항종양 면역반응 유도를 위한 수지상세포의 최적 활성화 조건

남병혁,조월순,이기원,오수정,강은영,최유진,도은주,홍숙희,임영진,김기욱,정민호,Nam, Byung-Hyouk,Jo, Wool-Soon,Lee, Ki-Won,Oh, Su-Jung,Kang, Eun-Young,Choi, Yu-Jin,Do, Eun-Ju,Hong, Sook-Hee,Lim, Young-Jin,Kim, Ki-Uk,Jeong, Min-Ho 한국생명과학회 2006 생명과학회지 Vol.16 No.6

수지상세포는 종양면역에서 필수적인 강력한 CTL 반응을 개시할 수 있는 유일한 세포이다 . 특히 외인성 종양항원에 대한 CTL 반응 유도는 활성화된 수지상세포의 IL-12 분비를 통한 CD4+ helper T세포의 cross-priming을 필요로 한다. 그러나 최근에 활성화된 수지상세포는 $Th_1$ 면역반응을 유도하지만 활성화 시간이 경과함에 따라 오히려 $Th_2$반응을 유도 할 수 있다. 따라서 본 연구에서는 OVA를 종양항원 모델로 설정하여 종양특이적인 CTL 반응을 형성하기 위한 최적의 수지상세포 활성화 조건을 조사하였다. 마우스 골수세포에 서 수지상세포로의 분화는 항원제시 기능을 위한 표면분자의 발현 측면에서 볼 때 배양 6일-7일 정도가 적합하였다. 수지상세포의 IL-12 생성능은 배양 6일 이상, OVA 항원 탑재 8시간 이상의 경우에 연이은 LPS 성숙자극으로 오히려 감소하는 경향을 보였다. 즉 배양 6일의 수지상세포에 OVA 항원 탑재를 8시간 수행한 경우(8-h DC)가 in vitro에서의 IL-12생성능, ex vivo에서의 세포내 $IFN-{\gamma}$를 발현하는 CD8+ T세포의 증가 및 OVA 특이적인 세포독성효과 등에서 가장 좋은 결과를 보였다. 또한 in vivo에서 종양 치료 및 예방효과에서도 8-h DC로 면역한 경우에 가장 우수한 종양형성 억제 효과와 생존기간 연장효과를 보였다. 현재 대부분의 수지상 세포를 이용한 항종양 백신에서 항원 탑재반응을 24시간 동안 수행하고 있으나, 본 실험의 결과로 볼 때, 8시간의 in vitro 항원 탑재가 보다 효과적인 종양특이적 CTL 반응과 항종양 면역반응을 유도함을 알 수 있다. 결론적으로 본 연구를 통하여 8시간 이상의 항원접촉은 수지상세포의 기능적 활성능력을 오히려 고갈시킬 수 있음을 제시한다. Dendritic cells (DCs) are the only antigen presenting cells (APCs) capable of initiating immune responses, which is crucial for priming the specific cytotoxic T lymphocyte (CTL) response and tumor immunity. Upon activation by DCs, CD4+ helper T cells can cross-prime CD8+ CTLs via IL-12. However, recently activated DCs were described to prime in vitro strong T helper cell type 1 $(Th_1)$ responses, whereas at later time points, they preferentially prime $Th_2$ cells. Therfore, we examined in this study the optimum kinetic state of DCs activation impacted on in vivo priming of tumor-specific CTLs by using ovalbumin (OVA) tumor antigen model. Bone-marrow-derived DCs showed an appropriate expression of surface MHC and costimulatory molecules after 6 or 7-day differentiation. The 6-day differentiated DCs pulsed with OVA antigen for 8 h (8-h DC) and followed by restimulation with LPS for 24 h maintained high interleukin (IL)-12 production potential, accompanying the decreased level in their secretion by delayed re-exposure time to LPS. Furthermore, immunization with 8-h DC induced higher intracellular $interferon(IFN)-{\gamma}+/CD8+T$ cells and elicited more powerful cytotoxicity of splenocytes to EG7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with 24-h DC. In the animal study for the evaluation of therapeutic or protective antitumor immunity, immunization with 8-h DC induced an effective antitumor immunity against tumor of EG7 cells and completely protected mice from tumor formation and prolonged survival, respectively. The most commonly used and clinically applied DC-based vaccine is based on in vitro antigen loading for 24 h. However, our data indicated that antigen stimulation over 8 h decreased antitumor immunity with functional exhaustion of DCs, and that the 8-h DC would be an optimum activation state impacted on in vivo priming of tumor-specific CTLs and subsequently lead to induction of strong antitumor immunity.

