new blood vessel formation and tend to be in hypoxic state. Hypoxic cancer cells adapt to hypoxic conditions by transforming its characteristics. On the other hand, one of the most important features of cancer cells is that carcinoma cells loses its inherent epithelial phenotype and acquires mesenchymal characteristics, called as epithelial-mesenchymal transition(EMT). It has been already well known that EMT contributes to tumor invasion and metastasis. The present study investigated whether hypoxia play a major role in induction of phenotypic changes of oral squamous cell carcinoma(OSCC). Furthermore, the mechanism of EMT in oral squamous cell carcinoma cells by hypoxia has been clarified. To mimic hypoxic condition, cobalt chloride and desferoxamine, well-known hypoxic mimetic agents, were used. This study shows that hypoxia suppresses the expression of E-cadherin(epithelial marker) and increases vimentin and N-cadherin( mesenchymal markers) in OSCC. In addition, α5 integrin protein, which is a receptor for fibronectin and an important molecule for tumor invasion, is prominently induced by hypoxia.

The aim of this study is to find out histomorphologic change and cellular activity of condyle resulted from unilateral mastication by comparison of cell proliferation and apoptosis activity. 30 adult rats were dived to 15 experimental group and 15 control group randomly. Right upper and l ower molars were gently extracted in experimental group, to make unilateral mastication environment. All subjects were sacrificed at 1 week, 2 weeks and 4 weeks by chloroform, and their tissues were prepare to observation. Streptovidin-biotin system for BrdU stanning, was used to determine cellular proliferative activity. TUNEL method was used to determine apoptotic activity. The result for cellular activity was recorded at both of anterior portion and posterior portion of condyle. Hematoxylin and Eosin stanning was used for histiomorphological change. The results were as follows. There were more change in superficial layer than deep layer of condyle in cellular activity. In anterior portion of condyle cartilage, cellular proliferative activity of experimental group was lower than control group and apoptotic activity of experimental group was higher than control group. And apoptotic activity of extracted side in experimental group is the most. In posterior portion of condyle cartilage, cellular proliferative activity of extracted side in experimental group was higher than non-extracted side and control group, And apoptotic activity of extracted side in experimental group was the low. As a result of histomorphological change, there was hyperplasia in posterior region o f extracted side c ondyle i n experimental g roup, but t here was n o change i n unextracted side i n experimental group. There was histomorphological hyperplasia in posterior condyle of experimental group as results of high cellular proliferative activity. There was mainly apoptotic change of anterior portion condyle in experimental group. But there was no histomorphologic change. In other words, there was hyperplasia by increasing of cellular proliferative activity in posterior portion of nonfunctional side condyle. In functional side condyle, there was no histomorphological change in functional condyle, but there was change in cellular activity.

Tumor cell biological factors, such as urokinase plasminogen activator(uPA) and its inhibitor plasminogen activator inhibitor- 1(PAI-1) play a role in tumor invasion, metastasis, and proliferation. These factors in patients with primary oral squamous cell carcinoma(Oral SCC) will be evaluated and correlated with clinicopathologic variables. However, relatively rarely has been known in oral squamous cell carcinoma in vivo and in vitro study . The purpose of this study were to investigate the protein expression of uPA and PAI-1 in oral SCC cell lines cell line compared to NHOK and to study migration and adhesion assay. All the cell lines were cultured under KBM bullet kit at 37℃ in a 5% CO2 incubator. We studied a possible association between cytosolic uPA and PA-1 concentrations in oral SCC cell line compared to NHOK using an enzyme-linked immunoassay(ELISA). Cell adhesion and migration assay were done in all the cell l ines. In migration assay oral SCC cell lines were about 70 folds higher than NHOK. In adhesion assay oral SCC cell line were about 7-12 folds higher than NHOK. uPA cy tosolic concentrations was about 15-19 folds and PAI-1 was 3 to 4.5 folds than that of NHOK. Both uPA and PAI-1 concentrations were correlated with migration and adhesion assay. High cytosolic concentrations o f uPA and PAI-1 were correlated with migration and adhesion assay . It suggested that these markers might be specific for oral SCC cell line and these results would be contributed to treatment and prognosis of human oral squamous cell carcinoma.

