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研究生：	何韋志 Ho, Wei-Chih
論文名稱：	介白素二十第二型接受器在肝疾病的研究 The Study of IL-20 receptor 2 in Liver Disease
指導教授：	張明熙 Chang, Ming-Shi
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	59
中文關鍵詞：	介白素-20第二型接受器、肝纖維化
外文關鍵詞：	IL-20R2, Liver fibrosis
相關次數：	點閱：85 下載：0
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肝臟疾病如脂肪性肝炎和肝纖維化是全世界造成罹病以及死亡的重要疾病之一。肝纖維化是指因為不斷有新的纖維蛋白形成，使得過多的細胞外基質沈積在肝臟中，最後可能造成肝硬化的產生。IL-20第二型接受器 (IL-20R2)除了可以和IL-20第一型接受器 (IL-20R1)形成二聚體，接受IL-19/20/24之外，另外也可以和IL-22第一型接受器 (IL-22R1)形成二聚體，接受IL-20/24。由於先前的研究顯示，IL-20在肝纖維化中是扮演一個促發炎的角色，而且IL-20R1基因剔除小鼠具有抵抗CCl4所誘導肝損傷的能力，因此，在本次研究將更進一步探討IL-20R2基因剔除小鼠在肝疾病中所扮演的角色。從結果發現，利用TAA誘導短期小鼠肝損傷模式後，IL-20R2基因剔除小鼠會產生比野生型小鼠更嚴重的肝損傷，例如：更高的血清麩草醋酸轉胺脢 (AST)，麩丙酮酸轉胺脢 (ALT)和膽紅素 (Bilirubin)數值，更大的肝臟壞死區域，較低的存活率以及增加促發炎激素的表現。IL-20R2基因剔除小鼠在由TAA或CCl4所誘導的長期小鼠肝損傷模式中也可以觀察到類似的現象。此外，在高脂食物所誘導的小鼠代謝性肝損傷模式中，IL-20R2基因剔除小鼠相較於野生型小鼠，同樣也會展現更高的血清麩草醋酸轉胺脢 (AST)和麩丙酮酸轉胺脢 (ALT)數值，另外還會出現更嚴重的脂肪性肝炎。因為出乎意料地發現IL-20R1與IL-20R2基因剔除小鼠之間所產生截然不同的效果，所以我們就想更進一步探討是否IL-24可能在肝臟疾病中扮演角色。在TAA誘導短期小鼠肝損傷模式中，利用肌肉內電擊的方式將pcDNA3.1-mIL-24質體送入老鼠體內，使其表現IL-24蛋白後，發現相較於空載體控制組（Empty Vector），可以顯著降低血清麩草醋酸轉胺脢 (AST)和麩丙酮酸轉胺脢 (ALT)數值，減少肝臟壞死的區域，增加老鼠的存活率以及降低促發炎激素的表現。利用直接給予老鼠腹腔注射IL-24蛋白的方式，在TAA所誘導的短期小鼠肝損傷模式中也可以觀察到相同的現象。綜合上述的實驗結果，我們認為IL-20R1與IL-20R2基因剔除小鼠之間會產生截然不同的效果是由於缺乏IL-24所誘發的訊息傳遞所造成，而IL-24在肝臟疾病中可能是扮演一個保護性的角色。