Radical Scavenging Activities of Phellinus pini

Byung Hyouk Nam(남병혁),Wol Soon Jo(조월순),Yong Cui(최영),Yoo Jin Choi(최유진),Jae Dong Lee(이재동),Min Ho Jeong(정민호) 한국생명과학회 2010 생명과학회지 Vol.20 No.3

Phellinus pini (CY001) 추출물에서의 페놀류성분의 농도는 P. pini (CY001)분획 g 당 GAEs ㎎으로 나타내었고, P. pini 의 EtOAc 분획(436.5 ㎎ GAEs/g)이 다른 분획에 비해 가장 높은 함량을 나타내었다. 환원력과 같은 항산화 활성을 screening 하기 위해 몇 가지 생화학적 시험이 수행되었고, 그것은 2, 2-diphenyl-1-picrylhydrazyl (DPPH) 라디칼 소거작용, NBT/XO superoxide system, DCF/AAPH peroxyl radicals 저해능 등이다. 여섯개의 버섯추출물 분획 중에서 EtOAc 분획이 DPPH, superoxide 라디칼, peroxyl 라디칼 소거작용이 뛰어났고, IC50 values는 각각 11.49 ㎍/㎖, 8.32 ㎛/㎖, and 1.91 ㎍/㎖이었다. P. pini (CY001)의 EtOAc 분획은 중요한 효소적 지질과산화 저해와 cytotoxicity 없이 RAW 264.7 macrophages의 LPS 유도 NO 생성을 효과적으로 감소시켰다. 또한 EtOAc 분획이 HepG2 cell의 타크린 유도 cytotoxicity에서 간 보호 작용을 나타내었다. 이러한 결과를 바탕으로 P. pini (CY001)가 라디칼 소거작용을 하는 성분을 함유한 천연 항산화제로써의 잠재력을 갖고 있는 것으로 사료된다. The concentration of phenolics in Phellinus pini (CY001) extracts, expressed as mg of GAEs per g of P. pini fractions, and the EtOAc fraction (436.5 ㎎ GAEs/g) of P. pini had a higher phenolic content than other fractions. Several biochemical assays were used to screen antioxidant properties such as reducing power, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, NBT/XO superoxide system and inhibition of DCF/AAPH peroxyl radicals. Among the six mushroom extracts, the EtOAc fraction from P. pini (CY001) showed the most potent DPPH radical, superoxide radical, and peroxyl radical scavenging activities, with IC50 values of 11.49 ㎍/ml, 8.32 ㎍/㎖, and 1.91 μg/㎖, respectively. The EtOAc fraction of P. pini (CY001) significantly inhibited enzymatic lipid peroxidation and effectively attenuated LPS-induced NO production of RAW 264.7 cells without cytotoxicity. We also found that the EtOAc fraction had a significant hepato-protectant effect on tacrine-induced cytotoxicity in HepG2 cells. These findings suggest that P. pini (CY001) may have potential as a natural antioxidant, which contains compound(s) with radical scavenging activity.

보문 : 밀리타리스 동충하초 열수추출물의 멜라닌 분비 억제능 효과

남병혁 ( Byung Hyouk Nam ),조월순 ( Wool Soon Jo ),최유진 ( Yoo Jin Choi ),이재윤 ( Jae Yun Lee ),강은영 ( Eun Young Kang ),정민호 ( Min Ho Jeong ),이재동 ( Jae Dong Lee ) 한국균학회 2010 韓國菌學會誌 Vol.38 No.2

The present study aims to evaluate Cordyceps militaris water extract (CMWE) with a view to develop melanogenesis inhibitors. Inhibitory activities of CMWE against tyrosinase, L-DOPA(L-3,4-dihydroxyphenylalanine) oxidation, and melanin biosynthesis in B16 mouse melanoma cells were investigated. CMWE, at 5000 μg/ml, inhibited tyrosinase activity of 71% and DOPA oxidation of 40% as reacting with L-DOPA. Furthermore, B16 mouse melanoma cell survived over 50% from low to high dose on MTT assay, and CMWE markedly inhibited (>50%) melanin synthesis at 5000 μg/ml. The inhibitory effect of CMWE on melanogenesis was attributed to enhancement of tyrosinase degradation. Key enzyme of melanin biosynthesis is tyrosinase which catalyses a beginning step from tyrosine to DOPA quinine and melanin formation step, respectively. These results indicated that CMWE may be a potential source of novel whitening agents for cosmetic or therapeutic application.

