생약복합물의 지방세포형성억제 기전규명을 위한 전사체 분석

국문 초록 (Abstract)

생약복합물인 SH21B는 황금(Scutellaria baicalensis Georgi), 행인(Prunus armeniaca Maxim), 마황(Ephedra sinica Stapf), 석창포(Acorus gramineus Soland), 포황(Typha orientalis Presl), 원지(Polygala tenuifolia Willd), 하엽 (Nelumbo nucifera Gaertner)의 혼합(비율 3:3:3:3:3:2:2)으로 이루어졌다. SH21B는 예로부터 한의학에서 비만의 치료에 사용되어 왔으나 자세한 분자적 메커니즘과 효능에 대한 연구는 이루어지지 않았다. 본 연구진은 선행연구를 통해 SH21B가 지방세포의 분화에서 adipogenesis (지방세포형성)와 관련된 유전자를 조절하여 중성지방의 축적을 억제함을 밝혔다. 본 연구에서는, microarray 기술을 이용하여 adipogenesis의 in vitro 모델인, 3T3-L1 세포에서 SH21B에 의한 지방세포형성 억제의 분자적 기작을 보다 상세하게 연구하고자 하였다. 전지방세포, 분화된 세포 그리고 SH21B에 의해 분화가 억제된 세포의 각각의 유전자 발현을 분석하기 위해 각 시료들에서 total RNA를 분리하여 cDNA를 합성한 후 microarray에 적용시켰다. 그 결과, 각각의 시료들의 비교에서 2배 이상의 유의한 발현 변화를 가지는 2,568개의 유전자를 확보하였다. 이 유전자들에 대해 Hierarchical clustering과 K-means clustering 분석을 진행하였고 서로 다른 양상을 가지는 9개의 군집(cluster)들을 분류하였다. 그 중, SH21B의 첨가에 의해 뚜렷하게 감소(cluster 4, cluster 6 및 cluster 9)하거나 반대로 뚜렷하게 증가(cluster 7와 cluster 8)하는 양상을 보이는 군집들을 따로 선별하여 그 군집들에 포함되어 있는 유전자들을 분석하였다. 선택된 5개의 군집에는 지방세포형성과 세포증식에 관련된 유전자가 다수 포함되어 있었다. Cluster 4, cluster 6 그리고 cluster 9에는 peroxisome proliferator activated receptor gamma γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), sterol regulatory element binding transcription factor 1 (SREBF1), adiponectin (ADIPOQ), fatty acid synthase (FASN), lipoprotein lipase (LPL) 등의 지방세포형성 유도 및 관련 인자와 B-cell leukemia/lymphoma6 (BCL6), retinoblastoma 1 (RB1), cyclin-dependent kinase inhibitor 2C (CDKN2c), ras homolog gene family, member B (RHOB) 등의 많은 세포증식 억제 유전자가 포함되었다. 이와는 반대로, cluster 7과 cluster 8에는 β-catenin, cyclin D1 (CCND1), WNT1 inducible signaling pathway protein 2 (WISP2) 등과 같은 지방 세포형성 억제 조절자와 MARCKS-like1 (MARCKSL1), colony stimulating factor 1 (CSF1), discoidin domain receptor family, member 2 (DDR2), leukemia inhibitory factor receptor (LIFR) 등의 세포증식을 유도하는 조절자가 다수 포함되었다. 결론적으로, 이러한 결과들은 SH21B가 지방세포형성과 관련된 조절자 및 세포증식과 관련된 조절자들의 유전자 발현을 조절하여 지방세포형성을 억제함을 제시한다.

다국어 초록 (Multilingual Abstract)

SH21B is a natural composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). In our previous study, we reported that SH21B inhibited adipogenesis and fat accumulation in 3T3-L1 cells through modulation of various regulators in the adipogenesis pathway. The aim of this study was to analyze the transcriptome profiles for the anti-adipogenic effects of SH21B in 3T3-L1 cells. Total RNAs from SH21B-treated 3T3-L1 cells were reverse-transcribed into cDNAs and hybridized to Affymetrix Mouse Gene 1.0 ST array. From microarray analyses, we identified 2,568 genes of which expressions were changed more than two-fold by SH21B, and the clustering analyses of these genes resulted in 9 clusters. Three clusters among the 9 showed down-regulation by SH21B (cluster 4, cluster 6 and cluster 9), and two clusters showed up-regulation by SH21B (cluster 7 and cluster 8) during the adipogenesis of 3T3-L1 cells. It was found that many genes related to cell proliferation and adipogenesis were included in these clusters. Clusters 4, 6 and 9 included genes which were related with adipogenesis induction and cell cycle arrest. Clusters 7 and 8 included genes related to cell proliferation as well as adipogenesis inhibition. These results suggest that the mechanisms of the anti-adipogenic effects of SH21B may be the modulation of genes involved in cell proliferation and adipogenesis.

