HHD Mice를 이용한 대장암세포유래 펩타이드 특이적 CD8⁺ T 세포의 입양전이

정헌순,안인숙,도형기,도명술,Jung, Hun-Soon,Ahn, In-Sook,Do, Hyung-Ki,Lemonnier, Francois A.,Tirosh, Boaz,Tzehoval, Esther,Vadai, Ezra,Eisenbach, Lea,Do, Myoung-Sool 대한면역학회 2004 Immune Network Vol.4 No.1

Background: 1-8D gene is a member of human 1-8 interferon inducible gene family and is shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from 1-8D gene were shown to have immunogenicity against colon cancer. Methods: To study tumor immunotherapy of these peptides we established an adoptive transfer model. $D^{b-/-}{\times}{\beta}2$ microglobulin (${\beta}2m$) null mice transgenic for a chimeric HLA-A2.1/$D^b-{\beta}2m$ single chain (HHD mice) were immunized with irradiated peptide-loaded RMA-S/HHD/B7.1 transfectants. Spleens were removed after last immunization, and splenocytes were re-stimulated in vitro. Lymphocytes from vaccinated HHD mice were transferred together with IL-2 to the tumor bearing nude mice that were challenged S.C. with the HCT/HHD/B7 colon carcinoma cell line that was found to grow in these mice. Results: Peptide 3-5 was found to be highly effective in CTL activity. Adoptively transferred anti-peptide 3-5 cytolytic T lymphocytes caused significant retardation in tumor growth. Conclusion: This study shows that peptide 3-5 can be the most effective candidate for the vaccine of adoptive immunotherapy against colon cancer.

Active Immunization Study of Colon Cancer Derived 1-8D Peptide in HHD Mice

정헌순,안인숙,도형기,Francois A. Lemonnier,송국현,도명술 대한면역학회 2005 Immune Network Vol.5 No.3

Background: 1-8D gene is a member of human 1-8 interferon inducible gene family and was shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from human 1-8D gene were shown to have immunogenicity against colon cancer. Methods: To study tumor immunotherapy of three peptides we established an active immunization model using HHD mice. Db / β2 microglobulin (β2 m) null mice transgenic for a chimeric HLA-A2.1/Db β2 m single chain (HHD mice) were challenged with B16/HHD/1-8D tumor cells and were immunized with irradiated peptide-loaded RMA- S/HHD/B7.1 transfectants. In therapy model tumor growth was retarded in HHD mice that were injected with 3-5 peptide-loaded RMA-S/HHD/B7.1. In survival test vaccination with 1-8D-derived peptide protects HHD mice from tumor progression after tumor challenge. Results: These studies show that peptide 3-5 derived from 1-8D gene can be the most effective candidate for the vaccine of immunotherapy against colon cancer and highlight 1-8D gene as putative colon carcinoma associated antigens. Conclusion: We demonstrated that RMA-S/HHD/ B7.1 loaded with 1-8D peptides, especially 3-5, immunization generates potent antitumor immunity against tumor cells in HHD mice and designed active immunization as proper immunotherapeutic protocols.

HHD Mice를 이용한 대장암세포유래 펩타이드 특이적 CD8+ T 세포의 입양전이

도명술,정헌순,안인숙,도형기,Francois A. Lemonnier,Boaz Tirosh,Esther Tzehoval,Ezra Vadai,Lea Eisenbach 대한면역학회 2004 Immune Network Vol.4 No.1

Background: 1-8D gene is a member of human 1-8 interferon inducible gene family and is shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from 1-8D gene were shown to have immunogenicity against colon cancer. Methods: To study tumor immunotherapy of these peptides we established an adoptive transfer model. Db / β2 microglobulin (β2m) null mice transgenic for a chimeric HLA-A2.1/Db-β2m single chain (HHD mice) were immunized with irradiated peptide-loaded RMA-S/HHD/B7.1 transfectants. Spleens were removed after last immunization, and splenocytes were re-stimulated in vitro. Lymphocytes from vaccinated HHD mice were transferred together with IL-2 to the tumor bearing nude mice that were challenged S.C. with the HCT/HHD/B7 colon carcinoma cell line that was found to grow in these mice. Results: Peptide 3-5 was found to be highly effective in CTL activity. Adoptively transferred anti-peptide 3-5 cytolytic T lymphocytes caused significant retardation in tumor growth. Conclusion: This study shows that peptide 3-5 can be the most effective candidate for the vaccine of adoptive immunotherapy against colon cancer. (Immune Network 2004;4(1):31-37)

