全國中小學科展

醫學與健康科學

Remedies Recovered from Roof Top Resources

Moss from a roof top was used to treat ear infections in my grandfather’s village. This remedy sparked my curiosity and so I began researching. I was bewildered to discover that the resistance to antibiotics has been labelled as a “Catastrophic Threat” and has been ranked in the same category as terrorism and climate change. Governments globally are urging scientists to identify and produce new antibiotics and reassess novel approaches1. This project aims to evaluate two objectives through the use of several integrated technologies and modified methods: (a) To determine if the extracts, solutions and raw materials derived from Heart wood portion of Picea glauca, Populus tremuloides, Salix spp, Betula papyrifera, Pinus contorta, Quercus alba, Thuja occidentalis, Climacium dendroides, Dicranum fuscescens and Kieselgur, will inhibit the growth of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Escherichia coli. (b) To scientifically reassess my grandfather’s traditional method of treating ear infections using roof moss. The Heartwood portion of each tree was removed using a hammer and mallet. The Heartwood was then burnt to derive the ash and a miter saw was used to make sawdust. The moss was collected, dried and labelled. A Methanol Extraction was performed on all saw dust samples and moss using a Soxhlet Extractor for 24 hours. The ash solutions were diluted, filtered, and neutralized to pH 7. The solvents were evaporated in a Rotary evaporator and the residual material was stored in round bottom flasks. The Kirby Bauer method was modified and a Well Infusion method was devised for the biological assay. The Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Escherichia coli were plated using a 0.5 McFarland Standard. Paper filter discs containing 20uL of each extract solution and raw material were precisely placed onto the inoculated plates and incubated for 24 hours. The preliminary results were initially unfavourable, as data could only be collected and analysed for one species; Thuja occidentalis (White Cedar). However, these results were extremely encouraging when the zones of inhibition were measured and analyzed. Confidence Intervals were calculated at 95% and the T-Tests were calculated at a 0.05 alpha level, which indicated significance when compared to the control. The Chi Square values were greater than the critical value of 7.8 and therefore the thorough statistical analysis indicates that the results were not due to chance alone. Literature has indicated that certain components of trees do indeed have antibacterial properties, however there is very limited research specific to the Heartwood portion. Furthermore, I discovered that the Heartwood portion of the White Cedar tree does have certain antibacterial properties that definitely justify further testing. In addition, a combination of examining my grandfather’s possessions and analyzing present data, I can confidently support my grandfather’s traditional method. In conclusion, the use of the Heartwood portion of the White Cedar to combat bacterial infection warrants further exploration. Remedies Recovered from Roof Top Resources may be the solution to this catastrophic threat.

探討rVP1在肺癌細胞上Integrin路徑及Wnt/β-catenin路徑的作用機制

經基因重組技術純化的口蹄疫病毒鞘蛋白rVP1被證實在癌細胞內會經由Integrin路徑對癌細胞的轉移及凋亡產生影響。本研究所探討的是rVP1對肺癌幹細胞H1299及肺癌細胞A549中Integrin路徑與Wnt/β-catenin路徑的作用機制,目的除了要了解兩路徑在肺癌細胞中的影響,也希望能找出兩路徑的交互作用機制。 本研究發現肺癌幹細胞H1299中β-catenin會因加入rVP1而表現量下降,與肺癌細胞不同;肺癌細胞A549中Wnt/β-catenin路徑上游的膜上受體Frizzled-8因rVP1作用而表現量下降;而不論在肺癌幹細胞H1299及肺癌細胞A549中Grb2都不會因rVP1作用而表現量有所改變;Wnt/β-catenin路徑下游產物MIG-7則會因加入Wnt3aligand而表現量下降。 由實驗結果推測癌症幹細胞本身與癌細胞特性不同,可能使得Wnt/β-catenin路徑產生的效果不同;Grb2的變化量則需要更多實驗求證。Integrin路徑會影響到Wnt/β-catenin路徑的上游生成,兩者確實有相互影響。

FAT10 Haplotypes as a Potential Biomarker for Cancer

Cancer is the second leading cause of death today[1], accounting for nearly 1 in 6 deaths worldwide. Despite this, diagnosis and treatment models for cancer are limited and as such, new methods to identify and treat susceptible patients are required urgently. HLAF- adjacent transcript 10 (FAT10) is an oncogene that is strongly implicated in the development of inflammation-associated cancers[2]. Previous research on this highly polymorphic gene has identified 2 haplotypes – the reference haplotype, which is found in both cancer patients and healthy individuals, as well as an additional haplotype that is occurs at higher frequency in cancer patients and is associated with higher odds of cancer. In this study, it was hypothesised that the cancer-associated FAT10 haplotype can better promote tumorigenicity and could thereby serve as a useful biomarker for cancer. Here, we functionally characterize the 2 FAT10 haplotypes to understand how they influence some of the hallmarks of cancer. The cancer-exclusive haplotype was observed to enhance hallmarks of cancer, namely uncontrolled cell growth, resisting cell death and anchorage-independent growth as compared to the reference haplotype. Moreover, we uncovered the differential gene expression patterns induced by each haplotype. Molecules involved in cell adhesion and proliferation, as well as transcription were upregulated by the cancer-associated haplotype and hence could have contributed to the increased tumourigenic potential of the cancer haplotype.

