臺灣博碩士論文加值系統

論文摘要內容：
主旨：牛樟芝是臺灣特有種真菌。先前研究已表明其粗萃取物或特定之分離純化產物具有潛在之生化活性。近期，從牛樟芝菌絲體固態發酵產物中分離純化出一種多醣體物質，並定名為ACP-1，且經本次研究發現，牛樟芝多醣體ACP-1具有顯著之抗發炎能力。在許多免疫細胞發炎反應研究中，NF-κB信號傳遞路徑及其調節具有高度之重要性，故在本次研究中，著重於探討牛樟芝多醣體ACP-1在小鼠巨噬細胞株RAW264.7中，對於由NF-κB所引發的發炎反應調節作用。
方法：脂多醣 (LPS)主要係用於引發NF-κB 發炎訊號。實驗開始時，給予小鼠巨噬細胞RAW264.7不同濃度之ACP-1，爾後加入脂多醣進行刺激。激發後的NF-κB發炎訊號表達及調節，著重探討有三大環節：一、其細胞因子/ 趨化因子之基因表達；二、與發炎相關之長鏈非編碼核醣核酸(lncRNA)基因表達；三、細胞外吐小體(exosome)與發炎作用之相關性
結果：小鼠巨噬細胞在脂多醣的刺激之下，激發NF-κB之訊號傳遞，包括細胞質中之抑制子-IκB之降解，使得蛋白質次單位-P65加以被釋放，進而從細胞質轉位進入細胞核之中，故細胞在牛樟芝多醣體ACP-1作用之下，降低了抑制子-IκB之降解以及蛋白質次單位-P65之轉位。此外，藉由NF-κB基因轉錄之啟動子報導基因測定法加以確認ACP-1抑制NF-κB基因表達之功能性。故在脂多醣所誘發的特定發炎作用相關之基因中，ACP-1能抑制如RANTES, IFN-β1, lncRNA-ch1a, 以及lncRNA-ch1b; chr2 等基因。且發現ACP-1作用於細胞後，能抑制脂多醣所刺激外吐小體之釋放。此外，在ACP-1作用與脂多醣刺激皆同時存在之情況下，長鏈非編碼核醣核酸-10a(LncRNA-chr10a)之表達不但未因ACP-1而抑制，反而明顯高於僅以脂多醣刺激組，且在ACP-1的單獨作用之下亦為明顯增加。故再以基因轉錄之啟動子報導基因測定法加以確認ACP-1可能直接與lncRNA-chr10a 基因表達相關 。
結論：本次研究結果表明ACP-1能抑制小鼠巨噬細胞RAW264.7藉由脂多醣所引發之NF-κB 訊號傳遞，進而減少與其相關之發炎反應。ACP-1可能致使特定基因表達及參與發炎作用之調節，如長鏈非編碼核醣核酸-10a(LncRNA-chr10a)。藉由本研究，對ACP-1的抗發炎功能提出新的見解之外，未來的研究方向將更針對ACP-1如何能抑制NF-κB信號傳遞之詳細機轉，以及長鏈非編碼核醣核酸之基因轉錄的關聯性進行深入探討。

The Contents of Abstract in This Thesis:
Aims: Antrodia camphorata (AC) is an indigenous mushroom species in Taiwan. Previous studies have demonstrated various biological functions of the crude extracts or isolated components from cultured AC. We recently purified a polysaccharide component (ACP-1) from the fermented AC, which shows strong anti-inflammation activity. Given the importance of NF-κB signal pathway in the regulation of inflammation in most of immune cells, in this study, we investigated the effects of ACP-1 on NF-κB-mediated inflammatory responses in murine RAW264.7 cells.
Methods: LPS was used to activate NF-κB signaling. RAW264.7 cells were treated with various doses of ACP-1, followed by LPS (1μg/ml) stimulation. Activation of the NF-κB signaling was monitored and inflammatory responses were characterized by measuring: i) expression of cytokine/ chemokine genes; ii) expression of inflammatory long non-coding RNA (lncRNA) genes; and iii) inflammatory release of exosomes.
