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標題: | C型肝炎病毒非結構性蛋白質NS5A參與細胞之複製型老化 Involvement of hepatitis C virus NS5A protein in replicative senescence |
作者: | Hui-Chia Chen 陳慧珈 |
指導教授: | 張鑫(Shin C. Chang) |
關鍵字: | C型肝炎病毒,非結構性蛋白質 5A,人類端粒?逆轉錄,複製型老化, hepatitis C virus (HCV),non-structural protein 5A (NS5A),human telomerase reverse transcriptase (hTERT),replicative senescence, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 目前全球約有七千一百萬人受到C型肝炎病毒 (hepatic C virus;HCV) 的感染,因此HCV感染屬於全球性的健康議題。統計顯示約有85% 急性感染患者會發展成慢性肝病,且10~20% 慢性肝病患者會形成肝硬化,有些患者最終可能惡化成肝細胞癌 (hepatocellular carcinoma;HCC) 而導致死亡。本實驗室先前研究發現肝細胞癌細胞株表達HCV的非結構性蛋白質NS5A時,會造成人類端粒酶逆轉錄酶 (human telomerase reverse transcriptase;hTERT) mRNA表現量下降,故推測端粒酶活性可能也會下降。而已知當細胞的端粒酶活性受到抑制時,細胞最終會走向複製型老化,故本研究想進一步探討NS5A是否會誘導肝細胞癌細胞走向複製型老化。首先檢測HCV的非結構性蛋白質對於hTERT mRNA表現量的影響,由RT-qPCR的結果得知,在表達非結構性蛋白質NS3-5B的HCVR細胞株和表達NS5A的細胞株中hTERT mRNA表現量均會下降,而在表達NS3的細胞株中則不受影響,此結果與先前實驗室的研究成果一致。利用誘導型表達NS5A的不同代數細胞進行細胞老化的相關實驗,由senescence-associated β-galactosidase staining 分析得知表達NS5A的細胞相較於控制組會提前走向複製型老化;且誘導細胞老化的分子p27和p21的表現量上升,其中已知p21參與誘導複製型老化。並檢測NS5A誘導細胞走向複製型老化與hTERT的相關性,結果顯示各代數細胞中的hTERT mRNA表現量下降,且在過度表達hTERT細胞中NS5A誘導的老化細胞數量會減少。此外也在穩定型表達NS5A的細胞確認了NS5A誘導複製型老化的現象。由本研究得知NS5A會抑制hTERT且誘導肝細胞癌細胞走向複製型老化。另外,NS5A會誘導p21的表現。雖已知p21會參與在誘導複製型老化的路徑中,但仍需進一步確認p21在NS5A誘導老化中是否為重要的因子。此外,也得知NS5A會促進p27的表現,推測NS5A所造成的細胞壓力可能會誘導壓力誘導型早期老化,此亦需進一步探討。 Globally, an estimated 71 million people have hepatitis C virus (HCV) infection, so it is a health issue in the world. HCV infection causes acute hepatitis, 85% of the cases become chronic, causing chronic hepatitis, cirrhosis (in 10~20% of cases after 10~20 years), and hepatocellular carcinoma (HCC). Previous studies in our laboratory found that HCV non-structural protein 5A (NS5A) caused a decreased hTERT mRNA level in a hepatoma cell line, so it was proposed that telomerase activity may also decrease in NS5A-expressing cells. It is known that a decreased telomerase activity may induce replicative senescence. Therefore, whether NS5A protein could induce cellular senescence by repressing hTERT expression was explored in this study. The effects of NS proteins on hTERT expression were first examined. RT-qPCR data showed that both HCVR cells, which express HCV NS proteins from NS3 to NS5B, and NS5A-expressing cells downregulated hTERT mRNA level. This result is consistent with our previous study. In addition, the phenomenon was not observed in NS3-expressing cells. Next, whether NS5A could induce replicative senescence was examined by using NS5A inducible-expressing cells cultured to different passage numbers. Results from senescence-associated β-galactosidase staining showed that NS5A induced replicative senescence in hepatoma cells earlier than the control group. In addition, the expression of p27 and p21, molecules to induce cellular senescence, were increased. The correlation between NS5A-induced replicative senescence and hTERT was further examined. The data showed that hTERT mRNA level decreased in every passage of the NS5A-expressing cells. The level of senescent cells induced by NS5A was repressed when hTERT was overexpressed, suggesting that NS5A induces cellular senescence by repressing hTERT expression. Replicative senescence was also observed in cells constitutively express NS5A. In conclusion, NS5A repressed hTERT and induced replicative senescence. In addition, NS5A induced p21 expression. Although p21 is known to induce replicative senescence, whether p21 is the predominant mediator in NS5A-induced senescence needs to be further confirmed. Moreover, NS5A also increased p27 expression. Whether NS5A is involved in stress-induced premature senescence needs to be further investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70291 |
DOI: | 10.6342/NTU201803404 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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