The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line
碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 98 === Hepatitis C virus (HCV) is a single-stranded positive sense RNA virus that belongs to the family Flaviviridae of the genus Hepacivirus. The World Health Organization (WHO) estimated there are more than 170 million people infected by HCV, about 2.9% of the worl...
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ndltd-TW-098CSMU52290122015-10-28T04:07:07Z http://ndltd.ncl.edu.tw/handle/36442261446372897204 The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line 探討金絲桃素抑制Ava.5細胞株中C型肝炎病毒複製之機制 Kuo-Li 袁國醴 碩士 中山醫學大學 醫學分子毒理學研究所 98 Hepatitis C virus (HCV) is a single-stranded positive sense RNA virus that belongs to the family Flaviviridae of the genus Hepacivirus. The World Health Organization (WHO) estimated there are more than 170 million people infected by HCV, about 2.9% of the world population. Approximately 80% of HCV infections result in chronic infection that can lead to severe liver diseases, such as cirrhosis and hepatocellular carcinoma. Recommended clinical care for HCV infections is a combination therapy of peginterferon alpha-2a and ribavirin. Chronic infection often leads to resistance to treatment. In clinical research, interferon alpha-2a and ribavirin showed significant side effects in patients. Finding effective drugs with fewer side effects to improve clinical treatment for HCV infection is an area of considerable interest. In order to understand the pathway that HCV replicates in host cells, scientists recently developed several types of cell lines containing HCV subgenomic replicon. A synthetic HCV subgenomic RNA replaced the HCV structural protein region of the neomycin- phosphotransferase gene and encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES). HCV RNA 5’-untranslated region (UTR) is an internal ribosomal entry site (IRES) promoting HCV downstream gene, such that HCV replicon can be efficiently expressed in these special cell lines. The Ava.5 cell line that expressed the HCV1b replicon was used to create a platform for testing anti-HCV drugs, and studying the biochemical mechanism of anti-HCV drugs. First , an MTS assay was used to measure cytotoxicity after cells were treated. Where concentrations did not induce cell death, HCV subgenomic RNA was examined by reverse transcription polymerase chain reaction and western blot to check whether drugs inhibited HCV replication. Finally, it was confirmed that hypericin can inhibit HCV1b NS5A RNA and NS3 protein. In order to understand the biochemical mechanisms that inhibit HCV replication, treated hypericin and interferon alpha-2a were combined in Ava.5 cells. There was no synergistic effect between hypericin and interferon alpha-2a to inhibit HCV1b NS3 protein. On the other hand, hypericin can inhibit Phospho- PKC αThr638/βIIThr641, but interferon alpha-2a does not. The mechanism of hypericin inhibited HCV NS3 protein different from interferon alpha-2a. After treatment with Ca2+-dependent PKC inhibitor Gö 6976, HCV NS3 protein was inhibited by Gö 6976 in the Ava.5 cell line. Accordingly, it is believed that Ca2+-dependent PKC signaling transduction regulated NS3 protein replicated. In this study, it was confirmed that hypericin is an excellent inhibitor of HCV replicon in Ava.5 cells. For HCV patients resistant to IFN-α treatment, there is an additional option to treat HCV patients with hypericin. Jiunn-Liang, Ko 柯俊良 2010 學位論文 ; thesis 110 zh-TW |
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碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 98 === Hepatitis C virus (HCV) is a single-stranded positive sense RNA virus that belongs to the family Flaviviridae of the genus Hepacivirus. The World Health Organization (WHO) estimated there are more than 170 million people infected by HCV, about 2.9% of the world population. Approximately 80% of HCV infections result in chronic infection that can lead to severe liver diseases, such as cirrhosis and hepatocellular carcinoma. Recommended clinical care for HCV infections is a combination therapy of peginterferon alpha-2a and ribavirin. Chronic infection often leads to resistance to treatment. In clinical research, interferon alpha-2a and ribavirin showed significant side effects in patients. Finding effective drugs with fewer side effects to improve clinical treatment for HCV infection is an area of considerable interest. In order to understand the pathway that HCV replicates in host cells, scientists recently developed several types of cell lines containing HCV subgenomic replicon. A synthetic HCV subgenomic RNA replaced the HCV structural protein region of the neomycin- phosphotransferase gene and encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES). HCV RNA 5’-untranslated region (UTR) is an internal ribosomal entry site (IRES) promoting HCV downstream gene, such that HCV replicon can be efficiently expressed in these special cell lines. The Ava.5 cell line that expressed the HCV1b replicon was used to create a platform for testing anti-HCV drugs, and studying the biochemical mechanism of anti-HCV drugs. First , an MTS assay was used to measure cytotoxicity after cells were treated. Where concentrations did not induce cell death, HCV subgenomic RNA was examined by reverse transcription polymerase chain reaction and western blot to check whether drugs inhibited HCV replication. Finally, it was confirmed that hypericin can inhibit HCV1b NS5A RNA and NS3 protein. In order to understand the biochemical mechanisms that inhibit HCV replication, treated hypericin and interferon alpha-2a were combined in Ava.5 cells. There was no synergistic effect between hypericin and interferon alpha-2a to inhibit HCV1b NS3 protein. On the other hand, hypericin can inhibit Phospho- PKC αThr638/βIIThr641, but interferon alpha-2a does not. The mechanism of hypericin inhibited HCV NS3 protein different from interferon alpha-2a. After treatment with Ca2+-dependent PKC inhibitor Gö 6976, HCV NS3 protein was inhibited by Gö 6976 in the Ava.5 cell line. Accordingly, it is believed that Ca2+-dependent PKC signaling transduction regulated NS3 protein replicated. In this study, it was confirmed that hypericin is an excellent inhibitor of HCV replicon in Ava.5 cells. For HCV patients resistant to IFN-α treatment, there is an additional option to treat HCV patients with hypericin.
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author2 |
Jiunn-Liang, Ko |
author_facet |
Jiunn-Liang, Ko Kuo-Li 袁國醴 |
author |
Kuo-Li 袁國醴 |
spellingShingle |
Kuo-Li 袁國醴 The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line |
author_sort |
Kuo-Li |
title |
The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line |
title_short |
The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line |
title_full |
The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line |
title_fullStr |
The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line |
title_full_unstemmed |
The Mechanism of Inhibiting HCV Replication by Hypericin in Ava.5 Cell Line |
title_sort |
mechanism of inhibiting hcv replication by hypericin in ava.5 cell line |
publishDate |
2010 |
url |
http://ndltd.ncl.edu.tw/handle/36442261446372897204 |
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