Salmonellosis is the commonest zoonosis worldwide that generally causes enterocolitis and foodborne poisoning which represents a considerable public health burden. Salmonella spp. are potential enteric pathogens and intracellularly replicates in host cells resulting in chronic infections. The medical treatments for salmonellosis have been difficult yet and had a serious problem including the increasing emergence of antibiotic resistance. The present report was designated to investigate the antibacterial effects of Saururus chinensis Baill ethanol extract (SCEE) on pure culture and infection with Salmonella enterica serovar Typhimurium (S. typhimurium) in murine derived macrophage RAW 264.7 cells. In determination of antibacterial activity of SCEE against S. typhimurium, bacterial viability was markedly decreased compared to the control. Also, SCEE significantly induced morphological change (p<0.05) of RAW 264.7 cells. In infection assay of S. typhimurium in RAW 264.7 cells pretreated with 100㎍/㎖ of SCEE, which is a non-cytotoxic concentration, bacterial uptake ability of macrophage was increased corresponding with morphological change, whereas bacterial survival rates within macrophage were markedly reduced compared with untreated control. Furthermore, nitric oxide (NO) production in SCEE-treated cells was slightly increased until 2 h but showed a tendency of decrease after 4 h until 24 h post infection compared with untreated control with S. typhimurium infection. Taken together, these findings demonstrated that SCEE has the antibacterial activity for S. typhimurium and the protective effects against S. typhimurium infection through activating murine macrophage independent on NO, suggesting that SCEE may be beneficial on the disease caused by intracellularly replicating pathogens as a safe alternatives of conventional chemotherapies.

본 연구는 우리나라 주요 재배종인 Muscari armeniacum ‘Early Giant’ 품종을 사용하여 엽절편체로 부터 직접적으로 신초재생과 체세포배 발생에 미치는 생장조절제의 효과를 구명하였다. 무스카리의 엽조직으 로부터 캘러스 과정을 거치지 않은 직접 신초형성은 2,4-D 0.1 mg·L−1가 함유된 배지에서 가장 좋았다. 반면, 체세포배 발생은 생장조절제를 첨가하지 않는 대조구와 IPA 0.1~1.0 mg·L−1가 함유된 농도의 배지에서 비교적 양호하였다. 무스카리의 엽조직으로부터 재생된 자구를 기외로 이식했을 때 맹아율은 모든 처리구에서 80%이상 으로 높았으며 특히 NAA 0.1mg·L−1, IPA 1.0~3.0mg·L−1 배지에서 재생된 자구의 생장이 양호하였다.

살모넬라증은 대표적인 인수공통전염병의 하나로서 세포내 기생하며 질병을 유발하며 장염과 식중독 등을 유발하여 공중보건학적으로 심각한 문제를 야기하고 있다. 본 연구는 삼백초의 수용성 추출물을 (SCWE) 이용하여 숙주세포에 대한 안전성, S. typhimurium 균에 대한 항균효과 및 대식세포 내 균 증 식억제 기능을 규명하였다. 본 실험을 통하여 SCWE 1, 10 및 100 μg/ml 농도로 첨가한 배지에서 RAW 264.7 체포와 24 시간 반응 후 평가해본 결과 세포독성이 인정되지 않았으며, S. typhimurium 균에 대하여 시간 경과에 따라 항균효과가 증가되는 것을 확인하였고, SCWE 처리에 의해 대식세포의 형태적 변화가 증가되는 것으로 나타났으며 (p<0.05), 살모넬라균의 탐식능력 및 대식세포 내 균 증식 억제 능 력이 비 처리군에 비해 현저히 증가되는 것이 확인되었다. 또한 SCWE 처리 한 대식세포에 살모넬라균 감염을 수행하였을 때 대식세포의 nitric oxide (NO) 산생능력이 비 처리 군에 비해 저하되는 것으로 나타나, 살모넬라균에 의한 대식세포의 세포독성을 억제하는 것으로 나타났다. 종합적으로 SCWE의 숙 주세포에 대한 안전성, 살모넬라균에 대한 항균효과 및 대식세포 내 균 증식 억제 효과가 있는 것으로 나타나 SCWE를 이용한 세포내 기생세균의 치료제 개발이 가능할 것으로 판단된다.

