臺灣博碩士論文加值系統

單純疹病毒(HSV)可以在像是愛滋病患或器官移植的免疫缺陷病人造成疾病。它可以被區分成兩個血清型，分別為HSV-1及HSV-2。因為這兩個血清型造成的病徵及對藥物的感受度是不同的，所以有分型的必要性。另外，在一些經常使用acyclovir(ACV)的病人，常會產生抗藥性病毒株。目前用來區分血清型及偵測藥物抗性的方法分別為免疫螢光染色法(IFA)及溶斑減少法(PRA)，而這兩個方法必須經人工方式肉眼觀察細胞病變(CPE)並且需耗費較長的時間。所以，我們希望能以指標細胞株來改善這些問題。
我們建立了兩株可以被單純疹病毒所感染的指標細胞株(BHK及CHO指標細胞株)。這兩株指標細胞株的建立方式為穩定轉染送入一質體含有兩個報導基因，分別為分泌性鹼性去磷酸(SEAP)及綠色螢光蛋白(GFP) 並且由單純疹病毒的啟動子所控制誘發。所使用的啟動子是來自單純疹病毒第二型的ICP10基因，它可以在感染早期被激活。所選用的兩個細胞株是幼倉鼠腎纖維細胞(BHK)及中華倉鼠卵巢細胞(CHO)，BHK細胞無論是HSV-1或HSV-2都可以感染，而CHO細胞則是只被HSV-2感染。
我們亦建立一套敏感和專一性極高的聚合鏈反應(PCR)方式來偵測並區分HSV血清型，以便可以確認指標細胞株的結果。以這PCR方式分析了140株檢體，預測陽性率及預測陰性率均為100%。
這兩株指標細胞株可以直接利用倒立螢光顯微鏡觀察，以區分血清型。我們共測試了35個已知血清型的臨床檢體，及和臨床實驗室進行雙盲試驗測試159株檢體，發現預測陽性率為100%及預測陰性率為99.3%，並發現有一株檢體其血清型難以判讀。BHK指標細胞株在被病毒感染後會產生高量的SEAP釋放於上清液中，所以可以直接以化學冷光方式偵測SEAP表現量。以104 溶斑形成單位 (PFU)以下的病毒量感染24小時後所表現出的SEAP表現量具有極佳的線性關係而且靈敏度可以偵測到1PFU。因為SEAP表現量代表溶斑數目，所以BHK指標細胞株可以發展成一套測試藥物感受度試驗的方法。以此方法測試了32株抗ACV的單純疹病毒株證明了這方法可以精準地確認病毒的藥物感受度。這個方法只需要10 PFU量的病毒在48小時後即可得知結果，然而傳統方法(PRA)則需要25 PFU量的病毒，且需耗時7天以上。
所以，這套指標細胞株的系統可以及時區分血清型，而且可以快速篩選病毒的藥物感受度，最後亦具有發展成自動化的潛力。

Herpes simplex virus (HSV) causes serious diseases in immunocompromised patients, such as those with AIDS or receiving transplants. There are two serotypes, HSV-1 and HSV-2, for which a correct typing is essential due to their distinct modes of pathogenesis and different responses to antiviral administration. Moreover, prolonged treatment with antiviral drug, with acyclovir (ACV) being the most frequently used, has led to the appearance of ACV-resistant mutants. Currently existing methods for serotyping and determination of antiviral resistance, namely the immunofluorescence assay and plaque reduction assay (PRA) respectively, have been suffered from subjective observation for the cytopathic effect (CPE) and lengthy turn-around time. Thus we sought to resolve the problems by developing indicator cell lines as follows.
Two indicator cell lines (BHK and CHO) that responds to infection by herpes simplex virus (HSV) were established. These cell lines were constructed by stable transfection of cells with an expression cassette that contains dual reporter genes, the secreted alkaline phosphatase (SEAP) and the green fluorescent protein (GFP), under the control of an HSV-inducible promoter. The promoter was from the upstream region of the HSV-2 ICP10 gene, an early gene promoter that is activated in the early phase of HSV infection. The baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines in the light of the findings that the former is permissive for both HSV-1 and -2, whereas the latter is only susceptible to infection by HSV-2.
A sensitive and specific PCR assay was designed for detection and typing of HSV to confirm the results assayed by the indicator lines. The PCR assay was used to analyze a total of 140 specimens, achieving 100% predictive positive value and 100% predictive negative value.
These indicator lines permitted differential diagnosis of HSV-1 and -2 by observation of GFP-positive cell under an inverted fluorescent microscope. The result was substantiated with 35 identified clinical specimens, as well as 159 unidentified specimens in the double-blind clinical evaluation. The data revealed an 100% predictive positive value and a 99.3% predictive negative value, with a specimen refractory to typing. In addition, the BHK indicator cell line secretes high level of the SEAP in the supernatants following infection, as measured by a chemiluminescence-based assay. The detection system is sensitive to an HSV titer as low as a single plaque-forming unit (PFU), with a linear range up to 104 PFU inoculum after infection for 24 h. The BHK indicator cell line was then utilized to develop an assay for determination of antiviral susceptibility given that the induced SEAP activity appeared to reflect the numbers of plaque. Evaluations of the stable line with 32 representative ACV-resistant HSV isolates demonstrated that their drug susceptibilities were accurately determined. The protocol required only 10 PFU virus input in a 48 h format , which otherwise takes 25 PFU inoculum for at least 7 days by a standard PRA.
In summary, this novel indicator cell system is a useful means for serotyping of HSV in real-time, as well as a rapid screening method for determination of anti-HSV susceptibilities, with the potential to development of an automated assay.

略字表 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·Ι
中文摘要 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 1
英文摘要 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3
第一章 緒論
第一節 單純疹病毒的基本特性 · · · · · · · · · · · · · · · · · ·5
第二節 研究緣由與目的 · · · · · · · · · · · · · · · · · · · · · · · ·15
第三節 實驗設計 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·19
第二章 研究材料與實驗方法
第一節 單純疹病毒的培養與定量 · · · · · · · · · · · · · · ·23
第二節 指標細胞株之建立· · · · · · · · · · · · · · · · · · · · · · · ·27
第三節 建立聚合連鎖反應(PCR)方法以區分單純疹
病毒血清型 · · · · · · · · · · ·39
第四節 指標細胞株之臨床試驗 · · · · · · · · · · · · · · · · · ·44
第五節 抗藥性單純診病毒株之分離及新穎
藥物感受度試驗之建立· · · · · · · · · · · · · 45
第三章 實驗結果
第一節 指標細胞株之建立 · · · · · · · · · · · · · · ·· · · · · 48
第二節 BHK＆CHO指標細胞株之特性測試 ·· · · · · 50
第三節 建立聚合連鎖反應(PCR)方法以區分
單純疹病毒血清型 · · · · · · · · · · ·53
第四節 指標細胞株之臨床試驗 ·· · · · · · · · · · · · · · · · ··55
第五節 以BHK指標細胞株進行藥物感受度試驗 · · ·57
第四章 討論 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · 58
第五章 表 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·67
第六章 圖 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·74
第七章 附錄 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·96
第八章 參考文獻 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·99
圖 表 目 錄

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