全國中小學科展

生物化學

藉由雙硫化物交聯製成的植物核酸奈米粒子可當作穀胱甘肽誘發的抗癌藥物載體

由於人工合成的化學材料在包覆藥物進行人體醫療測試時,不可分解性會帶來體內累積的毒性,搜尋各類化合物的結構後,發現核酸上的含氮鹼基具有一級胺結構,可和天然交聯劑京尼平或DTSSP進行反應,因此,本研究抽取水果的核酸進行實驗,利用界面活性劑乳化的過程中將阿黴素(DOX),包覆進入交聯後的核酸水膠,穿透式顯微鏡的結果顯示交聯後的水膠結構是球狀,平均粒徑大約是120奈米,核酸本身帶負電,水膠有包覆藥物的界面電位稍微減少負電,利用人類正常肺上皮細胞進行水膠共同培養,2毫克/毫升的水膠溶液在連續培養48小時後,並不會造成細胞死亡,奈米水膠在pH值5.5釋放DOX的量比pH值7.4還要高出27%,有奈米水膠包覆DOX的情況下,更能有效率進入癌細胞內與引起癌細胞毒性,本水果核酸製成的奈米水膠在生物體可分解的無毒包覆材料上,於未來可提供一個新的藥物載體參考。

以蛋白質工程開發新穎酵素於高尿酸檢測及降解藥物

尿酸氧化酶參與嘌呤代謝,然人類尿酸氧化酶基因已退化,易使過量尿酸沉積於關節造成痛風,近年來微生物源尿酸氧化酶之酵素工程改良,逐漸被應用於尿酸檢測與降解藥物,因此具極高研發價值。 本研究針對微生物源尿酸氧化酶進行基因體探勘,篩選出抗輻射奇異球菌(Deinococcus radiodurans)及耐熱雙球桿菌(Thermobispora bispora)源尿酸氧化酶基因,以蛋白質異源表現與金屬螯合層析法純化取得重組尿酸氧化酶,進行酵素動力學、熱穩定分析、結構解析、金屬離子耐受性分析與尿酸檢測應用。在最佳反應條件下,抗輻射奇異球菌源酵素於30 ℃之Km與Kcat為679.03 μM, 30.33 s-1;耐熱雙球桿菌源酵素於70 ℃之Km與Kcat為191.31 μM, 12.85 s-1。此外,我們已解析耐熱雙球桿菌源尿酸氧化酶結構,發現其羧基端之特異性構型可能與熱穩定性有關。本研究以此兩種尿酸氧化酶為酵素工程改良標的,盼未來能研發作為快速篩檢與臨床治療之生物替代藥物。

An In-Depth Patch-Clamp Study of HCN2 Channel (Year II): Discovery of Novel Biomarkers and Therapy for Ih Current Suppression in Autism Spectrum Disorders

The main goal of this study was to address a variety of topics concerning the role of the Ih current in HCN channels of SHANK Wild-Type and Knock-Out Thalamus Neurons (as described further below). This research explored the cellular effects of sedation (like Dexmedetomidine) and laser light stimulations on the Ih current of neurons, as well as discovering novel biomarkers for detecting Autism Spectrum Disorder. This study also showed that methods (like utilizing laser therapy with and without various photosensitizers) have the potential in raising depressed Ih currents of SHANK Knock-Out neurons.

細胞核 CTEN 蛋白質對 Wnt 訊息傳導路徑之影響及細胞癌化之調控

正常細胞的 C-terminal tensin-like (CTEN) 蛋白質位於細胞質且含量低,但研究發現 CTEN 在大腸癌細胞中會大量表現並累積於細胞核內,進而提升癌細胞遷移與侵襲的能力;而 Wnt 訊息傳遞路徑在癌細胞中常有過度活化的現象。因此本研究主要探討 CTEN 於細胞核內的累積和 Wnt 訊息傳遞路徑的互動關係,進而調控細胞癌化的機制。分析 CTEN 分布有差異之細胞株的轉錄體發現,在較多 CTEN 累積於細胞核的大腸癌細胞株中,其 Wnt6、Wnt11 兩基因的表現量上升,研究指出 Wnt6、Wnt11 皆會影響大腸癌細胞的遷移與侵襲。另外,CTEN 會與 Wnt 訊息傳遞路徑中的 β-catenin 結合,因此本實驗利用啟動子帶有 Wnt 訊息下游轉錄因子結合位的質體,測定其下游 β-catenin/TCF/LEF 轉錄因子的轉錄活性。結果顯示 CTEN 累積於細胞核中會提高 β-catenin/TCF/LEF 之轉錄活性,進而增加 Wnt6、Wnt11 等下游基因的表現量,影響大腸癌細胞的遷移與侵襲。

