非侵入性造影在癌症治療及抗癌藥物研發篩選模式之建立
Non-invasive imaging of gene expression for cancer gene therapy and discovery of anti-cancer drug
中文摘要
運用泡疹病毒第一型所攜帶的胸線嘧啶激酶(herpes simplex virus -1 thymidine
kinase，HSV1-tk)合併正子電腦斷層掃描(positron emission tomography，PET)分子核 子醫學造影圖譜模式的建立，對於往後發展基因治療及研發新藥上是一項新的技術。肝癌是東 南亞國家年發病率最高、復發率亦高的惡性腫瘤之一。近年來對於肝癌的診斷有很大的進步，治 療方式，大部分都以外科切除手術、局部酒精、化學藥治療劑注射、動脈拴篩及傳統放射治療為 主，但往往病人預後通常不佳。因此抗癌藥物的研發及發展新的治療模式，是當務之急。
本實驗中，利用已經有轉植HSV-tk 基因的 IMEA 7R.1 肝癌細胞株(IMEA 7R.1-tk)，做了初步 的測試；在ex vivo 實驗中我們可以發現在加入前趨藥物 GCV (prodrug)三天後，IMEA 7R.1-tk 在濃度 10μM 時，細胞存活率只剩下 5%，而對於沒有基因轉植的 IMEA 7R.1 細胞株，
卻沒有影響。同樣的在分子核醫造影上，HSV1-tk 可當作一個報導基因(reporter gene)，可將 放射性同位素標識的標記受質131I-FIAU、18F-FHBG(胸腺嘧啶-thymidine 的衍生物)磷酸化 而聚積在細胞中。經γ-counter 計讀細胞放射活性累積顯示，IMEA 7R.1-tk 的確能將 131I- FIAU、18F-FHBG 聚積在細胞中。由 microPET 及 SPECT 我們也順利追蹤到腫瘤存在於活體中 的位置及治療成效。分子核醫造影系統模式之建立，可以追蹤基因產物在體內表現的分佈、時間 及數量，以及確認基因治療之效果。在臨床前抗癌藥物的研發篩選，也可當作一個positive control。
為了增加基因治療之可行性及改進病毒載體之不良性，本實驗又另外建立一套慢病毒
(lentivirus)感染細胞的新基因轉植系統，能將基因轉植到分裂及不分裂細胞的進行治療。慢病 毒載體(lentiviral vector)是一種人類免疫缺乏症候群病毒(human immunodeficiency virus -1，HIV-1)的載體系統，利用改良型的 lentivirus 作為哺乳類動物細胞基因轉植的載體 (vector)，不僅能大大提高病毒載體的安全性，還能減少發炎及免疫排斥反應。其與一般轉染 (transfection)如反轉錄病毒(retrovirus)、腺病毒(adenovirus) ，不同的是 lentivirus 能感 染分裂(dividing)及不分裂(nondividing)的細胞，其所攜帶的基因是直接嵌入標的細胞的染色 體中表現，而非短暫存在於細胞中，集合了前兩病毒之優點。第一階段，是生產具有高效價 (high titer)之 lentivirus。我們利用 packaging helper plasmid (pHP) 提供病毒結構 gag/pol、env、TAT、REV，並將攜帶病毒包裝訊息(psi，ψ)及綠色螢光蛋白(green fluorescent protein，GFP)基因的病毒載體 pTY-EFGFP DNA，分別接上帶有不同 promoter 的泡疹病毒胸腺嘧啶激酶(HSV1-tk)基因；如 AFP-tk 具有肝癌組織特異性的 α- Fetoprotein 的啟動子，AFP-tk)及 P-tk(泡疹病毒胸腺嘧啶激酶本身的啟動子)。Env plasmid(VSV-G)可製造病毒外殼醣蛋白，取代 HIV 的 env，產生假型慢病毒載體
(pseudotype lentivirl vectors)，感染更廣泛的細胞。利用共同感染(co-transfection)的方 式，將pHP 、pTY-EFGFP(含 P-tk 及 AFP-tk)及 Env plasmid (pHEF-VSV-G)三種 plasmid，
同時轉染human rhabdomyosarcoma cells，以產生病毒。將所生產之病毒感染 HepG2(人
類肝癌細胞)，再以流式細胞儀測定感染率(即細胞表達螢光蛋白之比率)，並收集含有螢光的細 胞，此細胞即同時存在有tk gene 的細胞。
細胞分析實驗，發現具有HSV1-tk gene 的細胞相較於不含者細胞，對於 nucleotide analogue 如 131I-FIAU 核醫藥物，具有差異性的吸收(differential uptake)。在基因治療核 醫分子造影上，可作為報導基因(report gene)。含 HSV1-tk gene 的 HepG2 的吸收情形，也 證實了，與5'長端重複(LTR)中的促進者(promoter)方向相反(revrese orientation) 的HSV-tk 基因(內部基因)，比正向(forward orientation)表現的更好。此肝癌組織特異性 AFP-tk 及 P-tk 基因，將來分別可專一性的治療肝癌及其他癌症，並且運用於核醫照影上。
英文摘要
Clinical monitoring of gene targeting, gene transfer, and gene expression requires the appropriate combination of “reporter gene”(herpes simplex virus -1 thymidine kinase, HSV1-tk) and “marker substrate”(131I-FIAU,18F-FHBG) forming stable metabolites trapped in cells for the gene expression. A variety of imaging technology is being investigated as tools for studying gene expression for cancer gene therapy leading to the discovery of anti-cancer drugs. Hepatoma hepatocellular carcinoma (HCC) is one of the major human malignancies with poor prognosis and high recurrence in eastern Asia. Despite the development of novel treatment modalities for HCC, such as intra- arterial chemo-embolization, percutaneous intratumoral ethanol injection,
