臺灣博碩士論文加值系統

D型肝炎是由D型肝炎病毒伴隨著B型肝炎病毒共感染所引起，是病毒性肝病中最為嚴重的一種，病患肝臟可能出現肝硬化、肝衰竭、肝癌等症狀。儘管全球約有4800-6000萬人口感染D型肝炎，但是目前D型肝炎的臨床治療策略並非直接針對D型肝炎病毒，而是利用抗B型肝炎病毒藥物來間接抑制D型肝炎病毒。首選藥物是干擾素α (interferon-α)，利用干擾素α提升病患的免疫能力來毒殺病毒，但是此療程需要持續一年以上才能有效抑制D型肝炎病毒，且許多病患容易對干擾素類藥物產生副作用，因此D型肝炎的治療仍需要發展。鈉離子牛磺膽酸共轉運蛋白(Sodium taurocholate co-transporting polypeptide, NTCP)是肝細胞膜上所特有的穿膜蛋白，在正常情況下，NTCP可藉由鈉離子協同運輸，將血液中的膽酸送入肝臟。近幾年，NTCP被發現是B型肝炎病毒與D型肝炎病毒感染的肝臟細胞受器，NTCP會辨識大型B型肝炎病毒表面抗原(L-HBsAg)的pre-S1區域並與之結合，因此NTCP被作為治療D型肝炎病毒感染的新目標。
我們在此次研究中發現，在表現NTCP的人類肝臟細胞中，ergosterol peroxide (EP; 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol)有抑制D型肝炎病毒感染的能力。EP為固醇衍生物，存在於多種藥用真菌中，具有抗癌、抗病毒、抗發炎等功效，其化學結構與膽酸相似。過去有研究指出膽酸可藉由NTCP來抑制D型肝炎病毒感染。從細胞實驗結果中可以觀察到，與其他EP結構類似物相比，EP有較好抑制D型肝炎病毒的能力，另外也發現EP藉由干擾病毒進入時的fusion/endocytosis步驟來達到抑制D型肝炎病毒感染的效果。此外，我們利用表現人類NTCP蛋白的基因轉殖小鼠來評估EP對D型肝炎病毒的效果是否與細胞實驗結果相符。首先，實驗結果證明表現人類NTCP蛋白基因轉殖小鼠的肝臟確實大量表現人類NTCP蛋白且可成功被HDV感染，並觀察到以腹腔注射給予EP之後，小鼠肝臟內的D型肝炎病毒表現量下降。同時，小鼠體重、肝功能生化指數並沒有顯著變化，證明EP在基因轉殖小鼠體內的安全性。總括而言，我們的研究分別在體內與體外試驗中證實EP可以作為一個有發展潛力且用於抑制D型肝炎病毒的天然化合物。

Hepatitis D caused by coinfection or superinfection of hepatitis delta virus (HDV) in hepatitis B patients increases the risk of liver cirrhosis, liver failure, and hepatocellular carcinoma. Although there are up to 60 million hepatitis D patients worldwide, the standard treatment for hepatitis D patients is not specific. By using interferon-α (IFN-α) to enhance patients' immune responses, patients are obliged to take the therapy persistently but are liable to experience IFN-related side effects. Human sodium taurocholate co-transporting polypeptide (hNTCP) is a transmembrane transporter on the hepatocyte plasma membrane, it takes up bile acids in the normal situation. Recently, hNTCP has been identified as an entry receptor for HDV and HBV. This discovery provides researchers a new therapeutic option targeting the entry step of HDV infection.
Here, we showed that ergosterol peroxide (EP; 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol) had inhibitory effects on HDV infection in hNTCP-expressing immortalized human hepatocytes cells, differentiated HuS-E/2 cells induced by DMSO. EP, a steroid derivative found in many medicinal mushrooms, has a chemical structure resembling to bile acids. Interestingly, a previous study has revealed that bile acids have antiviral activity against HDV infection. Compared with other EP analogs, EP had a better potency on inhibiting HDV infection. Then, we observed that EP acted on the fusion/endocytosis step in HDV infection. Besides, we translated the results from cells to HDV-infected hNTCP transgenic mice driven by the loxP-cre system. In hNTCP transgenic mice, HDV RNA expression decreased upon EP treatment in a dose-dependent manner. Meanwhile, mouse body weight was of no statistical difference between groups and biochemical indices suggested the safety of EP treatment. Our study proved the natural compound EP as a potential agent against HDV in vitro and in vivo.

