• Journal of Acupuncture Research
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• v.22 no.3
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• pp.169-183
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• 2005

Objective : The purpose of this study was to investigate the anti-caner effect of cobrotoxin on the prostatic cancer cell line (PC-3).The goal of study is to ascertain whether cobrotoxin inhibits tile cell growth and cell cycle of PC-3, or the expression of relative genes and whether the regression of PC-3 cell growth is due to cell death or the expression of gene related to apoptosis. Methods : After the treatment of Pc-3 cells with cobrotoxin, we performed 형광현미경, MTT assay, Western blotting, Flow cytometry, PAGE electrophoresis and Surface plasmon resonance analysis to identify the cell viability, cell death, apoptosis, the changes of cell cycle and the related protein, Adk, MAP kinase. Results : 1. Compared with normal cell, the inhibition of cell growth reduced in proportion with the dose of cobrotoxin(0-16nM) in PC-3. 2. Cell viabilities of 0.1, 1, 4nM cobrotoxin treatment were decreased and those of 8, 16nM were decreased significantly. 3. S phase of cell cycle was decreased at the group of 1, 2, 4, 8, 16nM cobrotoxin, but M phase was increased at 0.1, 1, 2, 4, 8, 16nM cobrotoxin. 4. Cox-2 expression after cobrotoxin was peaked at 12hours and was decreased significantly after 6, 12, 24 hours. 5. The expression of Cdk4 was decreased dose-dependently at 1, 2, 4, 8nM cobrotoxin and was decreased siginificantly at 4, 8nM Cyclin D1 was decreased at 1, 2, 4, 8nM and Cycline E was not changed. Cycline B was decreased at 1, 2, 4, 8nM dose-dependently and was decreased siginificanlty at 2, 4, 8nM. 6. The expression of Akt was decreased at 1, 2, 4, 8nM dose-dependently and was decreased significantly at 2, 4, 8nM. 7. ERK was increased at 1, 2nM and decreased at 4, 8nM, p-ERK was increased at 1, 2, 4 nM, but decreased at 8nM. JNK and p-JNK were increased at 1, 4, 8 nM. p38 was increased at 2nM p-p38 was increased at lnM but decreased significantly at 2, 4, 8nM. 8. The nucli of normal cells were stained round and homogenous in DAPI staining, but those of PC-3 were stained condense and splitted. Apoptosis was increased dose-dependently at 2, 4, 8, 16nM and increased significantly at 2, 4, 8, 16nM. 9. Bax wasn`t changed at 1, 2, 4, 8nM and Bcl-2 was decreased significantly at 1, 2, 4, 8nM. Caspase 3 and 9 weren`t changed at 1, 2, 4nM but were decreased significantly at 8nM. Conclusions : These results indicate that cobrotoxin inhibits the growth of prostate Cancer cells, has anti-cancer effects by inducing apoptosis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.1
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• pp.63-75
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• 2009

The aim of this study was to elucidate the antimelanogenic effect of Dokhwalkisaeng-tang(Duohujisheng-tang) in B16F10 melanoma cells. Dokhwalkisaeng-tang(DKT) was used to develop the effective prescription of inhibition of melanin production. We determined inhibitory effects of DKT on melanin-release, melanin production, and tyrosinase activity in B16F10 melanoma cells. And to explicate the action-mechanism of DKT, melanin-related gene expressions were determined using RT-PCR and real time RT PCR technique in B16F10 melanoma cells. DKT inhibited melanin-release, melanin production in B16F10 melanoma cells considerably. DKT inhibited tyrosinase activity in vitro and in B16F10 melanoma cells. DKT inhibited the expression of tyrosinase, TRP-1, TRP-2 in B16F10 melanoma cells. DKT inhibited the expression of PKA, PKC, MMP-2 and MITF in B16F10 melanoma cells. On the other hand, DKT increased the expression of ERK-1, ERK-2, AKT-1 in B16F10 melanoma cells. From these results, we propose that DKT may have effect on the antimelanogenesis.

• Journal of Adhesion and Interface
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• v.17 no.4
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• pp.149-154
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• 2016

In this study, natural polymer-based virus neutralizing agent was developed in an attempt to replace the conventional sterilization method for mammalian cell culture. A catechol conjugated heparin was synthesized by using EDC chemistry, and it show unique binding ability to virus which has heparin affinity (adenovirus, adeno-associated virus). To evaluate neutralization ability of catechol conjugated heparin, adeno-associated virus was used for test model, instead of using a pathogenic virus. The catechol conjugated heparin exhibited resistance to high concentration of salt and complete inactivation of adeno-associated virus. The result suggests that the catechol conjugated heparin, which is biocompatible and efficiency, may replace conventional sterilization method for mammalian cell culture.

