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研究生： 呂意嫻
Lu, Yi-Sian
論文名稱： 介白素-20在瀰漫性大型B 細胞淋巴癌中的角色
The Roles of Interleukin-20 in Diffuse Large B-Cell Lymphoma
指導教授： 張孔昭
Chang, Kung-Chao
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫學檢驗生物技術學系
Department of Medical Laboratory Science and Biotechnology
論文出版年： 2022
畢業學年度： 110
語文別： 英文
論文頁數： 51
中文關鍵詞： DLBCL (瀰漫性大 B 細胞淋巴瘤) 、細胞激素 、IL-20 、免疫組織化學 、預後良好 、細胞週期調控
外文關鍵詞： DLBCL (diffuse large B-cell lymphoma), cytokine, IL-20, immunohistochemistry, favorable prognosis, cell cycle regulation
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    • 瀰漫性大 B 細胞淋巴瘤 (DLBCL) 是全球最常見的淋巴瘤類型，約佔所有淋巴 瘤病例的 40%。儘管細胞激素與淋巴細胞具有密切的免疫關係，但它們在淋巴瘤發病 機制的作用卻很少被提及。我們意外地在反應性生發中心 (GCs) 中發現了 介白素 (IL)-20 的過度表達;因此，我們假設 IL-20 對於淋巴瘤的生成扮演重要的角色。 在這項研究中，我們發現 IL-20 在 GC 衍生的淋巴瘤中頻繁表達，包括濾泡性淋巴 瘤(94%)、瀰漫性大 B 細胞淋巴瘤(DLBCL，48%)、伯基特氏淋巴瘤(81%)和 霍奇金氏淋巴瘤(50%)，以及小淋巴細胞淋巴瘤 (50%)、套細胞淋巴瘤 (57%) 和淋 巴結邊緣區淋巴瘤 (56%)。我們也發現 IL-20 在生發中心 B 細胞 (GCB) 中的表達 比在非 GCB 亞型中更頻繁(16/26 [62%] vs. 24/64 [38%]，p =0.038)並與較低的結 外侵犯率 (p=0.009)、骨髓侵犯率 (p=0.040) 和更好的總體生存率 (p=0.020) 相關。 從機制上來說，IL-20 過表達促進 G1 細胞週期停滯和隨後的 DLBCL 細胞凋亡，反 之亦然。此外，IL-20 過表達透過活化 Caspase-3、7、8、9 和下游切割產物進行內在 和外在細胞凋亡途徑。總結來說，IL-20 可能與淋巴瘤發生有關，透過調節細胞週期 並抑制 DLBCL 生長，可作為 DLBCL 患者的預後標誌物。

    Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma worldwide, accounting for approximately 40% of all lymphoma cases. Although cytokines have a close immunological relationship with lymphocytes, their role in the pathogenesis of lymphoma has been rarely addressed. We have accidentally found overexpression of IL-20 in reactive germinal centers (GCs); we, thus, hypothesized that IL-20 may play a role in GC- derived lymphomas. In this study, we found that IL-20 was expressed frequently in GC- derived lymphomas, including follicular lymphoma (94%), diffuse large B-cell lymphoma (48%), Burkitt lymphoma (81%), and Hodgkin lymphoma (50%), as well as a subset of small lymphocytic lymphoma (50%), mantle cell lymphoma (57%), and nodal marginal zone lymphoma (56%). We further found IL-20 expression was more frequently in germinal center B-cell (GCB) than in non-GCB subtype (16/26 [62%] vs. 24/64 [38%], p=0.038) and associated with a lower rate of extranodal involvement (p=0.009), bone marrow involvement (p=0.040), and better overall survival (p=0.020). Mechanistically, IL-20 overexpression promoted G1 cell cycle arrest and subsequent apoptosis of DLBCL cells and vice versa in vitro. In addition, overexpression of IL-20 induced either intrinsic and extrinsic apoptosis pathway by activating caspase-3,7,8,9 and downstream substrate cleavage and vice versa. We conclude that IL-20 may be involved in lymphomagenesis and plays an inhibitory role in DLBCL growth, probably through cell cycle regulation, and may be useful as a prognostic marker in patients with DLBCL.