Phytochemical screening and evaluation of antiangiogenic properties of sapinit (Rubus fraxinifolius) fruit crude extracts

Plants are potential low-cost alternatives for cancer treatment. Rubus fraxinifolius or “sapinit” has been found to possess phytochemicals with anti-cancer potential. This project aimed to evaluate the antiangiogenic properties of methanolic R. fraxinifolius fruit crude extracts through the chick chorioallantoic membrane (CAM) assay. Through phytochemical screening, leucoanthocyanins, phenols, and tannins were detected. For the CAM assay, 10, 20, and 30 μg/μL extracts, distilled water, methanol, and retinoic acid were applied on 60 ten-day-old chicken eggs. The CAM photographs were analyzed using ImageJ Software. The mean percent inhibitions (MPI) of total length and vascular density from both analyses were subjected to One-Way Analysis of Variance (ANOVA). The ANOVA for the MPI of total length, followed by a Tukey post hoc test, show that only retinoic acid treatment has significantly higher MPI (p = 0.0010). Meanwhile, the results for the MPI of vascular density show no significant differences between all groups (p = 0.1630). It is possible that the concentrations used in the study may not be the concentrations needed to achieve optimal antiangiogenesis. The results may also be due to the absence of phytochemicals that exhibit significant antiangiogenic properties such as alkaloids. Lower concentrations and isolated phytochemicals may also be tested.

New Approach to Food Packaging: Antimicrobial and Edible Materials

In addition to adequate and balanced nutrition in the protection of human health, the reliability of consumed foods is also of great importance. When foods contact with the environment, they are undergoing many microbiological, physical and chemical changes such as moisture loss, aroma exchange, oxidation and contamination with microorganisms. So this changes reduce quality and shorten the shelf life. During cutting and processing of chicken meat, especially contamination on the surface causes deterioration of the meat starting from the surface and thus short shelf life. Though contamination of chicken meat surface is inevitable, growth of contaminant microorganisms can be inhibited and microorganisms can be killed. In recent years, increased risks of infection due to antibiotic-resistant microorganisms have forced the discovery of new and natural antibacterial materials. It is a new and advantageous approach to avoid environmental pollution caused by the use of food packaging and safety of food, prolongation of product shelf life by natural, edible, antibacterial biomolecules in packaging products. In the scope of the project, in order to produce an edible alginate gel containing the antimicrobial peptide nisin, which has an antibacterial property to prevent microbial growth on the surface of chicken meat, following steps was done;  Synthesis of calcium alginate and nisin immobilized calcium alginate beads  Optimization of immobilizing nisin in calcium alginate beads,  Characterization of nisin immobilized calcium alginate beads by ATR-FTIR Spectrum and SEM Analysis,  Determination of the antimicrobial activity of white meat product chicken which is coated by nisin immobilizing calcium alginate gel.

以新型CRISPR-Cas9技術優化粒線體基因剪輯

本研究開發一個新型的CRISPR-Cas9技術剪輯粒線體DNA,提供粒線體基因所造成疾病的一個有潛能之治療方式。在本研究中,我們將嵌入粒線體標的訊號序列後的 Cas9 蛋白質和 sgRNA 分子送入粒線體內,將 CRISPR-Cas9系統套用於粒線體中,並達成剪輯粒線體基因之目的。我們將 Cas9 蛋白質和 sgRNA 分子鑲嵌於同一質體上,有效導入CRISPR-Cas9系統於粒線體內,並觀察到剪輯之標的基因ND4含量下降了 32%,達到粒線體基因編輯之目標。雖然前人曾用ZFN (Zinc-finger Nuclease)和TALEN (Transcription Activator-like Effector Nucleases)成功編輯粒線體基因,但由於製作過程繁瑣和經費昂貴等種種原因,並未被廣泛使用。我們開發的新型CRISPR-Cas9粒線體基因剪輯系統將可以提供一個相對簡易且價格低廉的粒線體基因剪輯平台。

