• 대한약침학회지
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• 제15권4호
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• pp.32-41
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• 2012

Objectives: Rehmannia glutinosa pharmacopuncture solution (RGPS) was investigated to determine both its anti-allergic inflammatory effects on mast cells and its detailed mechanism of actions. Methods: We investigated whether RGPS suppress cytokines, enzymes, $Fc{\varepsilon}RI$ expression and $Fc{\varepsilon}RI$-mediated signaling in RBL-2H3 cells stimulated with anti-DNP IgE/DNP-HSA. The suppressive effects of RGPS on the levels of cytokines such as IL-$1{\beta}$, IL-6 and GM-CSF were measured using emzyme-linked immunospecific assay (ELISA). The mRNA expression levels of cytokines, enzymes (HDC2, COX-1, COX-2 and 5LO) and $Fc{\varepsilon}RI$ ${\alpha}{\beta}{\gamma}$ subunits were measured using reverse transcription polymerase chain reaction (RT-PCR) method. The activation of $Fc{\varepsilon}RI$-mediated signaling was examined using Western blot analyses. Results: RGPS suppressed production of proinflammatory cytokines (IL-$1{\beta}$, IL-6, and GM-CSF) in stimulated RBL-2H3 cells significantly (p < 0.05). RGPS also suppressed mRNA expression of inflammatory enzymes (HDC2, COX-1, COX-2, 5LO). In addition, mRNA expression levels of $Fc{\varepsilon}RI{\alpha}$, $Fc{\varepsilon}RI{\beta}$and $Fc{\varepsilon}RI{\gamma}$ were lowered by treatment with RGPS. Finally, RGPS prevented phosphrylation of Lyn, Syk, LAT, Gab2, PLC ${\gamma}1/2$, PI3K, Akt, cPLA2 and $I{\kappa}B{\alpha}$. Conclusions: RGPS effectively suppresses mast cell activations such as degranulation and inflammatory response via down-regulation of the $Fc{\varepsilon}RI$-mediated signaling pathways in IgE/Ag-stimulated mast cells.

• KSBB Journal
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• 제24권6호
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• pp.591-597
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• 2009

심장이상증상의 상태에서 분비되는 심장혈관호르몬인 BNP의 혈중 농도측정은 심장기능부전환자의 진단에 유용하게 이용 되고 있다. 혈청 또는 혈장에서의 BNP 농도측정은 샌드위치형태의 면역학적 검정법을 이용할 수 있으며, 이때에 항 BNP 항체를 BNP 탐지 인자로 사용할 수 있다. 특히, 탐지 인자로의 사용을 위해서는 항원항체 반응부분을 링커로 연결하여 전체 항체에 비해서 경제적이며 효율 면에서도 뛰어난 scFv의 사용이 용이하다고 판단하였고, 이에 scFv의 취약점인 대장균에서의 불용성 형태의 발현을 최소화 시키고 기능적 발현을 극대화하여 심장질환 진단용 센서의 센서 칩 항체로 사용할 수 있도록 항 BNP scFv 항체의 발현을 대장균 세포질 내에서 유도 하였다. 대장균 세포질내에서의 scFv 제작을 위하여 항 BNP scFv 항체 유전자를 pColdⅣ 벡터와 pET22b (+)벡터에 각각 클로닝한 후 발현 하였으며, 그 결과 대량 생산의 장점이 있는 강력한 T7 promoter를 지닌 pET22b (+) 벡터를 이용하여 낮은 온도에서 단백질의 발현을 느리게 유도할 때 적절한 단백질 접힘 현상이 일어나 기능적인 scFv의 발현이 극대화됨을 확인하였다. 또한 IPTG의 투여에 있어서 그 농도가 높아지면 빠른 단백질 유전자의 전사를 도와 발현율이 증가하지만 과도한 농도의 IPTG 투여는 세포내의 독성을 일으켜 단백질의 생산을 저해할 수 있다는 결론에 도달하였으며, 연구를 통하여 개발한 항 BNP scFv 항체가 오직 BNP 항원과의 결합특이성을 가진다는 사실도 확인 가능 하였다.