The objective of this study was to evaluate the effects of co-culture of bovine oocytes with cumulus cells on in vitro maturation and development following in vitro fertilization in bovine oocytes. Bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DO) were co-cultured with the cumulus cells in TCM199 for 20~22 hr, and evaluated the nuclear type of oocyte. After in vitro maturation, oocytes were coincubated for in vitro fertilization with frozen-thawed spermatozoa selected by 65% percoll in DM-Heparin and DM-Caffeine for 15~18 hr. Presumptive zygotes were cultured for 48 hr in CR1aa in vitro culture medium with 10% FBS, and evaluated the cleavage rates. The results confirmed that the highest percentage of metaphase II (M-II) stage was observed in COCs (30.1±3.5%, 24.2±1.8%) as compared to DO (7.1±1.3%, 17.4±13.9%) (p<0.05). In addition, the increased cleavage rates were obtained from COCs (69.6±2.1%, 75.6±2.9%) when compared to DO (21.6±7.5%, 29.5±12.6%) (p<0.05). In conclusion, this study suggested that cumulus cells secreted positive factors during in vitro maturation of oocytes and early embryonic development after in vitro fertilization of bovine oocytes.

칠성초에 역병 저항성을 도입한 칠복1호에 베트남 도입 풋마름병 저항성 계통을 교배하여 육성한 및 에서부터 및 까지 역병-풋마름병 복합 저항성 선발을 2009년도와 2010년도에 걸쳐 수행하였다. 매 세대 역병을 접종하여 저항성을 평가하여 선발하고 선발개체에 풋마름병을 접종하여 감염되는 개체는 도태하였다. 역병에 대한 저항성은 선발과 함께 현저히 향상되었으며, 선발계통들은 역병 저항성으로 판매되고 있는 교배종 '무한질주'와 비슷한 수준의 저항성을 나타내었다. 선발개체를 칠복CMS-A라인에 교배를 하여 의 임성을 보고 화분친의 CMS-Rf유전자형을 검정하였다. 대부분Nrfrf로 고정되고, 칠복 KC995, 칠복 KC1009 조합의 일부 개체가 이형(heterozygote) 상태인 것으로 확인되었다.

The regeneration of periodontium is the goal of periodontal therapy. Many periodontologists try to achieve this goal by using guided tissue regeneration(GTR) method. However, these procedures always include several disadvantages. Recombinant human bone morphogenetic protein-2 (rhBMP-2) stimulated ectopic bone formation when it was implanted in rat muscles with insoluble bone matrix by differentiating muscle cells into chondrocytes and osteoblasts. The purpose of this study was to evaluate the osteoinductive potential of the mixture of rhBMP-2 (5 μg/ml) and heparin (0.25 or 25 μg/ml ) at the critically sized rabbit calvarial defects. And this study aimed to reveal that heparin also acts to enhance the bone forming activity of rhBMP-2. The 12 rabbits (4-month-old; NewZealand White) were used in the present study. 5 μg/ml of rhBMP-2 and 0, 0.25 or 25 μg/ml of heparin were mixed and blotted into anorganic bovine bone and filled cranial defects. The animals were sacrificed following a time schedule (1, 3, and 6 weeks). Sections were made in 7 μm thicknesses, stained with H&E and Masson's trichrome method, and examined under a light microscope. The differences among each obtained percent value were evaluated by one-way analysis of variance. A p value of p<0.05 was considered statistically significant and an ANOVA test was performed to verify significant differences. To adjust for multiple comparisons when one-way analysis of variance showed a significant difference between groups (p<0.05), Scheffe`s post hoc test was used to identify which group differences accounted for the significant p-value. In control group, osteoinduction was not outstanding, however, in experimental groups, osteoinduction was significantly outstanding, and as the concentration of heparin mixed with rhBMP-2 increased, osteoinduction was increased. Mixtures of rhBMP-2 and heparin affect bone formation at initial bone formation, but that effect disappeared following a time lapse.