새로운 번데기 동충하초의 수집, 동정 및 간기능에 미치는 효과

이기원 ( Ki Won Lee ),남병혁 ( Byung Hyouk Nam ),조월순 ( Wool Soon Jo ),오수정 ( Su Jung Oh ),감은영 ( Eun Young Kang ),최영 ( Yong Cui ),이재윤 ( Jae Yun Lee ),천상철 ( Sang Cheol Cheon ),정민호 ( Min Ho Jeong ),이재동 ( Jae Do 한국균학회 2006 韓國菌學會誌 Vol.34 No.1

굼벵이 유래 밀리타리스 동충하초 열수 추출물의 유전독성평가

조월순(Wol Soon Jo),남병혁(Byung Hyouk Nam),최유진(Yoo Jin Choi),오수정(Su Jung Oh),강은영(Eun-Young Kang),이상호(Sang Ho Lee),정민호(Min Ho Jeong) 한국독성학회 2007 Toxicological Research Vol.23 No.3

Water extract of Cordyceps militaris grown upon Protuetja dreujtarsis (CMPD) was examined for the genetic toxicity-bacterial mutagenicity, chromosome aberration, and micronucleus formation. For mutagenicity assay, bacterial reversion test with Salmonella typhimurium TA98, TA100, TA1535, TA 1537, and E. coli WP2uvrA were performed. The extract at the concentrations of 50~5,000 ㎍/plate did not induce mutagenicity at all. Chromosome aberration test was performed by using Chinese lung (CHL) cells. There was no significant chromosome aberration in CHL cells with S-9 mixture at the concentrations of 312.5~1,250 ㎍/㎖ of the extract and without S-9 mixture at the concentrations of 1.2~19.5 ㎍/㎖ of the extract. For micronucleus test, ICR mice were treated with the extract at the dose of 0.5, 1, and 2 g/㎏. The frequencies of the micronucleated polychromatic erythrocytes (MNPCE) in bone marrow preparations of the extract-treated group were not increased compared to the untreated control group. Taken together, our results show that water extract of CMPD did not induce any harmful genotoxicity.

굼벵이 유래 밀리타리스 동충하초 열수추출물의 간기능개선 효과 및 단회독성 평가

조월순(Wol-Soon Jo),남병혁(Byung-Hyouk Nam),오수정(Su-Jung Oh),최유진(Yoo-Jin Choi),강은영(Eun-Young Kang),홍숙희(Sook-Hee Hong),이상호(Sang-Ho Lee),정민호(Min-Ho Jeong) 한국식품과학회 2008 한국식품과학회지 Vol.40 No.1

굼벵이 유래 밀리타리스 동충하초 열수추출물(CMPD extract)의 안전성 자료를 확보하기 위하여 단회경구투여 독성시험을 실시하였으며, CCl₄의 경구투여로 유도된 간손상 실험동물모델로부터 시험물질(CMPD extract)의 간보호효과를 확인하였다. 단회경구투여 독성시험결과, 시험물질(CMPD extract)의 최고농도(2,000 mg/kg body weight)에서 독성을 나타내지 않아 LD<SUB>50</SUB> 값을 그 이상으로 결정하였다. 또한 독성물질(CCl₄)로부터 간손상이 유발된 SD rat에 시험물질(CMPD extract)을 투여한 후 혈청으로부터 간손상과 관련한 지표물질인 GOT, GPT, TG, TC, LDL 및 HDL 활성도를 측정하였으며, 이와 함께 병리조직학적 소견을 확인하였다. 혈청 GOT는 손상군(G2)에 비해 G4, G5 시험물질투여군에서 유의한 감소를 나타내었으며, GPT의 경우 고용량 시험물질 투여군(G4)에서 유의한 감소를 나타내었다(p < 0.05). 또한 LDL 및 HDL 활성도는 손상군(G2)에 비해 유의하지는 않지만 시험 물질투여군(G4, G5, G6)에서 어느 정도 회복기미를 보였다. 병리조직학적 소견에서도 손상군(G2)의 경우 심각한 세포독성을 보였으나, 시험물질 고용량 투여군(G4)에서는 손상된 세포가 감소하였음을 확인하였다. 이상의 결과들은 굼벵이 유래 밀리타리스 동충하초 열수추출물(CMPD extract)이 CCl₄ 투여에 의해 유발된 급성간 손상에 대하여 간조직의 보호와 간세포의 기능유지에 유효한 물질임을 제시 하고 있다. This study was designed to evaluate the single dose toxicity and the protective effect of water extract of Cordyceps militaris grown upon Protaetia dreujtarsis (CMPD extract) on liver damage on carbon tetrachloride (CCl₄)-induced acute hepatotoxicity in Sprague-Dawley (SD) rats. The CMPD extract was once administered orally to both sexes of rats at dose of 2,000, 1,000 and 500 mg/kg body weight, the recommended maximum limit dose for acute toxicity. Neither significant toxic signs nor death was observed during the observation period. These results indicate that LD<SUB>50</SUB> (lethal dose of 50%) of CMPD extract is greater than 2,000 mg/kg body weight in SD rats. To investigate also the effect of hepatoprotection of CMPD extract, SD rats were orally treated with CMPD extract (50, 25 and 12.5 mg/kg body weight) or silymarin (25 mg/kg body weight) before and after administration of CCl₄ (2 mL/kg body weight, 20% CCl₄ in olive oil). Treatment with CMPD extract or silymarin could decrease the GPT (glutamic-pyruvic transaminase) and GOT (glutamic-oxaloacetic transaminase) levels in serum when compared with CCl₄-treated group. Therefore, the results of this study show that CMPD extract can be proposed to protect the liver against CCl₄-induced hepatic damage in rats.