• 대한약침학회지
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• 제7권3호
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• pp.59-71
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• 2004

Objectives : In order to investigate effects and immune improvement of distilled white-ginseng herbal extract, expression of Cox-1, Cox-2, and mRNA of Bcl-2 and Bax were analyzed in A549 cell in vivo. Survival time and expression of cytokine mRNA were measured for the mice with Sarcoma-180 induced abdominal cancer. Methods : Balb/c mouse was treated with distilled white-ginseng Herbal Acupuncture at Wisu($Bl_{21}$) and Chung-wan($CV_{12}$) to investigate anti-cancer effects and immune response. Results : 1. For expression of mRNA of Cox-1 using RT-PCR. the control group and the experiment groups show significant increase. For Cox-2, both experiment groups and the normal group showed significant decrease. For Bcl-2, experiment groups showed slight decrease compared to the control group. For Bax, no significant changes were shown between the control group and experiment groups 2. For survival time, all of experiment groups didn't show significant differences. 3. IL-2 productivity using Flow cytometry, experiment group I didn't show any significance, For II-4, all of experiment groups showed slight decrease compared to the control group. 4. For IL-2 productivity using ELISA, experiment group I showed slight decrease compared to the control group, experiment group II didn't show any significance. 5. For expression of cytokine mRNA using RT-PCR, significant increase of IL-2 and IL-4 were witnessed in experiment group I compared to the control group. Significant decrease of IL-10 was shonwn in all of experiment groups compared to the control group. Conclusion : According to the results, we can expect that distilled white-ginseng Herbal Acupuncture may be further effects in anti-cancer and immune improvement if increasing concentration.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Journal of Nutrition and Health
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• 제42권3호
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• pp.207-212
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• 2009

생체 내 (ex vivo) 실험에서 더덕의 물 추출물을 4주간 격일로 마우스 체중 kg 당 0, 50, 500 mg/kg B.W.의 농도로 마우스에 경구 투여한 후 비장세포 증식능, LPS에 의해 활성화된 복강 대식세포가 분비하는 염증성 사이토카인 (IL-1 ${\beta}$, IL-6, TNF-${\alpha}$) 의 생성량을 측정하였다. 그 결과 더덕 물 추출물은 ConA나 LPS로 자극하지 않은 경우 500mg/kg B.W. 농도에서 대조군에 비해 유의적으로 높은 비장 증식능을 보였다. LPS에 의해 활성화된 복강 대식세포의 사이토카인 분비량을 측정한 결과에서도 IL-1 ${\beta}$, TNF-${\alpha}$의 분비량은 마우스 체중 kg 당 500 mg/kg B.W. 농도의 투여군에서 유의적으로 높은 생성량을 보여주었으며, 50 mg/kg B.W.의 농도 투여군에서는 대조군에 비해 유의적인 차이를 보이지 않았다. IL-6의 경우 50 mg/kg B.W.과 500 mg/kg B.W. 두 농도 모두에서 유의적으로 높은 분비능을 보였다. 이상의 결과에 따르면 더덕 추출물의 비장세포 증식능과 사이토카인 분비 효과는 500 mg/kg B.W. 농도 투여시 가장 효과적으로 면역 세포와 면역 기관의 주요기능을 증진시킬 가능성이 있을 것으로 사료된다. 이러한 연구결과를 토대로 앞으로 더덕을 이용한 기능성 식품 개발에 기초 연구 자료로 활용되기를 기대된다.

• Journal of Microbiology and Biotechnology
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• 제30권10호
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• pp.1458-1466
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• 2020

Oligomeric proanthocyanidins (OPCs), classified as condensed tannins, have significant antioxidation, anti-inflammation and anti-cancer effects. This study was performed to investigate the anti-inflammatory effects of OPCs and the mechanism underlying these effects in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cells (MAC-T). Real-time PCR and ELISA assays indicated that OPC treatment at 1, 3 and 5 ㎍/ml significantly reduced the mRNA and protein, respectively, of oxidant indicators cyclooxygenase-2 (COX-2) (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.01) as well as inflammation cytokines interleukin (IL)-6 (p < 0.01), IL-1β (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.05) in LPS-induced MAC-T cells. Moreover, OPCs downregulated LPS-induced phosphorylation of p65 and inhibitor of nuclear factor kappa B (NF-κB) (IκB) in the NF-κB signaling pathway (p < 0.01), and they inhibited p65 translocation from the cytoplasm to the nucleus as revealed by immunofluorescence test and western blot. Additionally, OPCs decreased phosphorylation of p38, extracellular signal regulated kinase and c-jun NH₂-terminal kinase in the MAPK signaling pathway (p < 0.01). In conclusion, the anti-inflammatory and antioxidant activities of OPCs involve NF-κB and MAPK signaling pathways, thus inhibiting expression of pro-inflammatory factors and oxidation indicators. These findings provide novel experimental evidence for the further practical application of OPCs in prevention and treatment of bovine mastitis.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.201-208
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• 2013

