• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Clinical and Experimental Reproductive Medicine
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• 제48권4호
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• pp.322-336
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• 2021

Objective: Endometriosis is a chronic debilitating inflammatory condition characterized by the presence of endometrial tissues outside the uterine cavity. Pelvic soreness and infertility are the usual association. Due to the poor effectiveness of the hormone therapy and the high incidence of recurrence following surgical excision, there is no single effective option for management of endometriosis. Mesenchymal stem cells (MSCs) are multipotent stromal cells studied for their broad immunoregulatory and anti-inflammatory properties; however, their efficiency in endometriosis cases is still a controversial issue. Our study aim was to evaluate whether adipose tissue-derived MSCs (AD-MSCs) could help with endometriosis through their studied anti-inflammatory role. Methods: Female Wistar rats weighting 180 to 250 g were randomly divided into two groups: group 1, endometriosis group; established by transplanting autologous uterine tissue into rats' peritoneal cavities and group 2, stem cell treated group; treated with AD-MSCs on the 5th day after induction of endometriosis. The proliferative activity of the endometriosis lesions was evaluated through Ki67 staining. Quantitative estimation of interferon γ, tumor necrosis factor-α, interleukin (IL)-6, IL-1β, IL-10, and transforming growth factor β expression, as well as immunohistochemical detection of CD68 positive macrophages, were used to assess the inflammatory status. Results: The size and proliferative activity of endometriosis lesions were significantly reduced in the stem cell treated group. Stem cells efficiently mitigated endometriosis associated chronic inflammatory reactions estimated through reduction of CD68 positive macrophages and the expression of the proinflammatory cytokines. Conclusion: Stem cell therapy can be considered a novel remedy in endometriosis possibly through its anti-inflammatory and antiproliferative properties.

• Parasites, Hosts and Diseases
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• 제50권1호
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• pp.29-35
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• 2012

The aim of the study is to characterize the phenotypes of $CD4^+$ $CD25^+$ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naive C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI ($P$<0.01). The percentage of T regulatory cells ($CD4^+$ $CD25^+$) increased significantly ($P$<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-${\gamma}$, IL-4, and TNF-${\alpha}$, showed significant decreases ($P$<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.

• 한국식품영양학회지
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• 제22권4호
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• pp.542-547
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• 2009

Brine mineral water(BMW) has recently gained attention as a new water resource due to its biological activities. In this study, BMW from the Geumjin area(Gangneung-city, Korea) was evaluated for its growth inhibition, anti-metastasis and anti-angiogenesis activity against cancer cells. The in vitro cytotoxicity was measured by CCK assay, and the anti-metastasis activity was estimated by lung metastasis in vivo. The in vitro incubation of mouse splenic cells with BMW that had been diluted more than 4-fold showed no effect on the cell growth when compared to a control group. Additionally, BMW inhibited the growth of the EL-4, L5178Y-R and colon26-M3.1 cancer cell lines in a dose-dependent manner. In vivo evaluation of the anti-metastasis activity of BMW in BALB/c mice inoculated with the colon26-M3.1 cell line revealed dose-dependent inhibition in response to treatment with samples that were diluted by up to 9 times. Finally, treatment with BMW effectively suppressed the growth of vascular endothelial growth factor(VEGF) added human umbilical vein endothelial cells. Overall, these results suggest that BMW has anti-cancer activity.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.254-254
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• 1996

Eosinophils are known to be important effector cells in pathogenesis of asthma. The elucidation of mechanism by which eosinophil survival is regulated in vivo at sites of inflammation is critical tn our understanding of asthma pathogenesis. The maintenance of these cells at site of inflammation depends upon tile balance between its tendency to undergo apoptosis and tile local eosinophil-viability enhancing activity, Qualitative and quantative phenotypic differences have been observed between bronchoalveolar lavage (BAL) and peripheral blood (PB) eosinophils (EOS). We hypothesize that BAL EOS Possess altered functional feature compared to PB EOS. BAL and PB EOS were obtained from ragweed allergic asthmatics after segmental antigen challenge (SAC) at 24 hour or one week, and purified over percoll and CDl6 negative selection. Cells were cultured in duplicate in RPMI, 15% FCS and 1% penicillin/streptomycin without exogenous cytokines. Eosinophil purity and viability was >92%. BAL. EOS viability was 69${\pm}$4.4% versus 39${\pm}$1.6% for PB EOS (p<0.005) at 48 hour time point, and this difference was maintained through day 5 (32${\pm}$7.6% vs. 3.0${\pm}$ 1.4%, p<0.05), Among BAL EOS, those harvested one week after SAC appeared to have an prolonged survival compared to those harvested at 24 hour. Coculture of BAL and PB EOS resulted in significant viability enhancement than expecteed. Direct neutralization of GM-CSF activity, not IL-3 and EL-5, markedly decreased tile survival of BAL EOS in culture, and abrogated tile viability enhancing activity of their culture supernatants in a dose dependent manner. We conclude that BAL EOS activated in vivo possess enhanced viability compared to PB EOS. Mixing and neutralization experiments suggest a role for autocrine production of GM-CSF.