목차 (Table of Contents)

• 서론
• 재료 및 방법
• 결과
• 고찰
• 감사의 글
• References
• 초록

참고문헌 (Reference)

• 1 Bai, F., "p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors" 27 : 1495-1504, 2007
• 2 Cadigan, K. M., "Wnt signaling: complexity at the surface" 119 : 395-402, 2006
• 3 Sethi, J. K., "Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation" 48 : 1253-1262, 2007
• 4 Darlington, G. J, "The role of C/EBP genes in adipocyte differentiation" 273 : 30057-30060, 1998
• 5 Kokkinaki, M., "The molecular signature of spermatogonial stem/progenitor cells in the 6-day-old mouse testis" 80 : 707-717, 2009
• 6 Tontonoz, P., "Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor" 79 : 1147-1156, 1994
• 7 Lee, H., "SH21B, an anti-obesity herbal composition, inhibits fat accumulation in 3T3-L1 adipocytes and high fat diet-induced obese mice through the modulation of the adipogenesis pathway" 2009
• 8 Liu, A. X., "RhoB is dispensable for mouse development, but it modifies susceptibility to tumor formation as well as cell adhesion and growth factor signaling in transformed cells" 21 : 6906-6912, 2001
• 9 Fajas, L., "Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: implications for adipocyte differentiation and metabolism" 19 : 5495-5503, 1999
• 10 Bennett, C. N., "Regulation of Wnt signaling during adipogenesis" 277 : 30998-31004, 2002
• 11 Schoonjans, K, "PPARalpha and PPARgamma activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene" 15 : 5336-5348, 1996
• 12 Derouet, D., "Neuropoietin, a new IL-6-related cytokine signaling through the ciliary neurotrophic factor receptor" 4827-4832, 2004
• 13 Bajenaru, M. L., "Neurofibromatosis 1 (NF1) heterozygosity results in a cell-autonomous growth advantage for astrocytes" 33 : 314-323, 2001
• 14 Tang, Q. Q., "Mitotic clonal expansion: a synchronous process required for adipogenesis" 44-49, 2003
• 15 Zhao, J., "MARCKS-like protein, a membrane protein identified for its expression in developing neural retina, plays a role in regulating retinal cell proliferation" 408 : 51-59, 2007
• 16 Shimomura, I., "Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy" 12 : 3182-3194, 1998
• 17 Ross, S. E., "Inhibition of adipogenesis by Wnt signaling" 289 : 950-953, 2000
• 18 Cao, Y., "IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development" 107 : 763-775, 2001
• 19 Zeeberg, B. R., "GoMiner: a resource for biological interpretation of genomic and proteomic data" 4 : R28-, 2003
• 20 Shang, W., "Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes" 198 : 561-569, 2008
• 21 Shang, W., "Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression" 80 : 618-625, 2007
• 22 Liu, J., "Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin" 26 : 5827-5837, 2006
• 23 Corazzari, M., "Fenretinide: a p53-independent way to kill cancer cells" 331 : 810-815, 2005
• 24 Lockhart, D. J., "Expression monitoring by hybridization to high-density oligonucleotide arrays" 14 : 1675-1680, 1996
• 25 Freytag, S. O., "Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells" 8 : 1654-1663, 1994
• 26 Olaso, E., "Discoidin domain receptor 2 regulates fibroblast proliferation and migration through the extracellular matrix in association with transcriptional activation of matrix metalloproteinase-2" 277 : 3606-3613, 2002
• 27 Saxena, N., "Differential expression of WISP-1 and WISP-2 genes in normal and transformed human breast cell lines" 228 : 99-104, 2001
• 28 Wang, C., "Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation" 23 : 6159-6173, 2003
• 29 Fu, M., "Cyclin D1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment" 280 : 16934-16941, 2005
• 30 Hulit, J., "Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice" 24 : 7598-7611, 2004
• 31 Perseghin, G., "Cellular mechanism of insulin resistance: potential links with inflammation" 27 (27): S6-S11, 2003
• 32 Lee, H., "Antiobesity effect of baicalin involves the modulations of proadipogenic and antiadipogenic regulators of the adipogenesis pathway" 23 : 1615-1623, 2009
• 33 Ashida, H., "Anti-obesity actions of green tea: possible involvements in modulation of the glucose uptake system and suppression of the adipogenesis-related transcription factors" 22 : 135-140, 2004
• 34 Guo, X, "Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation" 251 : 45-53, 2000
• 35 Fajas, L., "Adipogenesis: a cross-talk between cell proliferation and cell differentiation" 35 : 79-85, 2003
• 36 Rosen, E. D., "Adipocyte differentiation from the inside out" 7 : 885-896, 2006
• 37 Sage, J., "Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry" 424 : 223-238, 2003