• Development and Reproduction
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• v.12 no.2
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• pp.159-168
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• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.385-393
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• 1997

다세포 생물에서 몸의 효율적 생존을 위한 각 기관의 homeostasis는 세포 증식과 사망에 의해 조절된다. 따라서, apoptosis라 명명된 세포사망은 정교한 기전에 의한 능동적이고 자발적인 사망기전으로써 몸의 정상적 유지를 위한 필수적인 현상이다. 발생기 세포나 신경세포 또는 흉선세포 분화 동안 과다한 세포의 제거가 apoptosis의 대표적인 예이며, 각종 호르몬에 의해 그 기능이 조절되는 난소세포에서도 apoptosis가 활발히 일어난다. Rat난소에는 태어날 때 수십 만개의 난포를 지니고 있는데, 이 중 단지 1%만이 배란에 사용되어질 뿐이고 나머지는 모두 사망하게 된다. 이러한 난포사망은 난소의 적절한 세포 수를 유지하기 위한 필수적 과정이며, 인위적으로 apoptosis를 억제하는 유전자인 bcl-2를 과다 발현시키면 난소암이 발생하는 연구결과가 이를 입증해주고 있다. 이처럼 중요한 난포 사망기전은 apoptosis라는 개념이 정립되면서 최근 들어 점차 그 연구가 활발해지고 있다. Apoptosis의 특징 중 뚜렷한 점은 DNA가 일정한 간격으로(180∼200 bp)잘려지는 DNA fragmentation현상으로, 이를 이용하여 DNA3'-end 부위에 방사선동위원소를 label한 후 이를 전기영동으로 분리하면 apoptosis를 손쉽게 측정할 수 있다. 난소의 기능은 시상하부호르몬인 LH와 FSH 뿐만 아니라 난소에서 분비되는 각종 난소국부호르몬들에 의해 조절된다. 특정한 발육단계의 난포는 특정한 호르몬에 의해 그 기능을 조절 받는데, 이러한 난소기능 조절기작은 매우 복잡한 경로를 지니고 있다. 이러한 복잡한 기작으로 인해 초기 연구에서첨 생체 내에서 밝히려는 연구 시도는 어려움에 부딪치게 되었다. 생체내 실험은 난소가 다양한 발육단계의 난포를 동시에 지니고 있어 특정한 발육단계의 난포 사망기전을 연구하기 어렵다. 또한 난포는 생체 내에서 다양한 호르몬을 동시에 분비하기 때문에 특정한 난소국부호르몬이 사망기전에 미치는 영향을 조사하기 힘든 점이 있다. 최근 들어 난포체외배양이 다양하게 개발되면서, 이러한 어려운 점을 극복할 수 있게 되었다. 본 논문은 각 발육단계의 난포를 절단해 체외배양하면서, apoptosis DNA 절단 현상을 이용하여 각종 난소국부 호르몬들이 난포발육단계별로 사망기전에 미치는 영향을 요약해 보였다. 난포는 발육하면서 점차 복잡한 호르몬 경로를 생존을 위해 필요로 한다. Prevulatory난포생존에 필요한 난소국부호르몬들은 early antral 단계의 난포에서는 그 미치는 영향이 감소되다가 preantral단계의 난포에서는 영향을 전혀 미치지 못했다. 단지 예외는 cGMP처리로써, 세포내 cGMP수준을 일정하게 유지시켜주는 것이 난포발육단계에 무관하게 생존에 중요한 인자로, 장래 연구는 난포 세포내의 cGMP수준을 조절하는 기작을 규명하는데 있을 것이다.

• The Journal of Internal Korean Medicine
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• v.26 no.4
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• pp.820-827
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• 2005

연구목적: 본 연구는 Poloxamer-407로 유발시킨 고지혈증에 대한 청간탕의 효과를 알아보기 위해 수행되어졌다. 실험방법: Poloxamer-407로 쥐에 고지혈증을 유발시킨후 청간탕과 Lipidil을 경구 투여하여 혈청 cholesterol, 고밀도 지단백, 중성지질을 측정하였으며, 지질대사와 관련된 ACAT, DGAT, $CYP7{\alpha}H$, LDL receptor의 gene expression을 RT-PCR를 통해 비교 분석하였다. 실험결과: 청간탕 투여군은 혈청 콜레스테롤을 각각 32% 와 65% (p<0.05)로, 혈청 중성지방을 각각 21% 와 51% (p<0.05)로 감소시켰다. 또한, 청간탕은 LDL 수용체와 $CYP7{\alpha}H$ 유전자 발현을 증가시켰다. 결론: 이상의 연구로부터 우리는 청간탕이 지질의 흡수, 저장을 억제하고 콜레스테롤의 분비를 촉진함으로써 고지혈증에 일정한 효과가 있음을 알 수 있었다.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.6
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• pp.1622-1627
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• 2004