• IMMUNE NETWORK
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• 제3권4호
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• pp.295-301
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• 2003

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. Methods: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard $^{51}Cr$-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with $mIFN-{\gamma}$ELISPOT. Results: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of $IFN-{\gamma}$ secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. Conclusion: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies.

• 대한약리학회지
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• 제32권1호
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• pp.75-82
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• 1996

사염화 탄소에 의한 과산화 지질 증가 및 간 손상에 calcium 및 Kupffer cell의 역할 및 calcium channel blocker의 간 손상에 대한 방어 효과를 연구하였다. 사염화 탄소는 (1 gm/kg, ig) 간의 malondialdehyde (nmole/gm liver) 및 혈중 AST와 ALT (lU/ml) 활성도의 현저한 증가를 나타내었다. 고 농도의 Retinol (250,000U/kg/day)로 인한 Kupffer cell의 활성 증가는 사염화 탄소에 의한 간 과산화 지질 증가 및 간 손상에 상승 작용을 나타낸 반면, $GdCl_3$ 전처리는 $CCl_4$로 인한 ALT의 증가를 감소시켰다. 한편 Retinol 처치군에 Diltiazem (10mg/kg/day)을 병행하여 처치한 결과, 사염화 탄소에 의한 혈중 AST 및 ALT의 증가를 Retinol 단독 처치군에 비하여 현저하게 억제시킬 수 있었다. 이 결과들이 Retinol 혹은 Diltiazem의 투여에 의한 사염화 탄소가 cytochrome P450에 의한 대사 활성 또는 GSH와 관련된 항산화 기전에 미치는 영향에 기인한 것인가를 규명하기 위하여 cytochrome P450, cytochrome P4502El 활성도, GSH reductase 및 GSH peroxidase 활성도를 측정하였다. 그 결과, Retinol 및 Diltiazem의 전처리는 이들 효소의 활성도에 미치는 영향은 대조군에 비하여 유의한 차이가 없었다. 이상의 실험 결과를 종합하여 보면, 사염화 탄소의 투여에 의한 간 손상은 세포내 calcium의 증가를 가져오며, 이는 이차적으로 Kupffer cell을 활성화 시켜 이미 손상된 간세포의 독성을 증가시켰으며, calcium channel blocker인 Diltiazem의 투여는 사염화 탄소의 간독성을 현저하게 감소시키는 효과를 나타내었다.

• Journal of Periodontal and Implant Science
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• 제26권2호
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• pp.448-468
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• 1996

