- 學位論文
利用可誘導式 tet-on-BikDD 調控系統剔除斑馬魚 心肌細胞
Conditional cell ablation in the cardiomyocytes of zebrafish using the tet-on-BikDD system
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摘要
細胞凋亡 (apoptosis) 所造成的細胞死亡，在目前癌症的基因療法上扮演很重要的角色。其中，細胞凋亡前驅 (pro-apoptotic) 蛋白 Bcl2 interacting killer (Bik) 的突變株 Bik, T33D/ S35D (BikDD) 已經證實在胰臟癌、肺癌等癌細胞的剔除上有很好的效果。因此，本實驗希望擴展 BikDD 誘導細胞凋亡的應用性，使其不僅是剔除細胞，且能夠經由誘導的方式調控特定時空的細胞凋亡，進而發展出能夠研究在胚胎發育中，特定細胞的功能與命運和組織與器官再生機制的調控性細胞剔除系統。首先，在斑馬魚中建立以細胞凋亡前趨變種蛋白 BikDD 結合誘導性的 tetracycline-controlled transcriptional activation system (tet-on) 調控系的基因轉殖斑馬魚 ZCtBIBCM 品系。此轉殖品系使用斑馬魚心肌細胞 (cardiomyocytes) 專一性啟動子 zebrafish cardiac myosin light chain (cmlc2 ) promoter，開啟 tet-on 調控系統上游轉錄活化因子 rtTA-M2 在斑馬魚心肌細胞中表現；接著，在 rtTA-M2 與 doxycycline (Dox) 結合後，活化態的 rtTA-M2 會活化 tet-on 調控系統下游的 tetO min CMV 雙向啟動子，開啟 BikDD 的表現，並誘使細胞走向凋亡的途徑。接著，在經 Dox 處理的轉殖品系 ZCtBIBCM 胚胎中，觀察到心臟萎縮、且外型呈細長管狀以及綠螢光蛋白 (green fluorescent protein, GFP) 標定的心肌細胞減少 70 % 等心臟缺失。進一步，使用全胚胎原位雜合實驗 (whole mount in-situ hybridization , WISH) 也可以偵測到 tet-on 調控系統下游所開啟的 BikDD 在心肌細胞中大量表現，並且以 terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay) 證實心肌細胞減少是由 BikDD 所誘發的心肌細胞凋亡所造成的結果。此外，根據在誘導物質 Dox 移除後， 51.2 % (43 / 84) 心臟已受損的斑馬魚胚胎，可以恢復成正常的心臟形態與功能，證實 tet-on-BikDD 細胞剔除系統具有可逆性。因此，本研究成功地在斑馬魚心肌細胞中，建立可以在空間上與時序上調控，並且具組織專一性與可逆性的 tet-on-BikDD 細胞剔除系統。期望 tet-on-BikDD 細胞剔除系統能應用在胚胎發育與再生醫學的研究上。
並列摘要
Apoptosis, the programed cell death, plays an critical role in cancer therapy currently. BikDD, a T33D/ S35D mutant of pro-apoptotic protein Bik, has been used to eliminate cancer cells in pancreatic and lung cancers. Therefore, we hope to expand the application of BikDD to not only in cancer therapy but also in the researches of embryonic development and regenerative medicine. Fisrt, we designed a temporally and spatially controlled tissue-specific cell ablation system, combining BikDD and inducible tetracycline-controlled transcriptional activation (tet-on) system, named tet-on-BikDD cell ablation system. Then, a zebrafish transgenic line ZCtBIBCM, which possessed tet-on-BikDD cell ablation system, was established. In ZCtBIBCM embryos, rtTA-M2 was driven by zebrafish cardiac myosin light chain2 (cmlc2) promoter, and after rtTA-M2 combined with doxycycline (Dox), the active rtTA-M2 activated tetO min CMV promoter droved BikDD in cardiomyocytes specifically. After Dox treated, tubular and collapsed hearts showed, and the GFP positive cardiomyocytes was decreased 70 % in ZCtBIBCM embryos. Furthermore, In whole mount in-situ hybridization (WISH), BikDD was detected in cardiomyocytes specifically. And the reduction of cardiomyocytes resulted from cell apoptosis induced by BikDD was proved in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, we have observed 51.2 % (43 / 84 ) of embryos with injured hearts can recover after Dox removed, that meant tet-on-BikDD cell ablation system was reversible. These evidences indicated that the tet-on-BikDD cell ablation system is effective for tissue-specifically, temporally and spatially controlled cell ablation in zebrafish, thereby might be a potential genetic tool to analyze tissue interactions in embryonic development as well as the mechanisms of regeneration.
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