Many methods have been developed for more efficient gene delivery and expression in human cells. A number of studies have been performed in achieving successful gene delivery and expression conditions. We investigated differential gene expression patterns after delivery adenoviral vector containing green fluorescent protein(GFP) gene into human cancer cell lines. We constructed recombinant adenoviral Ad-CMV-GFP containing CMV promoter and GFP gene. The efficiency of gene expression was assessed by observation GFP expressing cells using fluorescent microscopy after transfer of Ad-CMV-GFP in concentrations of 0.1μl. 1μl. 10μl. At first, we evaluated expression patterns of gene in several human cancer cell lines, gastric adenocarcinoma cell line AGS was showed high level of GFP expression compared with colorectal adenocarcinoma cell line HT-29. After transfer 0.1μl of Ad-CMV-GFP in AGS, we could found that GFP expression cells were observed in next day and highly increased 2 days. While, small number of GFP expressing cells were examined in HT-29 and SNU-C4. Therefore, these data showed that AGS was expressed the highest level of GFP and almost AGS cells seems to express GFP in concentration of 1μl of Ad-CMV-GFP. GFP expression pattern in HT-29 reveal that expression was low in next day after gene transfer but significantly increase expression level in 2 days. In case of SNU-C4, GFP expression increased with increasing concentration of Ad-CMV-GFP and t ransfer times. For examine effects of transfer times in small amount gene, we transfer in concentration of 0.1μl Ad-CMV-GFP and detected GFP expression patterns after 2 days or 4 days. As a result, expression level of GFP in AGS was increase about 2 fold after 4 days compared with 2 days, but any difference of GFP expression levels were not showed in HT-29 and SNU-C4. Our study suggested that adenovirus was very efficient gene transfer vector for gene expression in human cancer cell lines. In addition to, we also demonstrated that gene expression patterns was dependent on each human cell lines. Therefore, further studies will be needed to confirm the optimum conditions for efficient gene delivery and expression in each target cell lines with consideration to cellular properties.

This study was designed to investigate the protective effect of the combination of fucoidan and lutein against AAPH-induced oxidative stress in THP-1 cells. The combination of fucoidan and lutein existed significant antioxidant effect on AAPH-damaged THP-1 cells by using lipid peroxidation and cellular antioxidant capacity assay. Fucoidan(1㎍/㎖) and lutein(10㎍/㎖) did not affect at all the viability of THP-1 cells, but protected the AAPH-damage of THP-1 cells at the same concentration. The viability of THP-1 cells was 0% with 1 mM AAPH alone, the protective effect of fucoidan(1㎍/㎖) and lutein(10㎍/㎖) was 37% and 36%, respectively. The combination of fucoidan(1㎍/㎖) and lutein(10㎍/㎖) exhibited significant inhibitory effect of lipid peroxidation using TBARS assay and cellular antioxidant capacity using DCFH-DA assay. In lipid peroxidation, the TBARS value of 1mM AAPH alone was 0.8±0.03nM MDA, its of the combination of fucoidan(1㎍/㎖) and lutein(10㎍/㎖) was 0.2±0.05nM MDA. In cellular antioxidant capacity, the combination of fucoidan(1㎍/㎖) and lutein(10㎍/㎖) exhibited significant cellular antioxidant capacity of 76%, whereas quercetin(10μM) as positive control exhibited the cellular antioxidant capacity of 32%. These results indicate that the cotreatment of fucoidan and lutein protects against AAPH-induced THP-1 cell damage by inhibiting lipid peroxidation, increasing cellular antioxidant capacity.

Radiotherapy is one of the major therapies for cancer treatment. p53 acts as a central mediator of the cellular response to stressful stimuli, such as radiation. Recently it has been known that activation of the phosphatidylinositol- 3-kinase (PI3K) pathway is associated with radioresistance. In this study, we investigated whether X-irradiation up-regulates PI3K in a p53-dependent manner in human colon cancer cells. In order to study this phenomenon, we have treated p53-wild type and p53-mutant type HCT116 cells with X-ray. Treatment of wild type HCT116 cells with 8 Gy resulted in a marked increase in PI3K (p85), which paralleled an increase in PTEN, a counterpart of PI3K. However, these effects of X-rays in the p53-mutant cells were not observed. These results suggest that the X-irradiation- induced up-regulation of PI3K/PTEN pathway is p53-dependent.