上皮細胞黏著分子(EpCAM)促進大腸癌細胞中對於艾瑞莎(Gefitinib)之抗藥性研究 EpCAM enhances Gefitinib-induced drug resistance in colon cancer cells

上皮生長因子受體 (EGFR) 已被確認在人類上皮惡性腫瘤扮演重要角色,因此臨床上開發出許多針對EGFR之大腸癌、肺癌等的小分子標靶藥物,但治療期間所產生的抗藥性仍是一大瓶頸。 過去上皮細胞黏著分子(EpCAM)只被認為是胞間連接分子,現今則在癌幹細胞 (cancer stem cells,CSCs)等領域被研究。然而從文獻與先前實驗室的試驗,可以看見EpCAM促進癌症抗藥性的可能。 本研究發現在大腸癌中,艾瑞莎 (Gefitinib) 會透過轉錄因子FOXO3a促進細胞凋亡,而EpEX會經由抑制FOXO3a所促進的凋亡路徑,導致癌細胞產生Gefitinib抗藥性,且此抗藥性,也與EpICD下游之誘導性多能幹細胞相關基因 ( iPS-related genes )的表現有關,但其分子機制尚不清楚。 本研究以大腸癌與Gefitinib做為癌症與EGFR小分子藥物的模型,找出EpCAM可能造成的抗藥路徑,未來可應用在各類癌症之聯合治療 (Combination therapy) ,以克服癌症治療所產生的EGFR小分子藥物抗藥性。

探討MafF對LRH-1調控相關代謝路徑的影響

已知 liver receptor homolog-1(LRH-1)掌控著多種生理功能,在肝臟代謝中扮演著相當重要的角色,我們利用酵母菌雙雜交技術(Yeast two-hybrid)找出數種可能跟LRH-1產生交互作用的蛋白質,並從其中選擇v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F (MafF)作為主要研究對象,探討其與LRH-1之間的作用對代謝造成的影響。本研究中,我們利用免疫沉澱法證實MafF可與LRH-1形成複合體,而MafF並不影響LRH-1蛋白的表現量。隨後以啟動子活性檢測MafF-LRH-1複合體在細胞內之功能,發現MafF能促進由LRH-1所調控的small heterodimer partner (SHP)、glucokinase IV (Gck)及side chain cleavage enzyme (SCC)等代謝相關啟動子之活性。此外我們以GST-pull down發現MafF與LRH-1結合區域位於LRH-1的DNA 鍵結位(DBD)。綜合上述實驗結果,MafF能與LRH-1的DBD鑑結形成複合體,並促進啟動子活性,未來希望能進一步探究MafF對LRH-1所調控下游基因的影響,以了解MafF於肝臟膽酸合成及醣類代謝機制中所扮演的角色。

小花蔓澤蘭活性成分Dihydromilanolide誘導胃癌細胞凋亡與自噬性死亡

外來入侵種小花蔓澤蘭繁殖速度太快,使台灣本土生環境及多樣性受到破壞。我們研究發現,小花蔓澤蘭葉萃取物會誘導人類胃癌(AGS)細胞毒性;以HPLC分析及分離出小花蔓澤蘭葉萃取物的活性成分Dihydromilanolide (DHK),發現DHK會毒殺胞胃癌、卵巢癌、乳癌與血癌細胞,其中以胃癌(AGS)細胞毒殺性最強。此外,抗氧化劑N-acetylcysteine可減緩小花蔓澤蘭葉萃取物及DHK對胃癌細胞毒殺性,推測是透過活性氧化物(ROS)來毒殺胃癌細胞。我們亦發現,DHK可與抗癌藥物(Doxorubicin、Cisplatin或Paclitaxel)對胃癌細胞產生協同作用。DHK作用胃癌細胞會誘導Caspase-3增加、PARP蛋白裂解、促凋亡Bax增加及抑凋亡Bcl-2減少;產生酸性囊泡(AVOs)、促自噬LC3-II及Beclin-1蛋白增加,且加入自噬抑制劑 3-MA可保護DHK誘導胃癌細胞死亡。推論 DHK可誘發胃癌細胞凋亡(Apoptosis)及自噬性死亡(Autophagy)。總結,小花蔓澤蘭與活性成分Dihydromilanolide (DHK) 具抗胃癌功效且與抗癌藥物產生協同作用,可開發成抗癌的藥品或保健食品。