해수산 클로렐라(Chlorella ellipsoidea) 유기용매 추출물의 항염증 효과

최유진 ( Yoo Jin Choi ),조월순 ( Wol Soon Jo ),김현지 ( Hyoun Ji Kim ),남병혁 ( Byung Hyouk Nam ),강은영 ( Eun Young Kang ),오수정 ( Su Jung Oh ),이계안 ( Gye An Lee ),정민호 ( Min Ho Jeong ) 한국수산과학회 2010 한국수산과학회지 Vol.43 No.1

세포주기 변화에 따른 방사선 유도 암세포 사망의 조절기전

정수진(Soo-Jin Jeong),정민호(Min-Ho Jeong),장지연(Ji-Yeon Jang),조월순(Wol-Soon Jo),남병혁(Byung-Hyouk Nam),정민자(Min-Za Jeong),임영진(Young-Jin Lim),장병곤(Byung Gon Jang),윤선민(Seon-Min Youn),이형식(Hyung Sik Lee),허원주(Won Joo 대한방사선종양학회 2003 Radiation Oncology Journal Vol.21 No.4

목 적 : K562 세포의 방사선에 의한 세포사망은 mitoticcatastrophe 현상이 위주로 나타나지만 herbimycinA(HMA)에 의하여 apoptosis 반응이 촉진되는 반면 genistein에 의하여 두 가지 형태의 세포사망이 모두 억제된다.본 연구에서는 HMA와 genistein에 의한 K562 세포의 방사선 유도 세포 주기 조절 변화와 세포 사망 양상의 연관성을 조사하였다. 대상 및 방법 : 지수증식기의 K562 세포에 6 MV 선형가속기(Clinac 1,800 C, Varian)를 이용하여 200˜ 300 cGy/min의 선량률로 10 Gy를 균일하게 조사하였다. HMA와 genistein은 각각 250 nM와 25μM 농도로 방사선 조사 후 즉시 투여하였다. 실험에서는 세포 주기,조절인자의 발현 및 활성,노화 및 분화정도 등에 있어서의 시간에 따른 변화를 조사하였다. 결 과: 방사선 단독조사에서 K562 세포는 G2기의 정체를 보였으나 정상적인 p53을 가지는 세포와는 달리 지속적인 세포 주기의 정체를 보이지 않았다. G2 정체가 유지되는 동안 cyclin B1의 점진적인 증가를 관찰할 수 있었으며, 이는 염색체의 복제가 완료되지 않은 상태에서 M기로 진행하여 미성숙한 염색체 응축과 mitotic catastrophe 현상이 나타나는 것 과 일치 한다. 방사선 조사와 함께 HMA를 투여한 경우에는 G2 정체가 빠르게 해소되었으며 동시에 G1기에서 세포가 정체되는 양상을 보였다. 세포 주기조절인자 cdc2 kinase 활성증가와 cyclinE와 A 발현 및 CDK2 활성의 감소 등의 현상으로 설명되며,이는 apoptosis의 증가와 연관성을 갖는다.반면 genistein의 경우에는 cyclin B1과 cdc25C 발현 및 cdc2 활성 이 모두 감소하는등 G2정체를 계속 유지하였다.이와 함께 방사선에 의한 노화와 megakaryocyte로의 분화도 지속되는 것을 관찰할 수 있었다. 결 론: HMA와 genistein에 의한 K562 세포의 방사선 유도 세포사망의 변화는 세포주기 조절과 밀접하게 연관되어 있음을 확인하였다.이는 다양한 방사선 유도 세포사망의 기전을 이해하는 데 독창적인 모델을 제공하며,방사선을 이용한 암 치료법의 개발에 새로운 표적을 제공할 수 있을 것이다. Purpose: In our previous study, we have shown the main cell death pattern induced by irradiation or protein tyrosine kinase (PTK) inhibitors in K562 human myelogenous leukemic cell line. Death o f t he cells treated with irradiation alone was characterized by mitotic catastrophe and typical radiation-induced apoptosis was accelerated by herbimycin A (HMA). Both types of cell death were inhibited by genistein. In this study, we investigated the effects o f H MA and genistein on cell cycle regulation and its correlation with the alterations of radiation-induced cell death. Materials and Methods: K562 cells in exponentialgrowth phase were used for this study. The cells were irradiated with 10 Gy using 6 MeV Linac (200-300 cGy/min). Immediately after irradiation, cells were treated with 250 nM of HMA or 25 M of genistein. The distributions of cell cycle, the expressions of cell cycle-related protein, the activities of cyclin-dependent kinase, and the yield of senescence and differentiation were analyzed. Results: X-irradiated cells were arrested in the G2 phase of