利用核磁共振造影分析蛋白質水解酶抑制劑 HAI-1在腫瘤生長與轉移中扮演之角色

Abstract

肝細胞生長因子/分散因子（HGF/ SF）是一具有多重功能之細胞激素，在癌症生長及轉移中扮演重要的角色。單鏈HGF 前驅物（pro-HGF）必須經由蛋白質水解酶之活化形成異二聚體，才具有生物活性。目前已知包括肝細胞生長因子活化因子（HGFA）、凝血因子 XIIa、membrane-type serine protease-1（MT-SP1）又稱為 matriptase、尿激酶血纖維蛋白溶酶原活化因子（uPA）及組織血纖維蛋白溶酶原活化因子（tPA）、hepsin 皆可活化 HGF，其中活性又以 HGFA 為最。第一型肝細胞生長因子活化因子抑制劑（HAI-1）為新發現之 Kunitz-type 絲胺酸蛋白質水解酶抑制劑，屬於第一型膜蛋白，透過其第一個 Kunitz domain（KD-1）而具有抑制 HGFA、matriptase、hepsin 及胰蛋白酶等絲胺酸蛋白質水解酶之活性，因此於癌症發展上扮演重要角色，在治療上具有相當的發展潛力。

本研究中利用基因重組技術，構築含 KD-1 之分泌型 HAI-1（sHAI-1）與人類 IgG Fc區域的融合蛋白表現質體 sHAI-1-Fc，以增加在活體內之存留時間。利用真核細胞表現系統生產 sHAI-1-Fc 條件培養液以進行功能性分析，驗證其具有抑制胰蛋白酶之活性與和 matriptase 結合之能力。更進一步利用流體動力學基因遞送法將此基因遞送至 C57BL/6 小鼠，並於血清中測得重組蛋白之表達，以利將來於活體內分析在腫瘤生長及轉移中扮演之角色。 另外，也利用 B16F10 小鼠黑色素細胞癌細胞於 C57BL/6 小鼠建立肝臟腫瘤注射及肝臟轉移之動物模式，並透過預先對癌細胞進行氧化鐵磁振顯影劑標定，結合核磁共振造影（MRI）進行活體腫瘤分析，這項初步研究可能提供癌症治療研究上，一種非侵入性動物模式。

Hepatocyte growth factor/ scatter factor (HGF/ SF) is a pleiotropic cytokine which plays important roles in tumor growth and metastasis. To generate biologically active HGF, the conversion of single-chain HGF precursor (pro-HGF) to a heterodimeric form is essential. To date, several proteases including HGF activator (HGFA), factor XIIa, membrane-type serine protease-1 (MT-SP1)/matriptase, urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and hepsin have been supposed to involve in the activation of HGF. Among them, the HGFA exhibits the most potent activity. HGFA inhibitor type 1 (HAI-1), a type I transmenbrane protein, is a novel Kunitz-type serine protease inhibitor that inhibits HGFA, matriptase, hepsin and trypsin through its first Kunitz domain (Kunitz domain 1, KD-1). Thus, HAI-1 may play an important role in cancer progression and possess strong therapeutic potential. In this study, a recombinant secreted HAI-1 (sHAI-1) with KD-1 domain, fused to human IgG Fc region (sHAI-1-Fc) was constructed. The Fc portion of this fusion protein may enhance protein stability in vivo. Eukaryotic expression system was employed to produce the recombinant protein. By using conditioned medium containing sHAI-1-Fc, functional assays were performed. We demonstrated that sHAI-1-Fc fusion protein could inhibit the activity of trypsin and also have ability to bind matriptase. Further, the naked plasmid was introduced to mice by hydrodynamics-based gene delivery method. The recombinant proteins could be detected in serum of mice after administration. Thus, we may analyze the roles of sHAI-1 in tumor growth and metastasis in vivo in the future. In addition, experimental models for intrahepatic tumor inoculation and liver metastasis were set up by inoculating B16F10 mouse melanoma cell line into C57BL/6 mice. Furthermore, by prelabeling the cancer cells with iron oxide magnetic resonance contrast agent, we can monitor the tumor growth and cellular distribution by MRI detection. This preliminary study may provide a non-invasive tool for studies of cancer therapy.