radiotherapy and surgical resection, the prognosis of HCC still remains relatively poor. Therefore, new treatments and discoveries such as novel anti-cancer drug for HCC are very important.
In respect to cellular uptake, the accumulation of 131I-FIAU/18F-FHBG in IMEA 7R.1-tk+ cells is much higher than that of IMEA 7R.1-tk- cells after incubating for 4 h at 37℃. In in vivo studies, SPECT and microPET images after intravenous
injection of 131-FIAU and 18F-FHBG, respectively clearly reflect the relatively high level of radioactivities retained in the transduced hepatoma cells (IMEA 7R.1-tk+).
Transduction of IMEA 7R.1 HCC with HSV1-tk gene followed by treatment with the prodrug ganciclovir (GCV) has been shown to induce apoptosis. After 6 days of daily treatment with GCV, the imaging of the same HSV1-tk+ tumor-bearing mice showed obvious tumor regression. The results will facilitate the monitoring and evaluation of gene therapy in human subjects by defining the location, magnitude, and persistence of gene expression over time.
Lentiviral vectors are human immunodeficiency virus-1(HIV-1) based retroviral vectors with many advantages for gene therapy: the ability to infect both nondividing and terminally differentiated cells , the occurrence of long-term transgene expression, and the lower induction of an inflammatory immune response.
To establish a Lentivirus gene delivery system, vesicular stomatitis virus
glycoprotein(VSV-G)-pseudotyped HIV-1 based lentiviral vector was used to introduce transgenes with a broad target spectrum. In this study, we constructed two hybrid gene: one consisting of herpes simplex virus thymidine kinase(HSV1-tk) gene drivin by the 0.3kb humanα-fetoprotein(AFP) promoter, and the other under the control of its own promoter.(P-tk).
Then the hybrids were inserted into pTY-EGFP vectors (transducing plasmid), namely, pTY-EGFP-Ptk and pTY-EGFP-AFPtk gene. A three-plasmid expression system including a packaging defective helper construct (pHP), a plasmid coding for a heterologous (VSV-G)envelope protein and the transducing vector(pTY-EGFP-Ptk/
pTY-EGFP-AFtk), was used to generate pseudotyped HIV-1 particles by transient transfection of human rhabdomyosarcoma cells. The virus was used to deliver both genes into human hepatoma cell lines in vitro and in vivo.
The two reporter genes can be further used in our studies: HSV1-tk gene can be used with non-invasive PET imaging, while green fluorescence protein , EGFP, can be a marker used to select successfully transfected pTY-EGFP-Ptk and pTY-EGFP -AFPtk with FACS or detect by fluorescence microscopy. The results demonstrate that lentivirus-based vectors successfully transduce human hepatoma in vitro. By cellular accumulation assay, we prove that [pTY-EGFP-Ptk/ AFPtk]R infected HepG2 human hepatomacells was much higher than that in [pTY-EGFP-Ptk/ AFPtk] infected.
