利用超音波刺激肌肉來進行抗血管生成因子轉移以強化治療遠端腫瘤 – 動物實驗

Abstract

抗血管生成的基因治療主要是藉由抑制腫瘤內部血管新生來達到抑制腫瘤生長之治療方式。近幾年，因肝癌具有高新生血管之能力，故被認為適合用抗新生血管之方法來進行治療。另一方面，超音波被認為是一種新型且可有效率地將基因局部導引進入標定之腫瘤。本研究目的是研發超音波基因轉移技術，以抑制原位肝腫瘤，腦腫瘤和肺腫瘤的生長。 此論文主要分成兩個部分（1）為展現以肌肉組織來製造基因產物之獨特優點，將在兩個腫瘤模型和肌肉組織中，藉不同顯影劑濃度及超音波照射時間以比較其基因表現。並研究最佳的聲學參數以獲得高效率之基因轉殖。（2）需發展能順利將治療用基因送入目標細胞之策略。因此，我們必須在動物模型上測試發展多次性超音波基因轉殖的方式，利用超音波在肌肉來傳送以攜帶抗血管新生的因子，血管內皮抑制素（ED）或者鈣網蛋白（CRT），進而抑制遠端原位肝腫瘤，腦腫瘤和肺腫瘤之生長。 本研究第一部分乃在了解藉著改變顯影劑濃度和超音波照射時間對於超音波基因轉殖效率在原位肝腫瘤，腫瘤皮下和肌肉組織之影響；結果顯示肌肉組織在超音波基因治療上是個容易產生大量基因產物的位置，其效率遠高於利用肝細胞或肝腫瘤。其次，本文評估了利用肌肉進行超音波轉移內源性血管生成抑制因子來治療遠端之肝腫瘤、腦腫瘤，與肺腫瘤。結果顯示利用超音波配合隔週肌肉注射是一種有效的非病毒性技術，以抑制遠端腫瘤生長。此外，結合抗血管生成療法與免疫療法或傳統化療也有很大的潛力，可產生協同抗腫瘤之作用。而我們從組織免疫化學染色也可發現合併組合明顯有較多T細胞產生，降低腫瘤內的血管數目，以及增加腫瘤細胞凋亡。另外，結合多種抗血管生成因子可被用來克服腫瘤在抗血管生成治療上所產生之抗藥性。

Antiangiogenic gene therapy is a promising approach to inhibit neovascularization via angiogenesis, the growth of new blood vessels from preexisting ones. In recent years, hepatocellular carcinoma (HCC) is considered as one of the suitable targets for anti-angiogenic approaches due to its highly neovascularization. At the same time, ultrasound is believed to be a novel and effective tool to locally deliver gene into target tumors. The objective of this research work is to develop ultrasound-mediated gene transfer strategies to suppress the growth of the inoculated orthotopic liver, brain, and lung tumors. In this thesis, two major parts of work are included: (1) To demonstrate the unique advantages of using muscle as a manufacturer of gene products, the gene expression in two tumor models and muscle tissue was compared at different contrast agent concentrations and ultrasound exposure durations. Optimal acoustic parameters for efficient gene transfection were also studied. (2) There always remains a need for developing successful strategies for delivering therapeutic genes into target cells. Thus, the strategies of repeated ultrasound mediated gene transfection were tested in animal models. We employed genes encoded with an anti-angiogenic factor, endostatin (ED) or calreticulin (CRT), to suppress the growth of distal inoculated orthotopic liver, brain, and lung tumors after transfected in muscle by ultrasound. The first part of this thesis was to clarify the impacts of contrast agent concentrations and ultrasound exposure period on the efficiencies of ultrasound-facilitated gene transfection at orthotopic liver tumor, subcutaneous tumor, and muscle tissue. Results indicated that a large amount of gene products were produced at muscular tissue via ultrasound gene transfection. The efficiency was much higher than that at hepatocytes or liver tumor. Next, an ultrasound muscular transfection of endogenous angiogenesis inhibitors was evaluated to suppress the growth of distal liver, brain, and lung tumors. Results suggested that ultrasound and weekly muscular transfection may serve as an effective nonviral technology to inhibit the growth of distant orthotopic tumors. In addition, combining antiangiogenic therapy with immunotherapy or conventional chemotherapy may have great potential to induce synergistic antitumor effects. Immunohistochemical analysis also revealed that combination treatment recruited significantly higher number of T cells to infiltrate tumor tissue, reduced tumor vascularization, and enhanced apoptosis in the tumor region. Furthermore, simultaneous use of multiple antiangiogenic factors may be used to overcome the resistance of tumor to the antiangiogenic therapy.