To maintain its functional integrity, bone is continuously remodelled by a process involving resorption by osteoeclasts and formation by osteoblasts, In order to respond to changes in the physical environment or to trauma with the relevant action, this process is strictly regulated by locally synthesized or systemic fators, Prostaglandin $E_2(PGE_2$) is perhaps one of the best studied factors, having been known to affect bone cell function for several decades.$PGE_2$ has both anabolic and catabolic activities. Excess of $PGE_2$ has been implicated in a number of pathological states associated with bone loss in a number of chronic inflammatory conditions such as periodontal disease and rheumatoid arthritis. $PGE_2$ and other arachidonic acid metabolites have been shown to be potent stimulators of osteoclastic bone resorption in organ culture. The anabolic effects of $PGE_2$ were first noticed when an increase in periosteal woven bone formation was seen after the infusion of $PGE_2$ into infants in order to prevent closure of the ductus arteriosus. The cellular basis for the catabolic actions of $PGE_2$ has been well characterized. $PGE_2$increases osteoclast recruitment in bone marrow cell cultures. Also $PGE_2$ has a direct action on osteoclast serving to inhibit activity and can also indirectly activate osteoclast via other cells in the vicinity, presumably osteoblast. The cellular mechanisms for the anabolic actions of $PGE_2$ are not nearly so well understood. The purpose of this paper was to study the effects of $PGE_2$ and dibutyl(DB)cAMP on osteoblastic clone MC3T3El cells and on the generation of osteoclasts from their precursor cells. The effect of $PGE_2$ and DBcAMP on the induction of alkaline phoaphatase(AlP) was investigated in osteoblastic clone MC3T3El cells cultured in medium containing 0.4% fetal bovine serum. $PGE_2$ and DBcAMP stimulated ALP activity and MTT assay in the cells in a dose-dependent manner at concentrations of lO-SOOng/ml. Cycloheximide, protein synthesis inhibitor, inhibited the stimulative effect of $PGE_2$ and DBcAMP on ALP activity in the cells. $PGE_2$also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 500ng/ml. The effect of $PGE_2$ on the generation of osteoclasts was investigated in a coculture system of mouse bone marrow cells with primary osteoblastic cells cultured in media containing 10% fetal bovine serum.After cultures, staining for tartrate-resistant acid phosphatase(TRAP)-marker enzyme of osteoclast was performed. The TRAP(+) multinucleated cells(MNCs), which have 3 or more nuclei, were counted. More TRAP(+) MNCs were formed in coculture system than in control group. $PGE_2(10^{-5}10^{-6}M)$ stimulated the formation of osteoclast cells from mouse bone marrow cells in culture. $PGE_2(10^{-6}M)$ stimulated the formation of osteoclast cells from mouse bone marrow cells in coculture of osteoblastic clone MC3T3E1 cells This results suggest that $PGE_2$ stimulates the differentiation of osteoblasts and generation of osteoclast, and are involved in bone formation, as well as in bone resorption.

• 한국생활과학회지
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• 제17권5호
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• pp.1027-1035
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• 2008

Essentail oils and carrier oils are generally used for Aromatherapy. Therefore the toxicity, possibilities of irritations and sensitive reactions and injury of essential oils must be considered for clients and therapists. So that, in this studies a toxicity of jojoba and 4 species essential oils (fennel, mandarine, tea tree and cedarwood) were investigated by the measurement of MTT-assay and sirius red staining. Liver, kidney and brain tell were chosen for the cell viability assay and observation of morphological change. In the result, no cytotoxicity was observed on live., kidney and brain cell at concentration of 0.01 $\mu\el/m\el$ jojoba oil. And lysis and nucleus breaking were not observed at same concentration of jojoba oil on live., kidney and brain cell. fennel oil was showed 50% of cell viability and inhibited cell growth on liver, kidney and brain cell at relatively high concentration compared with the other oils. 50% of liver, kidney and brain cell viability and delayed cell growth of tea tree and mandarine oil were revealed at lower concentration than fennel oil. In cedarwood oil, 50% of liver cell viability at concentration of 0.00067 $\mu\el/m\el$ was showed, but cell viability and cell growth of kidney and beam cell were effected at the lowest concentration compared with other oils. So that, jojoba oil as using of carrier oil may be not harmful. And 3 essential oils from the fennel, tea tree and mamdarine may have very low toxicity, but cedarwood may be used carefully for inhalation. And over dosage of concentrated cedarwood oil should be not directly touched and exposured, and absolute essential oils must be diluted with carrier oils for topical and systematic massage.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제19권4호
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• pp.395-398
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• 1997

Ipriflavone(IP)은 골흡수 억제효과에 골형성 촉진효과를 지니는 약물로 보고되어 왔다. 이러한 IP의 특성때문에 골의 치유를 촉진시키기 위한 약물로서 구강외과 영역에서 쓰일 수도 있으리라 생각되었다. 이에 저자는 그동안 보고되어온 IP의 골 형성 촉진효과가 실제로 나타나는지를 확인하고 또한 어떤 농도에서 나타나는지를 알아보기위해 IP를 서로다른 농도로 하여 골세포주(MC3T3-El cell line)의 배지에 넣은후, 골 형성의 지표로 쓰일수있는 collagen합성정도를 보고자 하였으며 이 자료를 앞으로서 in vivo 동물실험 연구의 기초자료로 사용코자 하였다. 본 연구에서 IP의 골형성 촉진효과를 collagen 합성정도를 측정을 통해 확인하였고, 특히 IP이 $10^{-7}M$농도일때 현저한 collagen합성의 증가를 관찰 하였으며 앞으로의 동물실험등을 통해 구강외과 영역에서의 사용가능성에 대해 좀더 연구해 보고자 한다.