以桿狀病毒作為人類 Endostatin::Angiostatin 蛋白長期表現載體並運用在癌症基因治療

Baculovirus-mediated Long Term Expression of Human Endostatin::Angiostatin Fusion Protein for Cancer Gene Therapy

施詠紳(Yung-Shen Shih)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/碩士(2010年)

摘要

血管新生作用與實體腫瘤進一步的發展有著密切關係，而且在許多與癌症相關的疾病中，抗血管新生的療法具有良好的抑癌效果。在本研究中，我們利用桿狀病毒載體將人類endostatin和angiostatin複合蛋白 (hEA) 基因直接送入腫瘤細胞中，並結合Sleeping Beauty (SB) 基因轉位系統來達到長期抑制腫瘤所誘發的血管新生。首先，我們建立三株重組桿狀病毒：Bac-hEA/w是以巨細胞病毒早期啟動子來驅動hEA的表現，並且在hEA基因下游插入土撥鼠肝炎病毒後轉錄調節序列來增強治療蛋白的表現量；Bac-SB-T2-hEA/w是在上述基因表現匣兩端接上反向重複序列，以完成SB轉位子的建構，並將SB轉位酶基因表現匣構築在同一株病毒；Bac-SB-T2-luc/w是將Bac-SB-T2-hEA/w中的hEA基因以luciferase基因取代之。由動物實驗結果可知在施打病毒後前期，桿狀病毒載體本身具有顯著的抗腫瘤效果，但不會減少腫瘤血管數；因為hEA的長期表現能有效抑制腫瘤所誘發的血管新生 (p < 0.05) ，因此能更有效地延緩腫瘤生長。

關鍵字

血管新生 ； endostatin ； angiostatin ； 桿狀病毒載體 ； Sleeping Beauty ； 後轉錄調節序列

並列摘要

Angiogenesis is tightly held together with the further development of solid tumors, and antiangiogenic therapy remains highly exceptional promise as an inhibitor in several cancer-related diseases. In this study, the gene encoding human endostatin and angiostain fusion protein (hEA) was directly delivered to tumor cells by baculoviral vectors combined with Sleeping Beauty (SB) transposon system for long-term inhibition of tumor-induced angiogenesis. We first constructed three recombinant baculoviruses: Bac-hEA/w harboring the hEA gene under the transcriptional control of cytomegalovirus immediate-early (CMV-IE) promoter and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) inserted into downstream of hEA gene to enhance the expression of therapeutic protein, Bac-SB-T2-hEA/w harboring the same gene expression cassette flanked by inverted repeats (IR) as a whole SB transposon and the expression cassette of SB tansposase (i.e. in cis), and Bac-SB-T2-luc/w with luciferase gene substituting for hEA gene of Bac-SB-T2-hEA/w. In animal studies, baculoviral vectors themselves showed marked anti-tumor effect without reduction of tumor vessel density in early days after virus injection, and long-term expression of hEA demonstrated decrease in tumor-induced angiogenesis (p < 0.05) and consequently retarded the tumor growth more effectively.

並列關鍵字

angiogenesis ； endostatin ； angiostatin ； baculoviral vector ； Sleeping Beauty ； post-transcriptional regulatory element

參考文獻

Abdollahi A, Hahnfeldt P, Maercker C, Grone HJ, Debus J, Ansorge W, Folkman J, Hlatky L, Huber PE. 2004. Endostatin's antiangiogenic signaling network. Mol Cell 13:649-63.

Abe T, Hemmi H, Miyamoto H, Moriishi K, Tamura S, Takaku H, Akira S, Matsuura Y. 2005. Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus. J Virol 79:2847-58.

Airenne KJ, Hiltunen MO, Turunen MP, Turunen AM, Laitinen OH, Kulomaa MS, Yla-Herttuala S. 2000. Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Ther 7:1499-504.

Boyce FM, Bucher NL. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93:2348-52.

Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL and others. 2000. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288:669-72.