利用運輸通道蛋白建構報導基因系統與其相關應用

Abstract

報導基因開啟了生物學研究的一個新的世代，使得發育、癌症與分子生物學以及細胞治療有著突破性的進展。活體細胞能利用螢光顯微鏡、非侵入式活體影像系統、正子攝影與磁核造影來進行觀察。現今的活體細胞追蹤仍有限制，譬如說自體螢光干擾、影像擷取深度與報導基因的強弱。因此，我們著重於新的報導基因的開發。首先我們選出Apical sodium-dependent bile acid cotransporter (ASBT)、sodium taurocholate co-transporting polypeptide (NTCP)與organic anion-transporting polypeptides 1B3 (OATP1B3)來評估作為報導基因的可行性，因為這些基因所表現的蛋白主要負責物質的交換。並且，NCTP與OATP1B3能運輸近遠紅外光波段的靛氰綠(ICG)與核磁造影顯影劑的卜爾邁斯(Primovist) 。此外，因為ASBT與NCTP同屬於SLC10A家族所以ASBT也可能作為一個報導基因。因此，我們選擇ASBT、NTCP與OATP1B3來測試其作為報導基因的可行性。從體外細胞實驗結果可得知NTCP有最好的ICG運輸能力，OATP1B3次之，而ASBT最弱。接著我們保留NTCP與OATP1B3作進一步活體的細胞影像追蹤。出乎意料之外，OATP1B3攝取靛氰綠的能力比NCTP來的好並且從我們的體外與活體測試結果發現NCTP並無法攝取卜爾邁斯。整體實驗結果顯示OATP1B3為一個多功能且卓越的報導基因應用。 基於上述結論，我們進一步測試在癌症細胞追蹤與細胞治療上OATP1B3作為報導基因的可行性。帶有OATP1B3的HT-1080細胞腫瘤在注射靛氰綠後的九十六小時依然能在非侵入式影像系統中偵測出靛氰綠訊號，並且打入卜爾邁斯後也能利用核磁造影進行追蹤。我們更進一步將OATP1B3轉染至具有分化成胰島相似細胞能力的PANC-1上，而帶有OATP1B3的PANC-1腫瘤也能利用核磁造影進行追蹤。由此，我們對於OATP1B3作為報導基因應用於癌症生物學與細胞治療上更具有信心。 近來報導指出NTCP為B型與D型肝炎病毒的入口之一。因此，我們嘗試利用帶有NTCP的細胞建構藥物篩選平台。有潛力的治療藥物會與靛氰綠競爭NTCP這個入口所以可藉由競爭結果得知是否可作為治療藥物。若是靛氰綠訊號低者則表示其作為治療肝炎的效果可能較好。藉由這樣的特性，我們成功建構帶有NTCP細胞的肝炎藥物篩選平台。 綜合上述，OATP1B3可作為可信賴的報導基因應用於癌症生物學與細胞治療上；而NTCP則可以作為肝炎藥物篩選平台的應用。

Reporter genes as a tracking tool open a new window for the investigation of biology and make huge progress in development, tumor, molecular biology, and cell therapy. Cells can be visualized through fluorescent microscopy, noninvasive in vivo imaging system (IVIS), positron emission tomography (PET), magnetic resonance imaging (MRI), etc. Currently, in vivo cell tracking still has a limitation, such as the autofluorescence, the penetration depth, and the intensity of the reporter. Thus, we focus on the new candidates as reporter genes. Apical sodium-dependent bile acid cotransporter (ASBT), sodium taurocholate co-transporting polypeptide (NTCP), and organic anion-transporting polypeptides 1B3 (OATP1B3) are what we address since they are membrane transporters responding for the exchange of many molecules. Besides, we predict NTCP and OATP1B3 could be applicable reporter genes because they can transport indocyanine green, near-infrared fluorophore, and Primovist, MR contrast. Furthermore, ASBT could serve as a reporter gene since ASBT and NTCP are derived from the same family- the SLC10A transporter gene family. Thus, we testify the feasibility of ASBT, NTCP, and OATP1B3 as reporter genes. The highest transportability for ICG was NCTP, the following was OATP1B3, and ASBT was the last in vitro. We remained NCTP and OATP1B3 for in vivo investigation. Unexpectedly, OATP1B3 had better transportability for ICG than NTCP in vivo. Moreover, it seemed that NTCP couldn’t intake Primovist in our in vitro and in vivo experiments. In summary, OATP1B3 was a superior reporter gene. According to this concept, we further testified OATP1B3 as a reporter gene for tumor cell tracking and cell therapy. The xenograft of HT-1080 carrying OATP1B3 could be traced at least 96 h using IVIS after ICG administration and could be identified using MRI after Primovist injection. We further transduced OATP1B3 into PANC-1 cells which can be differentiated as pancreatic islet-like cells. The cellular functions of PANC-1 weren’t affected after OATP1B3 transduction. Moreover, the xenograft of PANC-1 carrying OATP1B3 could be visualized with MRI. We had more confidence about OATP1B3 as a reliable reporter gene for cell tracking in the fields of tumor biology and cell therapy. We attempted to establish a drug screening platform using NTCP-expressing cells since NTCP has been reported that it is an entry for hepatitis virus B and D (HBV and HDV). The candidate drugs for HBV or HDV could be selected if the ICG intensity was decreased because they compete for the same entry-NTCP. The drug screening platform using NTCP-expressing cells was established successfully through the competition between ICG and the candidate drugs. In conclusion, OATP1B3 could serve as a reliable reporter gene and applied for tumor biology and cell therapy. NTCP could use for HBV and HDV drug selection.