Nephrite has been widely used as a medicinal mineral resource to treat a numerous chronic diseases and to replenish vital essence and blood in the Korean traditional medicine. However, as of yet, there is little understanding of the pharmacological and biochemical mechanisms of its therapeutic effects as regards anti-inflammation. We therefore examined whether nephrite represses the expression of major inflammation mediators, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), in LPS-stimulated murine macrophage cell line, RAW264.7 by using RT-PCR. The powder suspension and water extracts of nephrite significantly inhibited the mRNA expression of the mediators, despite a little toxic effects on growth of RAW 264.7 cells within the concentration range tested. These experimental results suggested that the nephrite can be utilized as a functional mineral exerting the anti-inflammation medicinal effect.

• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.99-105
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• 2014

In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhibition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

• The Journal of Korean Medicine
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• v.24 no.2
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• pp.148-158
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• 2003

Backgrounds & Objectives: In many recent studies, molecular biological methods have been used to investigate the role of cytokines in pathogenesis and new therapeutic targets of asthma. Recently, as a method of research on the gene expression, they are applying another method which assays multiple gene expressions at the same time by the microarray. In this study, the antioxidant effect, the inhibitory effect against interleukin-4 and the effect on the CD/cytokine gene expression in PBMC (peripheral blood mononuclear cells) was evaluated by using cDNA microarray chip of Chungsangboha-tang. Methods: Experimental studies were performed for the antioxidant effect of Chungsangboha-tang on DPPH (1, 1-diphenyl-2-picrylhydrazyl) solution, for the IL-4-inhibiting effect on BALB/c mouse spleen, and for the gene expression effect on PBMC (peripheral blood mononuclear cells) with microarray. Results: Chungsangboha-tang showed antioxidant effect dose-dependently. Chungsangboha-tang inhibited interleukin-4 dose-dependently and showed significant difference in 10ug/ml and 100ug/ml of test groups. There was no 2 more times upregulated genes than in the control group by using cDNA microarray chip of Chungsangbohn-tang, but there were 140%-200% upregulated genes. There was no 2 more times downregulated genes than in the control group by using cDNA microarray chip of Chungsangboha-Tang, but there was 50%-75% downregulated genes. Conclusions: This study showed that Chungsangboha-tang has an antioxidant effect and inhibition of Interleukin-4, but further studies are necessary with microarray.

• Proceedings of the Materials Research Society of Korea Conference
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• 2010.05a
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• pp.43.1-43.1
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• 2010

조직공학의 중요한 요소로 작용하는 scaffold는 여러 가지 필수적인 조건들을 만족시켜야 한다. 대표적인 특징들로는 (1)생분해성 및 비독성, (2)넓은 표면적을 갖는 상호 연결된 내부 다공성 구조, (3)구조적 안정성, (4)세포부착 기질의 제공, (5)낮은 면역 반응성, (6)혈전 형성 억제, (7)친수성, (8)생체 기능성 등을 들 수 있다. 이러한 scaffold가 갖추어야 할 특성 중에서 세포 부착 기질 제공을 위하여 scaffold에 표면 개질을 통한 기능기를 도입하였다. 본 연구에서는 BCP scaffold의 구조적 안정성 부여를 위하여 PCL(polycaprolactone)을 infiltration 하였다. PCL은 소수성의 특징을 갖고 있어 세포와 상호작용 할 수 있는 생물학적 반응기가 없기 때문에 세포와의 친화성이 떨어진다. 세포의 친화성을 높여주기 위해 실리콘의 전구체인 TEOS(tetraethly orthosilicate)를 코팅하고, 그 위에 카복실기(carboxylic acid group)를 도입하였다. 또한 세포의 고정화를 높여주기 위해 fibronectin을 코팅하여 BCP/PCL scaffold의 세포 친화성을 높여주었다. 이와 같이 제조된 고기능성 BCP/PCL scaffold의 내부 구조와 특성을 Micro-CT로 확인하였고, 또한 실리콘 코팅 여부를 확인하기 위하여 SEM-EDS를 통해 관찰하였으며, FT-IR 관찰을 통해 카복실기 도입 여부를 확인 하였다. 또한 생체적합성 평가를 위해 MTT assay, 조골세포의 부착에 미치는 영향을 관찰하기 위해 SEM, 조골세포의 유전자 발현에 미치는 영향을 관찰하기 위해 RT-PCR을 통해 확인 하였다.