Results: LPS stimulation activated the NF-κB pathway in RAW264.7 cells, including the degradation of cytoplasmic IκB and cytoplasm to nuclear translocation of the NF-κB p65 subunit. Pre-treatment of cells with ACP-1 attenuated LPS-induced IκB degradation and p65 nuclear translocation. Inhibition of NF-κB activation by ACP-1 was further confirmed by a NF-κB luciferase reporter assay. Accordingly, a significant inhibition of LPS-induced expression of selected inflammatory cytokine and lncRNA genes, such as RANTES, IFN-β1, lncRNA-chr1a, and lncRNA-chr1b; lncRNA-chr2, were detected in cells pretreated with ACP-1. LPS-induced release of exosomes from RAW264.7 cells was also inhibited by ACP-1 treatment. Intriguingly, ACP-1 treatment further enhanced LPS-induced expression of lncRNA-chr10a, an lncRNA that can modulate transcription of inflammatory genes through unknown mechanisms. Moreover, ACP-1 treatment alone increased expression of lncRNA-10a in RAW264.7 cells. Our promoter reporter analysis using a luciferase reporter plasmid covering the lncRNA-ch10a gene promoter demonstrated that ACP-1 may directly activate the transcription of the lncRNA-ch10a gene.
Conclusions: Our results indicate that ACP-1 can inhibit LPS-triggered activation of the NF-κB pathway in RAW264.7 cells, resulting in attenuation of NF-κB-mediated inflammatory responses. ACP-1 may directly activate the transcription of specific genes, such as ncRNA-chr10a, to modulate inflammatory responses. Our results provide new insights into the anti-inflammation functions of ACP-1. Further studies should investigate the molecular mechanisms by which ACP-1 inhibits NF-κB activation and triggers lncRNA-chr10a gene transcription.

TABLE OF CONTENTS
TABLE OF CONTENTS VI
LIST OF TABLES IX
LIST OF FIGURES X
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 LITERATURE REVIEW 3
2.1 Antrodia camphorata 3
2.2 Nuclear Factor kappaB(NF-κB) signal pathway 4
2.3 Long non coding RNAs (LncRNAs) 6
2.4 Exosomes 9
2.5 Summary 11
CHAPTER 3 STUDY STRATEGIES 12
CHAPTER 4 MATERIALS AND METHODS 13
4.1 Cell culture 13
4.2 Preparation of ACP-1 13
4.3 Quantitative Reverse Transcription-Polymerase Chain Reaction 14
4.3.1 RNA extraction 14
4.3.2 Reverse Transcription (RT) of mRNA and LncRNAs 15
4.3.3 Quantitative-PCR for mRNA and LncRNAs 15
4.4 Western Blot 16
4.4.1 Whole cell Protein extraction 16
4.4.2 Nuclear protein isolation 16
4.4.3 Gel electrophoresis 17
4.4.4 Gel transfer 17
4.4.5 Blocking and antibodies detection 18
4.5 Luciferase reporter assay for cytokine or LncRNA promoter binding activities 18
4.5.1 Plasmid DNA isolation 18
4.5.2 Cell culture and transfection 20
4.5.3 Protein collection of Luciferase for assay 21
4.6 Exosomal isolation 22
4.6.1 Cell culture 22
4.6.2 Collection of exosomes 22
4.6.3 RNA extraction and qRT-PCR investigation 22
4.7 Statistical analysis 23
CHAPTER 5 RESULTS 31
5.1 Inhibitory effects of ACP-1 on LPS-induced activation of NF-kappaB signaling pathway in RAW264.7 cells. 31
5.2 Time course of inflammatory cytokine and lncRNA expression profile in RAW264.7 cells treated with ACP-1 as measured by qRT-PCR. 38
5.3 Effects of ACP-1 on LPS-induced expression profile of inflammatory cytokines and lncRNAs in RAW264.7 cells. 46
5.4 Exosomal shuttling of RANTES mRNA and LncRNA-chr10a by ACP-1 pretreatment in RAW264.7 cells in response to LPS stimulation. 50
5.5 ACP-1 promotes transcription of the lncRNA-chr10a gene in RAW264.7 cells 52
CHAPTER 6 DISCUSSION 55
CHAPTER 7 CONCLUSION 58
REFFERENCES 60
AUTOBIOGRAPHY OF AUTHOR 66

Bidere N, Ngo VN, Lee J. Casein kinase 1alpha governs antigen-receptor -induced NF-kappaB activation and human lymphoma cell survival. Nature 2009; 458:92-96.
