評估利用RNA interference 抑制B型肝炎病毒複製之可行性

陳美麟

doi:10.6342/NTU.2004.00785

3.238.70.175，Hello！

Close
Search Quick Search Advanced Search Publication Search	Personal Service Member Login Searching History Use Bonus Card SYMSKAN
Browse Browse by University of Graduate Thesis Browse by Pinyin of Graduate Thesis Browse by Conference Proceedings Topic Browse by Conference Proceedings Publisher Browse by Pinyin of Conference Proceedings Browse by eBook Category Browse by eBook Topic Browse by eBook Publisher
Customer Services Authorize Airiti Recommend a Journal

Search Symbol (Half-width)	Description of Search Symbols
Space	"AND" indicates the intertwining of key terms used in a search
Double Quotation Marks ("") ( " " )	Double quotation marks indicate the beginning and end of a phrase, and the search will only include terms that appear in the same order of those within the quotations. Example: "image process" ： " image process "
?	Indicates a variable letter. Entering two ? will indicate two variable letters, and so on. Example: "Appl?", search results will yield apple, apply… e , appl y … ( (often used to English word searches) )
*	Indicates an unlimited number of variable letters to follow, from 1~n. Example: Enter "appl*", search results will yield apple, apples, apply, applied, application…(often used in English word searches) e , appl es , appl y , appl ied , appl ication … ( (often used to English word searches) )
AND、OR、NOT	Boolean logic combinations of key words is a skill used to expand or refine search parameters. (1) AND (1) AND: Refines search parameters (2) OR (2) OR: Expands search parameters (3) NOT: Excludes irrelevant parameters

DOI stands for Digital Object Identifier （ D igital O bject I dentifier ） ,
and is the unique identifier for objects on the internet. It can be used to create persistent link and to cite articles.

Using DOI as a persistent link

To create a persistent link, add「http://dx.doi.org/」「 http://dx.doi.org/ 」 before a DOI.
For instance, if the DOI of an article is 10.5297/ser.1201.002 , you can link persistently to the article by entering the following link in your browser: http://dx.doi.org/ 10.5297/ser.1201.002 。
The DOI link will always direct you to the most updated article page no matter how the publisher changes the document's position, avoiding errors when engaging in important research.

Cite a document with DOI

When citing references, you should also cite the DOI if the article has one. If your citation guideline does not include DOIs, you may cite the DOI link.

DOIs allow accurate citations, improve academic contents connections, and allow users to gain better experience across different platforms. Currently, there are more than 70 million DOIs registered for academic contents. If you want to understand more about DOI, please visit airiti DOI Registration （ doi.airiti.com ）。

Data Source

Health & Medical Care > Basic Medical Science
College of Medicine > Graduate Institute of Microbiology

Bibliography Management Tool

Related Links

Report an Issue

Purchase Single Articles

Download PDF

評估利用RNA interference 抑制B型肝炎病毒複製之可行性

Evaluation on the Feasibility of Using RNA Interference for Inhibition of Hepatitis B Virus Replication

陳美麟 , Masters　　Advisor：董馨蓮　　

繁體中文 DOI： 10.6342/NTU.2004.00785

肝炎病毒； RNA interference

Abstract │ Reference (127) │ Altmetrics

Abstract 〈TOP〉

RNA interference (RNAi) 為一普遍存在於動物及植物細胞中的機制，由雙股RNA與其序列上有互補的目標mRNA結合之後，造成目標mRNA的分解。在哺乳動物細胞，可經由外送約21個核苷酸的小片段雙股RNA (siRNA)至細胞中而引發RNAi的產生。在本實驗中，我們試圖評估以siRNA作為治療B型肝炎病毒(HBV)感染的可行性。

在本實驗的第一部份，我們想瞭解是否能利用siRNA在肝癌細胞株中作為控制基因表現的工具。首先針對綠色螢光蛋白質(GFP)的基因序列設計了兩個siRNA，利用pSUPER載體表達siRNA，發現能夠在不同的肝癌細胞株(Hep G2, Huh 7, PLC/PRF5 and Hep 3B)中有效並具特異性地降低GFP的表現。接下來針對HBV基因體我們建構了六個表達siRNA的質體(命名為pSUPER/HBx1, pSUPER/HBx2, pSUPER/HBx3, pSUPER/TP, pSUPER/RT, pSUPER/ε)，將這些表達質體各別與pHBV48共同轉染至HepG2細胞中，以北方墨點法分析這些siRNA表達質體抑制pHBV48轉錄本表現的效果。六個pSUPER質體均能表現siRNA。然而，針對HBV基因體上相當具保守性、且與encapsidation有關的序列所設計的pSUPER/ε無法抑制HBV轉錄本的表現。pSUPER/TP，pSUPER/RT及pSUPER/HBx3則證明了在共同轉染的系統中能夠有效並特異地將HBV轉錄本分解。