the cell cycle but unlike the p53-positive cells, they were not able to sustain the cell cycle arrest. An accumulation of cells in G2 phase of first cell-cycle post-treatment and an increase of cyclin B1 were correlated with spontaneous, premature, chromosome condensation and mitotic catastrophe. HMA induced rapid G2 checkpoint abrogation and concomitant p53-independent G1 accumulation. HMA-induced cell cycle modifications correlated with the increase of cdc2 kinase activity, the decrease of the expressions of cyclins E and A and of CDK2 kinase activity, and the enhancement of radiation-induced apoptosis. Genistein maintained cells that were arrested in the G2-phase, decreased the expressions of cycl in B1 and cdc25C and cdc2 kinase activity, increased the expression of p16, and sustained senescence and megakaryocytic differentiation. Conclusion: The effects of HMA and genistein on the radiation-induced cell death of K562 cells were closely related to the cell cycle regulatory activities. In this study, we present aunique and reproducible model in which for investigating the mechanisms of various, radiation-induced, cancer cell death patterns. Further evaluation by using this model will provide a potent target for a new strategy of radiotherapy.

Anti-inflammatory and Anti-nociceptive Effects of the Methanol Extract of Fomes fomentarius

PARK, Young-Mi,KIM, In-Tae,PARK, Hee-Juhn,CHOI, Jong-Won,PARK, Kun-Young,LEE, Jae-Dong,NAM, Byung-Hyouk,KIM, Deog-Gon,LEE, Jin-Yong,LEE, Kyung-Tae WHO COLLABORATING CENTRE FOR TRADITIONAL MEDICINE 2004 東西醫學硏究所 論文集 Vol.2004 No.-

In an attempt to find bioactive natural products with an anti-inflammatory activity, we evaluated the effects of the methanol extract of Fomes fomentarius (MEFF) on in vivo anti-inflammatory and anti-nociceptive activities. MEFF (50, 100 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed MEFF analgesic activity, as determined by an acetic acid-induced writhing test and a hot plate test in mice. To investigate the mechanism of the anti-inflammatory action of MEFF, we examined the effect of MEFF on lipopolysaccharide (LPS)-induced responses in murine macrophages cell line RAW 264.7. MEFF potently inhibited the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophages. Consistent with these observations, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) levels were reduced by MEFF in a dose-dependent manner. Furthermore, MEFF suppressed nuclear factor-кB (NF-кB) activation in LPS-stimulated RAW 264.7 macrophages. These findings suggest that the anti-inflammatory and anti-nociceptive properties of the methanol extract of MEFF may result from the inhibition of iNOS and COX-2 expression through the down-regulation of NF-кB binding activity.