Liver diseases such as hepatic steatosis and liver fibrosis, are important causes of morbidity and mortality worldwide. Liver fibrosis is defined as the presence of excess extracellular matrix deposition due to new fibrin formation, which may finally result in cirrhosis of the liver. IL-20 receptor (R)2 can dimerize with IL-20R1 to form the receptors for IL-19, IL-20 and IL-24, whereas IL-20R2 also can dimerize with IL-22R1 to form the receptors for IL-20 and IL-24. Our previous study indicated that IL-20 acts as a proinflammatory cytokine in liver fibrosis. IL-20R1-deficient mice were protected from CCl4-induced liver injury. Therefore, we were aimed to investigate the role of IL-20R2-deficient mice in liver disease. However, we found that IL-20R2 deficiency induced more severe effects in liver compared to wild-type mice under short-term thioacetamide (TAA)-induced liver injury. We demonstrated higher serum AST, ALT, and bilirubin levels, increasing necrosis area of liver, reducing survival rate, and up-regulating pro-inflammatory cytokine expression in IL-20R2-deficient mice. The similar phenomenon was found in long-term TAA- and CCl4- treated IL-20R2-deficient mice. In high-fat diet-induced metabolic liver injury, IL-20R2-deficient mice also showed higher serum ALT and AST, and had more severe hepatic steatosis compared with wild-type mice. Because of the unexpected opposite effects between IL-20R1- and IL-20R2-deficient mice, we further investigated whether IL-24 may play a role in liver disease. In short-term TAA-induced liver injury model, intramuscular electroporation of pcDNA3.1-IL-24 plasmid DNA significantly reduced serum AST/ALT, decreased necrosis area of liver, increased survival rate, and down-regulated pro-inflammatory cytokine expression compared to intramuscular electroporation of pcDNA3.1 empty vector. The similar phenomenon was found in recombinant mIL-24 protein treated mice in short-term TAA-induced liver injury model. Taken together, our data supposed that the opposite effects between IL-20R1- and IL-20R2-deficient mice might attribute to the deficiency of IL-24-induced signaling pathway. Therefore, IL-24 may play a protective role in liver disease.

Chinese Abstract…………………………………………………………………I
English Abstract…………………………………………………………………III 
Acknowledgement…………………….…………………………………………V
Contents.………………………………………….……………………………..VII
Contents of table………………………………………………………………..IX
Contents of figure……………………………………….……………………….X
Abbreviations……………………………………………………………………XII

Introduction ……………………………………………………………….......1
I.	IL-20 …………………..…………….………………………………...……..1
II.	Liver disease………………………………………………………...………2
    1.	Liver fibrosis…………………………………………………..……………..2
    2.	Damaged hepatocyte in liver fibrosis……………………….……………3
    3.	Activated HSC in liver fibrosis…………………………………………….4
    4.	Animal model of liver fibrosis………………………………….…………..4
III.	IL-20 and liver disease.…………….....……………………………………5
IV.	Interleukin24…………………………………………………………………6

Specific Aims…………………..……………………………………………….7

Materials and methods….……………………………………………………..8
    1.	Mice……………………………………………………………………..…….8
    2.	Animal model of Thioacetamide (TAA)-induced liver injury…………...8
    3.	Animal model of CCl4 (Sigma-Aldrich)-induced liver injury…….…….8
    4.	Animal model of high-fat diet-induced metabolic liver injury…..…….9
    5.	Mouse IL-24 protein treatments……………..……………………………9
    6.	Sample collection and serum biochemistry……………………………..9
    7.	Mice body weight measurement…………………………….……………9
    8.	ELISA assay………………………......……………………………………10
    9.	Histology………………………...…………………………....……………10
  10.	RTQ-PCR……………………………………………………………………11
  11.	Plasmid DNA preparation………………….……………………………..11
  12.	Intramuscular electroporation……………………………………...……12
  13.	Detection of exogenous mIL-24 in mice serum by ELISA…………...12
  14.	Expression and purification of mouse IL-24…………………..………13
  15.	Statistical analysis…………………………………………………………14

Results.………………………….…………………………………….………..15
    1.	IL-20R2 deficiency induced more severe effects in liver compared to wild-type mice under short-term TAA-induced liver injury.……………......15
    2.	IL-20R2 deficiency induced more severe effects in liver compared to wild-type mice under long-term TAA-induced liver injury………………....16
    3.	IL-20R2 deficiency induced more severe effects in liver compared to wild-type mice under long-term CCl4-induced liver injury.…………..…...17
    4.	IL-20R2 deficiency induced more severe effects in liver compared to wild-type mice under high-fat diet-induced metabolic liver injury…….…17
    5.	Intramuscular electroporation of pcDNA3.1-IL-24 protected mice from short-term TAA-induced liver injury…………………………..…...……18
    6.	Recombinant mIL-24 protein treated mice were resistant to short-term TAA-induced liver injury…………………………………….....…..……. 19

Discussion………………………………………………………..……………….20

References…………………………….…………………………….……………24

Table……………………………………………………………………………….28

Figure and figure legends…………….………………………………………...33

Contents of Table

Table 1.	Schematic representation of IL-20R2 knockout construction..28 
Table 2.	A PCR genotyping strategy to distinguish WT and IL-20R2 KO   was established using specific primers………………..................………..29
Table 3.	Comparison of liver fibrosis between CCl4 and TAA-treated   mice………........................................................................................….30
Table 4.	Primer pairs used for amplifying transcrips………………….....31
Table 5.	Characteristics of Interleukin 24………………………......……..32
   