熊果酸抗胃癌細胞增生與順鉑的協同效應

胃癌的治療方式以手術及化學藥物治療為主。本研究目的是研究中草藥白花蛇舌草的主要成份熊果酸(Ursolic acid, UA)對於胃癌細胞增生的影響。研究使用兩種人類胃癌細胞株MKN45及SCM-1。藉UA處理胃癌細胞之後,由鏡檢可見細胞數目減少,SRB分析法也證明UA會降低細胞的存活率。利用流式細胞儀,證實UA會讓胃癌細胞凋亡。進一步由西方墨點法實驗顯示,UA會誘導促細胞凋亡蛋白Bax及Bak的上升,並且降低抗細胞凋亡蛋白Bcl-xl及Bcl-2的表現。UA也會抑制訊息傳遞途徑p-Stat3/c-Myc/Cyclin D1。實驗也發現UA合併使用化療藥物順鉑(cisplatin)會使胃癌細胞存活率降低。經由周塔氏藥物合併指數及流式細胞儀分析均與協同順鉑能抑制胃癌細胞的增生,西方墨點法也發現pro-caspase 9 被活化,皆與UA會促進順鉑引發凋亡效應有關,這些實驗證實UA是cisplatin治療胃癌的輔劑,這個結果對中草藥白花蛇舌草的抗癌效果提供了具體的實證。

TCA cycle perturbation induces renal mitochondrial dysfunction and enhanced oxidative stress in diabetic nephropathy-1H NMR-based untargeted metabolomics analysis for potential biomarkers and the effe

本研究藉由基因誘導之第二型糖尿病小鼠(Male B6.BKS-Leprdb mice; db/db mice)做為動物模型,並利用氫質譜儀與代謝體學之平台(1H NMR-based untargeted metabolomics)了解第二型糖尿病所導致的腎病變之疾病進程中的代謝途徑變化,及白胺酸的介入對於疾病進程的影響。研究發現,三羧酸循環及支鍊胺基酸代謝途徑中的代謝物,在基因誘導之第二型糖尿病小鼠尿液中有顯著下降,此發現指出糖尿病腎病變所造成的粒線體能量代謝功能下降。在肝臟組織萃取中,脂化膽固醇(esterified cholesterol)的上升及 β-胡蘿蔔素(β-carotene)、維生素A(vitamin A)及維生素A酸(retinoic acid)的下降顯示肝臟產生脂肪肝及其氧化壓力之上升。白胺酸的介入對於減緩腎臟粒線體受損及降低脂肪肝與氧化壓力並無顯著功效,此現象可歸因於支鍊胺基酸啟動mTORC1代謝途徑。本研究所標定之代謝物可被應用於第二型糖尿病及其所導致之腎病變的生物指標。

以體外培養模式驗證魚鱗可作為細胞移植載體用於修復眼角膜內皮

研究目的: 探討魚鱗可否做為細胞移植載體以及其表面改良之方法,並了解相關之調控機轉。 研究過程: 魚鱗表面分別以纖維黏連蛋白、層黏連蛋白、第四型膠原蛋白或FNC® coating mix等胞外基質塗佈後,觀察B4G12細胞(人類角膜內皮細胞株)的貼附與增生是否有改善,並以基因微陣列探討FNC塗佈後對B4G12細胞基因表現的影響。 研究結果: 魚鱗表面塗佈能幫助B4G12細胞的貼附與增生,以交聯劑固定魚鱗表面塗佈不會影響其效果。基因微陣列、定量PCR與西方墨點法實驗結果顯示表面塗佈處理能1.上調整合素途徑以及其下游Wnt途徑的活化,有利於細胞增生、2.上調醣類代謝相關酵素(PFKP),使細胞有更高之代謝活性、3.上調與液體運輸相關的第12型碳酸酐酶 (CA12)。抑制ILK可下調CA12的蛋白質表現,顯示CA12之調控受ILK途徑影響。 研究結論: 表面塗佈後魚鱗可做為細胞載體。塗佈可活化整合素途徑,促進B4G12細胞貼附與增生。整合素徑亦可能經由調控CA12表現調節水分運輸。此外,表面塗佈亦可能經由上調醣類代謝酵素增加細胞代謝活性。 應用性: 此研究提供了一種創新的細胞載體及其改進策略,或可促進角膜內皮細胞療法之實現。

對抗上皮細胞黏附因子之CAR-T細胞於癌症免疫治療之研究

以往人類對抗癌症只能依靠手術、化療、電療等治標不治本的手段殺死癌細胞,不僅效率差且有強烈的副作用,而CAR-T免疫療法透過活化病人自身免疫細胞來專一性的治療癌症。 本研究旨在發展能治療實質固態瘤的CAR-T免疫療法,透過活化由人血中所分離出來的T細胞,使之能辨識在癌細胞中大量表現的上皮細胞黏附因子(EpCAM),並建立一套體外細胞毒殺測試的模型來測試CAR-T殺死腫瘤細胞的能力,以篩選出具有發展潛力CAR-T細胞。 本研究從外周血單個核細胞(PBMC)中分離T細胞,透過基因重組技術與lentivirus來改造T細胞,使T細胞表現CAR基因,再利用Dynabeads Human T-Activator CD3/CD28活化,並以大腸癌細胞株HCT116當作癌細胞目標,測試CAR-T是否有能力殺死腫瘤細胞,另外也以不表現EpCAM的A549肺癌細胞株、H460肺癌細胞株進行毒殺能力測試,結果顯示此CAR-T細胞有高度專一性,證明此CAR-T細胞具有潛力成為新一代的CAR-T免疫療法。