Objective: To investigate changes in the invasive capacity of gastric cancer cells in vitro after expression inhibition of T lymphoma invasion and metastasis inducing factor 1 (Tiam 1) and underlying mechanisms. Methods: Using adhesion selection, two subpopulations with high ($M_H$) or low ($M_L$) invasive capacity were separated from the human gastric cancer cell line MKN-45 ($M_0$). Tiam 1 antisense oligodeoxynucleotide (ASODN) was transfected into $M_H$ cells with liposomes, and expression of Tiam 1 mRNA and protein was determined by RT-PCR and quantitative cellular-ELISA. Changes in the cytoskeleton, invasive capacity in vitro and expression of ras-related $C_3$ botulinum toxin substrate 1 (Rac 1), integrin ${\beta}1$ and matrix metalloproteinase 2 (MMP 2) between Tiam 1 ASODN transfected $M_H$ cells and non-transfected cells were observed by HE staining, cytoskeletal protein staining, scanning electron microscopy, Boyden chamber tests and cyto-immunohistochemistry. Results: A positive correlation existed between the expression level of Tiam l mRNA or protein and the invasion capacity of gastric cancer cells. After ASODN treatment ($0.43{\mu}M$ for 48 h), Tiam 1 mRNA transcription and protein expression in $M_H$ cells were decreased by 80% and 24% respectively (P < 0.05), compared with untreated controls, while invasive capacity in vitro was suppressed by 60% (P < 0.05). Morphologic and ultrastructural observation also showed that ASODN-treated $M_H$ cells exhibited smooth surfaces with obviously reduced filopodia and microspikes, which resembled $M_0$ and $M_L$ cells. Additionally, cytoskeletal distribution dramatically altered from disorder to regularity with reduced long filament-like structure, projections, pseudopodia on cell surface, and with decreased acitn-bodies in cytoplasm. After Tiam 1 ASODN treatment, the expression of Rac 1 and Integrin ${\beta}1$ in $M_H$ cells was not affected (P > 0.05), but that of MMP 2 in $M_H$ cells was significantly inhibited compared with untreated cells (P < 0.05). Conclusion: Over-expression of Tiam-1 contributes to the invasive phenotype of gastric cancer cells. Inhibition of Tiam 1 expression could impair the invasive capacity of gastric cancer cells through modulating reconstruction of the cytoskeleton and regulating expression of MMP 2.

• IMMUNE NETWORK
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• 제10권2호
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• pp.64-74
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• 2010

Background: The present study was undertaken to examine the immunological effects of pentabrominated diphenyl ether (penta-BDE) and decabrominated diphenyl ether (deca-BDE) on the immune system of the dams and the developmental immune system of the offsprings. Methods: In this study, mated female C57BL/6J mice were orally administered penta-BDE, deca-BDE or corn oil for 5 weeks, from gestational day 6 to lactational day 21. Results: The body weight of PND21 exposed to penta-BDE was significantly decreased relative to control mice, but that of post-natal day 63 (PND63) were recovered. Orally dosed dams with penta-BDE had significantly smaller absolute and relative spleen masses than control mice. Absolute and relative spleen and thymus masses of PND21 exposed to penta-BDE were significantly decreased over control. The exposure of dams and PND21 with penta-BDE reduced the number of splenocytes and thymocytes. As results of hematologic analysis, percentage WBC and percentage neutrophils increased in dams with deca-BDE. Splenic T cell proliferation in dams and PND21 exposed to penta-BDE was increased, and there were no significant difference in splenic B cell proliferation in all treatment groups. As results of flow cytometric analysis of splenocyte, percentage total T cell, Th cell and Tc cell in PND21 exposed to penta-BDE was slightly increased, and percentage macrophage in dams and PND21 exposed to deca-BDE was decreased. The ELISA results of antibody production show no significant difference in all treatment groups relative to controls. Conclusion: These results imply that PBDEs given to the dam were transferred to the offspring during gestation and lactation, and PBDEs transferred from the dam affect immune system of offspring.

