台灣欒樹胰蛋白酶抑制劑抑制大腸直腸癌細胞生長之分析

Inhibition of Growth in Human Colon Adenocarcinoma Cells by Koelreuteria formosana trypsin inhibitor

由台灣欒樹（Koelreuteria formosana）種子純化出胰蛋白酶抑制劑（Koelreuteria formosana trypsin inhibitor），簡稱KFTI，其分子量約19 kDa，由12.3 kDa及6.7 kDa兩條多胜鏈以雙硫鍵連接所組成，屬於Kunitz type蛋白酶抑制劑。KFTI對大腸直腸癌細胞（HT-29）生長之影響以MTT試驗觀察其細胞存活率，實驗結果得知其有效濃度為10μM且反應72小時才有明顯作用，之後利用流式細胞儀以annexin-V染色觀察發現在10μM 72小時的M2（凋亡細胞）有明顯上升（p<0.05），顯示KFTI在10μM具有使HT-29細胞產生細胞凋亡的能力。以西方墨點試驗檢測細胞凋亡（apoptosis）相關蛋白如caspase-3、PARP、Bax等，發現KFTI會使得HT-29生長抑制是因誘導細胞走向細胞凋亡的路徑。

A trypsin inhibitor (KFTI) was purified from the seed of Koelreuteria formosana. 15% SDS-PAGE analysis of KFTI revealed that it is constituted by two polypeptide chains (A-chain, Mr 12.3 kDa and B-chain, Mr 6.7 kDa), the molecular weight being ~19 kDa. The KFTI was found to be a thermostable Kunitz type protease inhibitors. KFTI is also conducted the experiment on the growth impact on cancerous cell of large intestine and rectum (HT-29). For instance, MTT is used to observe the survival rate of its cell, showing that the effective concentration has to be 10μM with 72 hours response before it can effect prominently. Afterward, flow cytometer will use annexin-V dye to observe if there is any prominent increase (p<0.05) with M2 (apoptosis) with 10μM in 72 hours, showing that KFTI has the capability to render HT-29 cell to generate apoptosis within in 10μM. Furthermore, western blot is exploited to test and examine related protein as caspase-3、PARP, and Bax regarding apoptosis, and it is found that the death results of cell is heading towards the path of apoptosis.