根據許多研究指出口腔鱗狀細胞癌(Oral squamous cell carcinoma, OSCC)的轉移與基質金屬蛋白酶-9(Matrix metalloproteinase-9, MMP-9)的表現有高度相關，此外，在臨床上，病人身上的基質金屬蛋白酶高度表現會促進癌細胞的侵襲與遷移。

在實驗室的過去研究裡，指出在口腔鱗狀細胞癌之細胞株中發現到乙型轉型生長因子-1(Transforming Growth Factor-β1)會經由Smad3/Smad4蛋白形成三聚體，並且進入細胞核且誘導MMP-9基因的表現。另外發現到四環黴素(Tetracycline)類型的強力黴素(Doxycycline, Dox)會經由Smad4蛋白來抑制TGF-β引起的MMP-9基因表現、經由電泳流動遷移分析(electrophoresis mobility shift analysis, EMSA)證實來自管柱純化得到的Smad4蛋白具備與DNA結合的功能，最後經由表面電漿共振(surface plasma resonance, SPR)證實純化後的Smad4蛋白會與Dox有顯著的交互作用。

我們想更進一步地觀察Smad4蛋白與Dox的共結晶，大量的Smad4蛋白是必需的。然而，在使用凝血酶(thrombin)切割GST-Smad4蛋白時，面臨結構上的立體障礙，因此無法在適當的溫度條件下，拿掉GST-tag (glutathione-S-transferase)。即便提升溫度到37℃與增加用量來改善切割效率，但GST-Smad4蛋白不可避免地會受到嚴酷溫度(37℃)反應而引發降解，使得蛋白回收產率不佳。

因此，為了凝血酶(38kDa)能克服GST(26kDa)與Smad4(61kDa)之間立體障礙，更能夠有效的在和條件下分離GST與Smad4蛋白，我們在表現質體上Smad4基因與GST基因之間，利用DNA cloning方式嵌入一小段Linker，解決立體障礙的問題。

在本實驗中，比起過去的研究，我們成功地優化了表現載體、立體障礙、破菌方法、管柱選擇、凝血酶的條件優化，來克服過去研究中產率不佳的問題。

然而，在Smad4的功能分析上，我們利用電泳流動遷移分析實驗，觀察Smad4蛋白與Smad-binding element之間的交互作用，但沒有觀察到交互作用的現象。因此，未來仍需要用SPR實驗觀察Smad4蛋白與Doxycycline來證實本研究Smad4的功能性。

Head and Neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer-related death in western societies and responsible for half a million deaths worldwide per year. Oral squamous cell carcinoma (OSCC) is a part of HNSCC. Then tumor metastasis is still the primary cause of death in cancer patients. In lots of studies, they indicated that OSCC cancer metastasis is highly relevant to matrix metalloproteinase-9 (MMP-9).

In our previous study, there was a data indicating that transforming growth factor-β (TGF-β) induced MMP-9 expression through Samd4 protein in OSCC cell line. Besides, we also acquired the purified Smad4 protein, observed the interaction between Smad4 protein and doxycycline via surface plasma resonance (SPR) biosensor and confirmed the function of Smad4 protein by electrophoresis mobility shift analysis (EMSA). However, in the process of purified Smad4 protein, we faced the challenge of the structural steric-hindrance on the cleavage of GST-Smad4 by thrombin. Despite of raising the temperature to improve cleaved efficiency at 37℃, it could not still escape from temperature-induced protein degradation so as to make the yield drop.

In this study, we want to thoroughly optimize cleavage efficiency of thrombin and raise the yield on GST-Smad4 for further purification of Smad4. Therefore, we tried to insert a DNA linker between GST gene and Smad4 gene by molecular cloning to allow that thrombin can cleave on GST-Smad4 protein structurally. At length, compared with past study, we successfully optimized the plasmid construct, rupture method, steric-hindrance, thrombin on the optimal conditions and the increase in yield.

However, on functional analysis of Smad4 protein, we observed the interaction between SBE element and Smad4 protein by EMSA analysis, the interaction results were not seen from the gel. In future, we will still confirm the Smad4 function by using SPR biosensor to observe the interaction between Smad4 and Doxycycline.

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