Bretz, N.P., Ridinger, J. Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-Like receptor signaling. Journal of Biological Chemistry Vol. 288, Issue 51, 20 December 2013, Pages 36691-36702
Chang CY, Huang ZN, Yu HH, Chang LH, Li SL, Chen YP. The adjuvant effects of Antrodia camphorata extracts combined with anti-tumor agents on multidrug resistant human hepatoma cells. J Ethnopharmacol 2008; 118:387-95.
Chen FE, Huang DB, Chen YQ, Ghosh G. Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Nature 1998; 391:410-413.
Chen JJ, Lin WJ, Liao CH, Shieh PC. Anti-inflammatory benzenoids from Antrodia camphorata. J Nat Prod 2007; 70:989-92.
Chen YJ, Cheng PC, Lin CN, Liao HF, Chen YY, Chen CC, et al. Polysaccharides from Antrodia camphorata mycelia extracts possess immunomodulatory activity and inhibits infection of Schistosoma mansoni. Int Immunopharmacol 2008; 8:458-67.
Cheng PC, Hsu CY, Chen CC, Lee KM. In vivo immunomodulatory effects of Antrodia camphorata polysaccharides in a T1/T2 doubly transgenic mouse model for inhibiting infection of Schistosoma mansoni. Toxicol Appl Pharmacol 2008; 227:291-8.
Costa, F.F. Non-coding RNAs: meet thy masters, Bioessays 32 (2010) 599-608.
Current Opinion in Immunology 2014, 140-146. Long noncoding RNAs in innate and adaptive immunity
embryogenesis, Nat. Rev. Genet. 12 (2011) 136-149.
Fitzgerald K-A and Caffrey D-R. Long noncoding RNAs in innate and adaptive immunity. Current Opinion in Immunology 2014, 26:140-146
Gibb, E.A., Brown, C.J., Lam, W.L. The functional role of long non-coding RNA in human carcinomas, Mol. Cancer 10 (2011) 38.
Guo J-H and Tseng W-T, 2013. A Polysaccharide ACP-1 Produced by Antrodia camphorata NPU-50 Mycelia Strain in Solid-State fermentation, 2013. Department of Food Science, NPUST No.1, Hsueh-Fu Rd, Neipu, Pingtung 91201, Taiwan, ROC.
Hacker H, Karin M. Regulation and function of IKK and IKKrelated kinases. Sci STKE 2006.
Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell 2008; 132:344-362.
Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev 2004; 18:2195-2224.
Hoffmann A, Levchenko A, Scott ML, Baltimore D. The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science 2002; 298:1241-1245.
Hseu YC, Chen SC, Yech YJ, Wang L, Yang HL. Antioxidant activity of Antrodia camphorata on free radical-induced endothelial cell damage. J Ethnopharmacol 2008; 118:237-45.
Jelena Škuljec, Hui Sun, Refik Pul, Karelle B#westeur042#nardais, Daniela Ragancokova, Darius Moharregh-Khiabani, Alexandra Kotsiari, Corinna Trebst, Martin Stangel. CCL5 induces a pro-inflammatory profile in microglia in vitro. Cellular Immunology 270 (2011) 164-171
Kang SM, Tran AC, Grilli M, Lenardo MJ. NF-kappa B subunit regulation in nontransformed CD4+ T lymphocytes. Science 1992; 256:1452-1456.