Contents of Figure

Figure 1.  IL-20R2 deficiency showed lower survival rate in short-term TAA-induced liver injury…………...…..…………………………….....……...33
Figure 2.	IL-20R2-deficient mice lost more body weight in short-term TAA-induced liver injury………………………………..…………….…….…..34
Figure 3.	IL-20R2-deficient mice showed larger necrosis area in short-term TAA-induced liver injury…….……………..……………………………..35
Figure 4.	IL-20R2-deficient mice had higher serum AST and ALT in short-term TAA-induced liver injury……………………………………..…………...36
Figure 5.	IL-20R2-deficient mice showed more severe jaundice and gallbladder enlargement in short-term TAA-induced liver injury………....37
Figure 6.	IL-20R2 deficiency induced more pro-inflammatory and pro-fibrogenic cytokines in short-term TAA-induced liver injury……..…….....38
Figure 7.	IL-20R2 deficiency induced more fibrosis under long-term TAA-induced liver injury……………..…………………………………..……………39
Figure 8.	IL-20R2-deficient mice showed no protective effect in serum TGF-β value in long-term TAA-induced liver injury..................................40
Figure 9.	IL-20R2-deficient mice had higher serum AST, ALT and bilirubin, and showed lower triglyceride and cholesterol in long-term TAA-induced liver injury…..............................................................................41
Figure 10.	IL-20R2 deficiency induced more pro-inflammatory and pro-fibrogenic cytokines in long-term TAA-induced liver injury………………..42
Figure 11.	IL-20R2 deficiency induced more fibrosis in long-term CCl4-induced liver injury………………....…………………………………………...43
Figure 12.	IL-20R2-deficient mice showed enlarged liver and spleen in long-term CCl4-induced liver injury……………………………..…………...44
Figure 13.	IL-20R2-deficient mice had higher serum TGF-β in long-term CCl4-induced liver injury……………….....……………………………….…..45
Figure 14.	IL-20R2-deficient mice had higher serum AST, ALT and bilirubin in long-term CCl4-induced liver injury…………………………………….....46
Figure 15.	IL-20R2 deficiency induced more severe hepatic steatosis and fibrosis in HFD-induced metabolic liver injury……………………….....…..47
Figure 16.	IL-20R2-deficient mice had higher serum AST and ALT in HFD-induced metabolic liver injury………………………………………...……….48
Figure 17.	Detection of exogenous mIL-24 in mouse serum after electroporation…...................................................................................49
Figure 18.	Intramuscular electroporation of pcDNA3.1-IL-24 increased the survival rate of mice in short-term TAA-induced liver injury……………...50
Figure 19.	Intramuscular electroporation of pcDNA3.1-IL-24 decreased body weight lose of mice in short-term TAA-induced liver injury………...51
Figure 20.	Intramuscular electroporation of pcDNA3.1-IL-24 decreased liver necrosis area of mice in short-term TAA-induced liver injury..……...52
Figure 21.	Intramuscular electroporation of pcDNA3.1-IL-24 decreased serum AST and ALT of mice in short-term TAA-induced liver injury…..…53
Figure 22.	Intramuscular electroporation of pcDNA3.1-IL-24 decreased pro-inflammatory and pro-fibrogenic cytokines in mice liver in short-term TAA-induced liver injury…………......……………………..…………………..54
Figure 23.	IL-24 increased the survival rate of mice in short-term TAA-induced liver injury…………………………………………………..…………..55
Figure 24.	Recombinant IL-24 protein-treated mice lost less body weight in short-term TAA-induced liver injury…………………………………….….56
Figure 25.	Recombinant IL-24 protein-treated mice showed smaller liver necrosis area in short-term TAA-induced liver injury…………………….…57
Figure 26.	Recombinant IL-24 protein-treated mice had lower serum AST and ALT in short-term TAA-induced liver injury……………………………..58
Figure 27.	Recombinant IL-24 protein-treated mice had lower pro-inflammatory and pro-fibrogenic cytokines in mice liver in short-term TAA-induced liver injury……….......................................................................59

                                    

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