Inflammatory Gene Expression Patterns Revealed by DNA Microarray Analysis in TNF-α-treated SGBS Human Adipocytes

도명술,정헌순,최봉혁,Leif Hunter,Stuart Langley,Laszlo Pazmany,Paul Trayhurn 연세대학교의과대학 2006 Yonsei medical journal Vol.47 No.5

We report here the use of human inflammation arrays to study the inflammatory gene expression profile of TNF-α- treated human SGBS adipocytes. Human preadipocytes (SGBS) were induced to differentiate in primary culture, and adipocyte differentiation was confirmed, using Oil Red O staining. We treated the differentiated adipocytes with TNF-α, and RNA from differentiated adipocytes with or without TNF-α treatment was hybridized to MWG human inflammation arrays to compare expression profiles. Eleven genes were up- or down-regulated in TNF-α-treated adipocytes. As revealed by array analysis, among 6 up-regulated genes, only eotaxin-1, monocyte chemoattractant protein-1 (MCP-1), and vascular cell adhesion molecule 1 isoform a precursor (VCAM1) were confirmed by real-time polymerase chain reaction (PCR). Similarly, among 5 down-regulated genes, only IL-1 family member 5 (IL1F5), a disintegrin and metalloprotease with thrombospondin motifs-1 preproprotein (ADAMTS1), fibronectin 1 isoform 1 preprotein (FN1), and matrix metalloproteinase 15 preprotein (MMP15) were confirmed by real-time PCR. There was a substantial increase (50-fold) in eotaxin-1 in response to TNF-α. Taken together, we have identified several inflammatory molecules expressed in SGBS adipocytes and discovered molecular factors explaining the relationship between obesity and atherosclerosis, focusing on inflammatory cytokines expressed in the TNF-α-treated SGBS cells. Further investigation into the role of these up- or down-regulated cytokine genes during the pathological processes leading to the development of atherosclerosis is warranted.

Effect of Retinoic Acid on Leptin, Glycerol, and Glucose Levels in Mature Rat Adipocytes In Vitro

Myoung-Sool Do,안인숙,정헌순,D. Vernon Rayner,홍성의 한국식품영양과학회 2004 Journal of medicinal food Vol.7 No.3

To elucidate the effects of retinoic acids (RAs) on adipogenesis and insulin sensitivity, we treated mature adipocytes with two different kinds of RA, 9-cis RA and all-trans RA. Both 9-cis and all-trans RA inhibited the secretion of leptin. However, the inhibition was significantly decreased at a higher dose of each RA. The inhibitory effect of 9-cis RA was synergistically enhanced by the addition of rosiglitazone, a synthetic ligand for PPAR. 9-cis RA also leads to adipogenesis in a dose dependent manner. On the contrary, all-trans RA does not increase adipogenesis in a dose dependent manner. To clarify the anti-diabetic effects of RA, glucose uptake was assessed by estimating glucose concentrations in the medium. 9-cis RA reduced glucose levels in the culture media but all-trans RA did not. In conclusion, all-trans RA does not alter adipogenesis and glucose uptake but does inhibit the leptin secretion. 9-cis RA, however, seems to increase both adipogenesis and glucose uptake through the activation of the RXR/PPAR heterodimer.