• Journal of Periodontal and Implant Science
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• 제24권3호
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• pp.661-670
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• 1994

It is well known that the application of dressings after periodontal surgery have benefits to provide the comforts to patient and to promote the healing process with action of bleeding control and temporary stabilization for the operated mobile teeth. But until recently the relationship between periodontal dressings and cells which are composed of periodontium has not been clear. The purpose of this study was to evaluate the cytotoxic effect of soluble extracts from the four different kinds of periodontal dressings, two of them were eugenol type (K.H.pack, Wondrpak) and the others were non-eugenol type (Coe-pak, Periocare), on the human gingival fibroblasts in vitro. Human gingival fibroblasts were primarily cultured from gingiva around third molar during the extraction for preventive purposes. Extracts solution were prepared with culture medium by means of imersing the consistent size of periodontal dressing made from plastic mold. Cell were inoculated into the 24 well plate with $3\;{\times}\;10^4\;cells/well$ of medium at $37\;^{\circ}C$, 100% of humidity, 5% of $CO_2$, incubator for 24 hours. After discard of the supernatant of medium, those cells were cultured with original, 1/2, 1/5, 1/10 diluted soluble extract for 24, 48 and 72 hours, and counted the number of cells using the hemocytometer at each designed time and concentration. Also, the cytotoxic effect of soluble extract was measured by Wataha's MTT assay method. In briefly, cells were inoculated and cultured into 96 well culture plate with $2\;{\times}\;10^4\;cells/well$ for 24 hours. Soluble extracts were applied to cultured cells and incubated for 48 hours at same condition. $50\;{\mu}l$ of MTT solution and DMSO were added into each well for the detection of absorbance with ELISA reader. The measured data were calculated by value of colorimetric assay for survival rate. The results were as follows ; In the case of eugenol type of dressing, original, 1/2 and 1/5 diluted extracts of K.H.pack showed very low survival rate. And original extract of Wondrpak showed strong cytotoxic effect and 1/2 diluted extract showed moderate cytotoxic effect. In the case of Non-eugenol type of dressings, only original extract of Coe-pak revealed strong cytotoxic effect and Periocare had little cytotoxic effect. It is concluded that eugenol type of dressings showed more cytotoxic effect than non-eugenol types. This study suggest that use of non-eugenol dressings after periodontal surgery is recommended.

• Journal of Periodontal and Implant Science
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• 제37권1호
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• pp.115-124
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• 2007

Silica is known as a promising osteoconductive material, and polycaprolactone is a bioactive and degradable material. The purpose of this study was to monitor the differentiation of MC3T3-E1 cells cultured on the layer-built silica/poly caprolactone non-woven fabric produced by electrospinning. Non-woven fabric (silica, polycaprolactone, PSP, SPS) was made by electrospinning and they were inserted in the 48 well cell culture plate. MC3T3-E1 cells were prepared by subculture. Cells were seeded to each well $1{\times}10^5$ concentration per well. Dulbecco's modified eagle medium with 10% FBS and 1% antibiotic-antimycotic solution was used. Confocal laser scanning microscope was taken 4 hours after incubation (95% air. 5% $CO_2$, $37^{\circ}C$). Cell proliferation was monitored by spectrophotometer on 1, 7, 14 days, and the morphology of the growing cells was observed by field emission scanning electron microscope. To monitor the differentiation of osteoblasts on the materials, MC3T3-E1 cells were incubated in 48 well culture plate after seeding with the density of $1{\times}10^5$ concentration. Then ELISA kit & EIA kit were used on to assess osteocalcin and osteopontin expression respectively. The other conditions were the same as above. MC3T3-E1 cells were proliferated well on all of the materials. There were no statistical differences among them. The osteopontin expression of silica, PSP, SPS was significantly higher than other groups on day 3 (p/0,05), but after that time, there were no statistically signigicant differences. The osteocalcin expression was significantly higher in silica and PSP than other groups on day 14. These findings show that PSP was as good as silica on the effect of osteoblast differentiation. The PSP non-woven fabric may have the possibility as bone graft materials.