    摘要 I ABSTRACT II 致謝 III ABBREVIATIONS VIII INTRODUCTION 1 Diffuse large B-cell lymphoma (DLBCL) 1 Interleukin (IL)-20 2 MATERIALS AND METHODS 5 Lymph node, tonsil, bone marrow, and lymphoma tissues 5 Cell culture 6 Immunohistochemical staining 6 Cell proliferation assay 7 Western Blotting analysis 7 Knock-down and overexpression of IL20 gene 7 PCR array 8 Apoptosis and cell cycle assays 8 Subcutaneous xenograft mouse model 8 Statistical analysis 9 RESULTS 10 Expression patterns of IL-20 in non-neoplastic lymph nodes, tonsils, and bone marrow 10 Expression patterns of IL-20 in lymphomas 10 Expression of IL-20 correlated with a lower frequency of extranodal involvement, bone marrow involvement, and a better prognosis of survival in DLBCL 11 Expression of IL-20 and IL-20 receptors in DLBCL cell lines 12 Knock-down of IL-20 attenuated G1 cell cycle arrest and apoptosis in DLBCL cells12 Overexpression of IL-20 enhanced G1 cell cycle arrest and apoptosis in DLBCL cells in vitro 12 Knock-down of IL-20 promoted proliferation in DLBCL cells and vice versa in vitro 13 The apoptosis-related genes showed a significant difference in PCR array 13 Knock-down of IL-20 in DLBCL cells inhibited either the intrinsic or extrinsic apoptosis pathway and vice versa 13 Overexpression of IL-20 in SU-DHL-5 cells caused an increase in the weight of tumors in NOD-SCID mice 14 DISCUSSION 15 REFERENCE 18 TABLES AND FIGURES 26 Table 1. IL-20 expression in various types of lymphoma 26 Table 2. Clinicopathologic difference of DLBCL based on IL-20 expression 28 Table 3. Clinicopathologic parameters affecting overall survival of patients with DLBCL 29 Table 4. The significant difference of genes in PCR array 30 Figure 1. Expression pattern of IL-20 in the lymph node, tonsil and bone marrow 31 Figure 2. Expression patterns of IL-20 in lymphomas 32 Figure 3. Survival plots show clinicopathologic factors affecting overall survival of DLBCL patients 33 Figure 4. Expression of IL-20 receptors─IL-20R1, IL-20R2, and IL-22R1 in lymphoblastoid cell line (LCL) and DLBCL cell lines 36 Figure 5. Knock-down of IL-20 attenuated G1 cell cycle arrest and apoptosis in DLBCL cells and vice versa 38 Figure 6. Knock-down of IL-20 promoted proliferation in DLBCL cells and vice versa in vitro 39 Figure 7. Knock-down of IL-20 downregulated expression of Fas and upregulated expression of Bcl2-L1 in DLBCL cells and vice versa 40 Figure 8. Knock-down of IL-20 in DLBCL cells inhibited either the intrinsic or extrinsic apoptotic pathway and vice versa 42 Figure 9. Overexpression of IL-20 caused an increase in the weight of tumors in SU- DHL-5 cells in NOD-SCID mice 43 APPENDIX 44 Appendix 1. Characterization of DLBCL cell lines 44 Appendix 2. Antibodies of immunoblot 45 Appendix 3. Sequence of cDNA for transfection of IL20 (IL20 cDNA) 46 Appendix 4. Sequence of short hairpin RNAs (shRNAs) against IL20 47 Appendix 5. Clinicopathologic difference of DLBCL based on GCB vs. ABC subtypes 48 Appendix 6. Survival curves of DLBCL patients with IL-20 expression (+) or without IL-20 expression (-) in ABC vs. GCB subtypes 49 Appendix 7. Western blot of IL-20 in six DLBCL cell lines tested 50 Appendix 8. This thesis has been accepted by Pathology in 2022 51

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