Lee TH, Lee CK, Tsou WL, Liu SY, Kuo MT, Wen WC. A new cytotoxic agent from solid-state fermented mycelium of Antrodia camphorata. Planta Med 2007; 73:1412-5.
Lenardo M, Pierce JW, Baltimore D. Protein-binding sites in Ig gene enhancers determines transcriptional activity and inducibility. Science 1987; 236:1573-1577.
Lenardo MJ, Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 1989; 58:227-229.
Liang B, Peng P, Chen S, Li L, Zhang M, Cao D. Characterization and proteomic analysis of ovarian cancer-derived exosomes. J Proteomics 2013; 80C:171-82.
Liu JJ, Huang TS, Hsu ML, Chen CC, Lin WS, Lu FJ, Chang WH. Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicol Appl Pharmacol 2004; 201:186-93.
Lu MK, Cheng JJ, Lai WL, Lin YJ, Huang NK. Fermented Antrodia cinnamomea extract protects rat PC12 cells from serum deprivation-induced apoptosis: the role of the MAPK family. J Agric Food Chem 2008.
Lu ZM, Tao WY, Zou XL, Fu HZ, Ao ZH. Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats. J Ethnopharmacol 2007; 110:160-4.
Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics 2010; 73:1907-20.
Nana-Sinkam, S.P., Croce, C.M. Non-coding RNAs in cancer initiation and progression and as novel biomarkers, Mol. Oncol. 5 2011; 483-491.
Natoli G, Chiocca S. Nuclear ubiquitin ligases, NF-kappaB degradation, and the control of inflammation. Sci Signal 2008; 1:pe1.
Neta R-R, Danny W. Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles. Cell 153, 1120-1133, May 23, 2013
Pauli, A. , Rinn J.L., Schier, A.F. Non-coding RNAs as regulators of
Ponting, C. P., Oliver, P.L., Reik, W. Evolution and functions of long noncoding RNAs, Cell 136 (2009) 629-641.
Raposo G, StoorvogelW. Extracellular vesicles: exosomes,microvesicles, and friends. J Cell Biol 2013; 200:373-83.
San Yun Press; 1985. p 116-7.
Sen R, Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein NF-kappa B by a posttranslational mechanism. Cell 1986; 47:921-928.
Stein, L.D. Human genome: end of the beginning, Nature 431 (2004) 915-916.
Su H, Bidere N, Zheng L. Requirement for caspase-8 in NF-kappaB activation by antigen receptor. Science 2005; 307:1465-1468.
Tanaka Y, Kamohara H, Kinoshita K, Kurashige J, Ishimoto T, Iwatsuki M, et al. Clinical impact of serum exosomal microRNA-21 as a clinical biomarker in human esophageal squamous cell carcinoma. Cancer 2013; 119:1159-67.
Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 2008; 110:13-21.
Tsai ZT, Liaw SL. The use and the effect of Ganoderma. Taichung, Taiwan:
Urban MB, Schreck R, Baeuerle PA. NF-kappaB contacts DNA by a heterodimer of the p50 and p65 subunit. EMBO J 1991; 10; 1817-1825.
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007; 9:654-9.
Vallabhapurapu S, Karin M. Regulation and function of NFkappaB transcription factors in the immune system. Annu Rev Immunol 2009; 27:693-733.
Wan-F, Lenardo M- J. The nuclear signaling of NF-κB: current knowledge, new insights, and future perspectives. Cell Research (2010) 20:24-33.
Wang GJ, Tseng HW, Chou CJ, Tsai TH, Chen CT, Lu MK. The vasorelaxation of Antrodia camphorata mycelia: involvement of endothelial Ca2t-NOcGMP pathway. Life Sci 2003; 73:2769-83.
Wapinski, O., Chang, H.Y. Long noncoding RNAs and human disease, Trends Cell Biol. 21 (2011) 354-361.
Zhang-Q, Lu-M, Wang-C, Du-J, Zhou-P, Zhao-M. Characterization of estrogen receptor a activities in polychlorinatedbiphenyls by in vitro dual-luciferase reporter gene assay. Environmental Pollution 189, 2014; 169-175