微生物源之甜菜素合成酵素的結構功能探討與潛在應用

甜菜黃素為植物內水溶性色素,以植物4,5-多巴雙加氧酶催化L-多巴形成甜菜醛胺酸,再與胺基化合物可得到甜菜素。甜菜素極佳的抗氧化性被認為可發展有效的抗癌保健。 目前甜菜素由植物萃取與植物酵素合成,近期研究發現某些微生物有可催化L-多巴轉換機制的4,5-多巴雙加氧酶,但資訊未完備。又偵測L-多巴可診斷初期帕金森氏症,此酵素的催化機制可檢測L-多巴。 本研究探索微生物源4,5-多巴雙加氧酶,經基因體探勘、基因選殖與蛋白表現後,將重組蛋白純化並探討結構與功能。目前已進行活性測試、得到蛋白最佳反應環境,並分析兩種蛋白質結構,比較兩種蛋白結構與活性之關聯性;以及進行酵素動力學實驗。亦已開始著手應用層面,成功設計並合成出雙加氧酶與酪胺酸酶之引子。 未來期待以結構資訊改良酵素;應用面用於測定醫療之L-DOPA,加入酪胺酸酶於細菌大量生產甜菜素。

激增殺手T細胞的水膠細胞平台於免疫療法的應用

本實驗研發新型水膠細胞優化技術,成功提供一個平台供應淋巴細胞激活信號(MHC-antigen complex)、共刺激配體及細胞因子刺激三大要素,能激增殺手T細胞。實驗中優化的水膠細胞能完整保留細胞膜的表面特性,經實驗證實無生物安全性的疑慮。另外本實驗也著重於探討人工合成抗原細胞強韌度對T細胞增生程度的影響。我們利用水膠細胞易改變細胞質強韌度且不影響生物相容性的優點,針對T細胞增生程度進行比較。本研究結果顯示,高細胞質強韌度(120kPa)的人工合成抗原細胞(aAPCs, Artificial antigen presenting cells)擁有高效率的T細胞激活潛力,相對於低細胞質強韌度(25kPa)的對照組可以提高約51.2%,並達到顯著差異,推測其和收縮性及突出性絲狀肌動蛋白增加以致訊號增強有關。本實驗的新型水膠細胞優化技術提供臨床醫學免疫療法一個高潛力的新技術平台。

探討粒線體如何參與調控細胞內鈣離子訊息傳遞

鈣池調控鈣離子內流(store-operated calcium entry, SOCE)是非興奮性細胞中最主要的鈣離子通道。當內質網缺乏鈣離子時,位在內質網上的STIM1(Stromal interaction molecule 1)便會改變構型與在細胞膜上的ORAI1(Calcium relaease-activated calcium channel protein1)接合,激活SOCE的路徑。近來的研究顯示,粒線體會影響SOCE的活性。已知平均有5%-20%的粒線體藉由連繫蛋白與內質網相連。又已知內職網在缺乏鈣離子時會移動至細胞膜附近,故我們認為粒線體有很大的機率藉由連繫蛋白與內質網一同移動至細胞膜並透過吸收鈣離子的機制來調控SOCE的活性。 在使細胞表現特定螢光蛋白的前提下,我們透過活體細胞攝影來觀察特定對象(粒線體、粒線體內鈣離子)的動態變化。 從實驗結果中我們發現:當SOCE被激活後,粒線體會移動至SOCE發生處且較靠近STIM1。又絕大部分移動至SOCE發生處的粒線體同時也會吸收鈣離子。 過去的研究已證實,當粒線體與內質網之間缺乏鈣離子時,SOCE的活性會降低,且當粒線體內膜的主要鈣離子通道MCU(Mitochondria calcium uniporter)缺乏時,亦會導致相同的結果。又從我們的實驗可知當SOCE被激活時,粒線體會移動至SOCE發生處並吸收鈣離子。綜合上述,我們可以推論以下機制,當細胞內的SOCE被激活時,粒線體會藉由連繫蛋白與內質網一同移動至SOCE發生處,同時以吸收鈣離子的方式來調控SOCE的活性以及細胞內的鈣離子濃度。