| 摘要: | 基因治療在近年來被應用廣泛於癌症治療中,非病毒VISA載體系統在最近被洪明奇團隊研發來增強標靶癌症細胞的能力,而在本篇論文,我們試著將絕緣子嵌入VISA載體系統去改善啟動子的互相干擾。首先我們選殖不同片段的絕緣子嵌入到VISA載體系統裡(Survivin 啟動子和G5E4T 啟動子間),並利用啟動子活性分析實驗來選擇最佳的絕緣子片段(包括1.2 kb,2x250 bp, 250 bp)來進行之後實驗,同時絕緣子的方向性也是我們考量的因素之一。由啟動子活性分析實驗我們得知反向絕緣子比起正向絕緣子還來得佳,且又以1.2 kb全長又更好。接著我們試著將VISA載體系統的螢光基因置換成具有毒殺細胞效果的trail基因,來測試絕緣子VISA載體系統的毒殺效果,實驗結果顯示在口腔癌和肺癌中利用冷光酵素活性的測定測得酵素活性比本身VISA載體系統高。接著我們將比較絕緣子VISA載體系統和VISA載體系統對於肺癌跟口腔癌細胞輻射線的增敏性,在2 Gy時可看到絕緣子VISA載體系統的確有增加肺癌跟口腔癌細胞輻射線的增敏性以及利用WST-1細胞增殖試劑來看絕緣子VISA載體系統對於抑制細胞增生是否有效果,由實驗結果發現的確對於口腔癌和肺癌都有明顯的抑制細胞增生，以及我們還看了一些相關的細胞凋亡的蛋白質(Cleaved-Caspase-8,9,3,PARP)。由我們的研究結果中可以發現，絕緣子的確可以增強VISA載體系統的基因表現並且還能增加VISA載體系統對於肺癌跟口腔癌細胞輻射治療的增敏性。絕緣子VISA載體系統也許可以成為新的基因治療的載體。
Recently, a VISA vector has been developed to enhance the transcriptional activity of therapeutic gene without impairing tumor-targeting efficacy. Herein, we further improve the effectiveness of gene expression of VISA vector by incorporating insulator elements between two adjacent promoters to minimize the promoter interference. To select the optimal length of the cHS4 insulator, we tested various survivin-VISA plasmids by inserting 1.2 kb full length, 250 bp core element, and two tandem repeats of core element of cHS4 insulator between survivin promoter and G5E4T promoter in forward (+) and reverse (-) orientation. The transgene expression among constructs were determined and compared in survivin-expressing CL1-5 human lung cancer cell line and a survivin-null normal epithelial 184A1 cell line. One copy of core element either within the proximal 250 bp or full length of cHS4 insulator displays no significant increased luciferase activity by survivin-VISA vector in the forward orientation. However, around two-fold higher reporter expression was observed in the two tandem repeats of core element than survivin-VISA vector. In contrast, reverse orientation of insulator fragment in the survivin-VISA vector enhanced luciferase activity about 2-3 folds regardless of the region of core element in CL1-5 cells but remained no effect on 184A1 cells, this result also shown in oral cancer. We then determine cell cytotoxicity by construct trail into survivin-VISA vector. Insulated-survivin-VISA-trail and survivin-VISA-trail were determined the in vitro cell killing effect in both lung and oral cancer, insulated-survivin-VISA-trail was shown increased in vitro cell killing effect compared to survivin-VISA-trail. Combine insulated-survivin-VISA trail and radiotherapy could also sensitize CL1-5 and SAS oral cancer cell. Our data presents for the first time that insulator can be incorporated into dual-promoter based vectors to enhance transgene production from each expression cassette in the same construct. We are currently evaluating this newly developed VISA system into targeted gene therapy for its therapeutic potential. |
