PILP-1、PILP-2及PILP-3為台灣雨傘節 (Bungarus multicinctus) 蛇毒中，成功篩選出的三個類蛋白酶抑制劑 (Protease inhibitor-like protein, PILP)，其結構與Kunitz-type蛋白酶抑制劑相似。 本研究為暸解PILPs是否具有抑制間質性金屬蛋白酶-2活性的能力。 藉由螢光受質探討間質性金屬蛋白酶-2活性的試驗中發現，PILP-3不同於PILP-1及PILP-2，具有抑制間質性金屬蛋白酶-2的活性，此外，在細胞的移動及侵犯的試驗中得知，PILP-3亦抑制人類神經母細胞瘤的移動和侵犯。 由Pull-down 及Dot blotting的試驗中發現PILP-3直接結合間質性金屬蛋白酶-2。 欲進一步了解PILP-3抑制間質性金屬蛋白酶-2之可能結合的功能性區域，利用與PILP-3序列相似度極高的PILP-2建構兩組突變，分別將PILP-3的氮端或碳端置換成相對應之PILP-2序列，即為PILP-3N及PILP-3C。 相較於PILP-3及PILP-3的氮端突變，PILP-3的碳端突變僅些微抑制間質性金屬蛋白酶-2的活性，並且對於結合間質性金屬蛋白酶-2的親合性也顯著地減弱。 綜合上述研究結果，PILP-3 或許可作為模板，設計專一性結合於間質性金屬蛋白酶-2之抑制劑。

Three protease inhibitor-like proteins (PILPs) identified from Bungarus multicinctus genome are structurally homologous with Kunitz-type proteinase inhibitor. The goal of the present study is to explore whether PILPs exhibit an inhibitory action on matrix metalloproteinase 2 (MMP-2) activity. Unlike PILP-1 and PILP-2, PILP-3 was found to inhibit MMP-2 activity as evidenced by specific substrate assay. Moreover, in vitro migration and invasion assays, and wound-healing assay showed that PILP-3 suppressed the migration and invasion of human neuroblastoma SK-N-SH cells. Pull-down assay and dot blotting-binding assay proved an interaction between PILP-3 and MMP-2. Nevertheless, PILP-3 did not affect either expression or secretion of MMP-2 in SK-N-SH cells. In terms of highly structural similarity between PILP-2 and PILP-3, two chimeric mutants in which amino acids at N-terminus and C-terminus of PILP-3 were substituted by those of PILP-2 were prepared. In contrast to N-terminus chimera, C-terminus mutant of PILP-3 was unable to inhibit MMP-2 activity and showed a reduction in binding with MMP-2. Taken together, our data suggest that PILP-3 may be a useful template for rational designing pharmaceutical agent in inhibiting MMP-2 activity.

目錄 ………………………………………………………… 1
中文摘要 …………………………………………………… 2
英文摘要 …………………………………………………… 3
縮寫表 ……………………………………………………… 4
緒論 ………………………………………………………… 5
材料 ………………………………………………………… 11
實驗方法 …………………………………………………… 14
結果 ………………………………………………………… 28
討論 ………………………………………………………… 32
圖表 ………………………………………………………… 37
參考文獻 …………………………………………………… 57

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