HepG 2 2.2.15為一能夠持續穩定表現HBV轉錄本及產生HBV病毒顆粒的細胞株，實驗的最後一部份，我們以HepG 2 2.2.15細胞為模式模擬慢性肝炎的患者，觀察siRNA是否能對於已存在於細胞中的HBV複製造成影響。我們採用兩種方法企圖提高細胞的轉染效率，其一是將pSUPER/TP(Hyg)，pSUPER/RT(Hyg)及pSUPER/HBx3(Hyg)各別轉染至HepG 2 2.2.15細胞後，經過Hygromycin篩選後所建立的pooled clones，雖然這些pooled clones能夠表現siRNA，但對於HBV轉錄本並沒有明顯的抑制效果。此外我們建構以lentiviral vector為骨架表達siRNA的質體，利用病毒感染的方式將質體送入HepG 2 2.2.15細胞中，發現可以將轉染效率提高至80~ 90 %以上，然而對於HBV轉錄本只有約30~ 40 %的抑制效果。綜合上述結果，對於siRNA是否為治療慢性肝炎一個良好的工具還存有不確定性，相關的問題及解決方法將會在內文中詳加討論。

Parallel Abstract 〈TOP〉

RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). In this study, we would like to evaluate the potential of using siRNAs for inhibition of hepatitis B virus (HBV) replication.

In the first part of the study, we investigated the feasibility of using siRNAs for controlling gene expression in hepatoma cell lines. Two anti-GFP siRNAs expressed by pSUPER vectors were able to efficiently and specifically knockdown GFP expression in several hepatoma cell lines (Hep G2, Huh 7, PLC/PRF5 and Hep 3B). Next, six siRNA expression vectors (designated as pSUPER/HBx1, pSUPER/HBx2, pSUPER/HBx3, pSUPER/TP, pSUPER/RT, pSUPER/ε) were constructed. Northern blot analysis was performed to evaluate the efficacy of degradation of HBV-specific transcripts after co-transfection of individual siRNA-expressing construct with pHBV-48 in Hep G2 cells. All six constructs expressed the siRNAs of interest. However, the effects on the HBV-specific transcripts varied. , pSUPER/εtargeting the conserved encapsidation sequences, failed to exert its effect. pSUPER/HBx3, pSUPER/TP, and pSUPER/RT were demonstrated to knockdown the HBV-specific transcripts specifically and efficiently in the co-transfection system.

Lastly, Hep G2 2.2.15 cell line, stably expressing HBV transcripts and producing HBV viral particles, was utilized to examine the effect of siRNAs on an established-infection system. Two approaches were utilized to overcome the poor transfection efficiency problem. One approach was enrichment of cells with the siRNA expression cassette through selection. Though siRNAs of interest were demonstrated to be expressed in selected Hep G2 2.2.15 cells, they showed limiting effects on HBV-specific transcripts. The other approach was to deliver the siRNA expression cassette using HIV-based lentiviral vectors. The transduction efficiency could be reached by 80 to 90 %, however, only 30 to 40 % of HBV-specific transcripts were knocked down. Taken together, our results raised an uncertainty in utilizing siRNAs for treatment of chronic HBV-carriers. Problems and solutions were discussed in the text.

Reference ( 127 ) 〈TOP〉

Allen, M. I., Deslauriers, M., Andrews, C. W., Tipples, G. A., Walters, K. A., Tyrrell, D. L., Brown, N., and Condreay, L. D. (1998). Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 27, 1670-1677.
連結：
Ambros, V. (2000). Control of developmental timing in Caenorhabditis elegans. Curr. Opin. Genet. Dev. 10, 428-433.
連結：
Barik, S. (2004). Control of nonsegmented negative-strand RNA virus replication by siRNA. Virus Res. 102, 27-35.
連結：
Bayer, M. E., Blumberg, B. S., and Werner, B. (1968). Particles associated with Australia antigen in the sera of patients with leukaemia, Down's Syndrome and hepatitis. Nature 218, 1057-1059.
連結：
Bisbal, C., Salehzada, T., Lebleu, B., and Bayard, B. (1989). Characterization of two murine (2'-5')(A)n-dependent endonucleases of different molecular mass. Eur. J. Biochem. 179, 595-602.
連結：

Altmetrics

〈TOP〉

E-mail ：

When an article is available to download, a notice will be sent to your mailbox address.

E-mail ：