Antiobesity Effect of Kochujang (Korean Fermented Red Pepper Paste) Extract in 3T3-L1 Adipocytes

안인숙,도명술,Su-Ok Kim,정헌순,Young-In Kim,Hye-Jung Kim,박건영 한국식품영양과학회 2006 Journal of medicinal food Vol.9 No.1

Kochujang (Korean fermented red pepper paste) is a mixture of fermented soybeans, wheat, and red pepperpowder. Kochujanghas been reported to reduce body fat gain and lipid levels of adipose tissues and serum in rats. We stud-ied the inhibitory effect of Kochujang on lipid accumulation and investigated the molecular mechanism of the action in 3T3-L1 adipocytes by measuring the expression levels of adipocyte-specific genes by real-time reverse transcription-polymerasechain reaction. When 3T3-L1 adipocytes were treated with Kochujangextract (KE), the sizes of adipocytes and leptin secre-tion were decreased. Hormone-sensitive lipase (HSL) was transcriptionally up-regulated at 4 hours, and glycerol secretion wasincreased at both 4 hours and 24 hours. Moreover, mRNA expression levels of both sterol regulatory element-binding protein1-c (SREBP-1c) and peroxisome proliferator-activated receptor-. (PPAR-.), which are critical transcription factors for adi-pogenesis, were markedly down-regulated. Tumor necrosis factor-. (TNF-.) is reported to impair pre-adipocyte differentia-tion and induce lipolysis and apoptosis. KE treatment of 3T3-L1 adipocytes decreased TNF-. mRNA levels, but had no ap-parent affect on apoptosis. Taken together, our study shows that Kochujangdecreased lipid accumulation in 3T3-L1 adipocytesby inhibiting adipogenesis through down-regulation of SREBP-1c and PPAR-. and by stimulation of lipolysis due to increasedHSL activity. TNF-. might not be involved in the reduction of lipid accumulation by KE.

Stem-loop RT-qPCR 분석법을 이용한 siRNA 치료제의 생체시료 분석법 검증 및 약물 동태학적 분석

김혜정(Hye Jeong Kim),김택민(Taek Min Kim),김홍중(Hong Joong Kim),정헌순(Hun Soon Jung),이승호(Seung Ho Lee) 한국생명과학회 2019 생명과학회지 Vol.29 No.6

본 연구는 siRNA 기반 치료제등의 핵산치료제 개발에 있어서 필수적인 약물의 생체내 흡수, 분포, 대사, 배설에 대한 동태의 확인을 위해 stem-loop RT-qPCR 법을 이용하여 보다 더 정확한 시험법을 확립하고자 하였다. siRNA에 특이적인 primer와 probe를 선별하여 siRNA 정량검출 시험법을 최적화하였다. siRNA 표준시료를 이용하여 최적화된 시험법을 적용하였을 때 siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과, 기울기 평균 -3.3, 결정계수 R2>0.99으로 확인되어 siRNA 표준시료와 Cp 값 간의 회귀성이 매우 높아 정량 분석이 가능한 시험법임을 확인하였고, 같은 표준시료를 이용한 stem-loop RT-qPCR의 검출한계(LOD)는 10 fM, 최소정량한계(LLOQ)는 100 fM이었다. 확립된 시험법의 신뢰성을 확인하기 위해 시험자를 다르게 하고, 시험법을 3회 반복하여 각각 진행한 결과, siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과 기울기와 결정계수 R2의 재현성(slope ± -3.2, 결정계수 R2>0.99)을 확인하였고, 표준 곡선으로부터 환산된 siRNA 표준시료의 회수율(recovery ± 20%)과 완건성이 우수함을 확인하였다. 확립된 stem-loop RT-qPCR을 생체내 존재하는 약물 검증에 적용할 수 있는지 확인하기 위하여 시험동물에 siRNA를 주입 후 시간별 혈액을 채취하여 확립된 시험법으로 시험을 진행하였고 약물 동태학적 분석을 통해 siRNA치료제의 혈액내의 안정성을 확인하였다. 따라서 본연구에서 개발된 stem-loop RT-qPCR분석법은 정확성, 정밀성 및 민감도가 높은 분석법으로 핵산치료제 개발 연구의 다양한 생체시료 분석 연구에 적용할 수 있을 것으로 기대한다. The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method—which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics—is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of R2>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ±20% of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.