Table of Contents
中文摘要................................i
Abstract .............................iii
Table of Contents..................... iv
List of Figures and Tables ...........vii
Chapter 1. Introduction ................1
1.1 Hepatitis delta virus (HDV) ........1
1.1.1 Virology .........................1
1.1.2 Epidemiology......................1
1.1.3 Replication cycle ................2
1.1.4 Clinical course of hepatitis D......... 3
1.1.5 Immune response of HDV infection .......3
1.1.6 Diagnosis........................ 4
1.1.7 Treatment ........................5
1.2 Sodium taurocholate cotransporting polypeptide (NTCP) ........................................6
1.2.1 The function of NTCP............. 6
1.2.2 NTCP-expressing hepatocytes and mice for HDV infection ........................................6
1.3 Ergosterol peroxide (EP) ...........7
Chapter 2. Specific aim................ 8
Chapter 3. Materials and Methods....... 9
3.1 Drugs ..............................9
3.2 Cell lines and cell culture ........9
3.2.1 HuS-E/2.......................... 9
3.2.2 HepG2.2.15 .......................9
3.2.3 HepG2-hNTCP-C4 ..................10
3.3 Cell viability assay ............. 10
3.4 HDV viral particle purification and quantification ...................................... 11
3.5 HDV infection in cell lines .......11
3.6 Animals-hNTCP transgenic mice .... 12
3.7 Mouse genotyping... .............. 12
3.8 Mouse HDV viral inoculation and ergosterol peroxide treatment ............................ 13
3.9 Western blot...................... 13
3.10 Antibodies....................... 14
3.11 Quantitative real-time polymerase chain reaction ...................................... 14
3.12 Biochemical analysis............. 14
3.13 Statistical analysis ............ 15
Chapter 4. Results ................... 16
4.1 Generation of HDV viral stock .... 16
4.2 Determination of EP dose.......... 16
4.3 EP inhibits HDV infection in differentiated HuS-E/2 cells ................................ 17
4.4 EP inhibited HDV infection in HepG2-hNTCP-C4 cells ...................................... 18
4.5 The effect of EP and its analogues on HDV infection in differentiated HuS-E/2 cel............ 18
4.6 The effect of EP on steps involved in HDV life cycle in differentiated HuS-E/2 cells ......... 19
4.7 The human NTCP transgenic mice model and mice genotyping ...................................... 20
4.8 The hNTCP expression and the susceptibility to HDV infection in hNTCP transgenic mice ... 20
4.9 EP suppressed HDV infection in hNTCP transgenic mice ...................................... 21
4.10 The effect of EP on liver hNTCP expression, body weight, and liver function in hNTCP transgenic mice ...................................... 21
4.11 The innate immune response of hNTCP transgenic mice ...................................... 22
Chapter 5. Discussion and conclusion ............. 23
5.1 Discussion ....................... 23
5.2 Conclusion ....................... 27
Supplementary Information............. 49
Reference ............................ 52

List of Figures and Tables
Figure 1. HDAg protein expression of pSVD2-transfected HepG2.2.15 cells. ................................ 28
Figure 2. HDV virus stock quantification. ........ 29
Figure 3. The cytotoxicity of EP in the differentiated-HuS-E/2 cell line. ................................... 30
Figure 4. The effect of EP on HDV replication in differentiated HuS-E/2 ........................... 32
Figure 5. The effect of EP on HDV infection in HepG2-hNTCP-C4 cells. ........................................ 33
Figure 6. The effect of EP and its analogues on HDV replication in differentiated HuS-E/2 cells............................................. 34
Figure 7. The effect of EP on HDV replicated steps in differentiated HuS-E/2 cells. .................... 37
Figure 8. Graphical illustration of hNTCP gene cassette in hNTCP transgenic mice and mice genotyping. ....... 39
Figure 9. The hNTCP expression and the susceptibility to HDV infection in hNTCP-cre transgenic mice. .......... 40
Figure 10. The effect of EP on HDV infection in hNTCP-cre transgenic mice. ................................. 42
Figure 11. Liver hNTCP expression, body weight, serum GOT, and GPT in hNTCP-cre transgenic mice. ............ 45
Figure 12. Inflammatory factor and interferon expression in the liver of EP-treated hNTCP-cre transgenic mice. .... 46
Figure 13. Graphical demonstration of mechanisms of EP action. .......................................... 48
Table. S1 Primer list..............................49
Figure. S1 EP inhibits the binding of the pre-S1 domain of LHBsAg and NTCP....................................51

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