全國中小學科展

醫學與健康科學

Synergistic effects of external factors on reprogramming of insulin-producing cells

第一型糖尿病是因自身免疫破壞使貝他細胞(ß-cell)數量減少,目前治療糖尿病最好的方式是利用胰島移植補充貝他細胞,而胰島來源缺乏導致全世界超過三千萬糖尿病患者無法用胰島移植治療糖尿病。在低等節肢動物的消化腔,肝胰組織存在同一個器官中,稱為肝胰臟(Hepatopancreas)。因此從生物演化角度看,肝胰兩種器官細胞親緣相近,且為擴大貝他細胞供應來源,我們試著測試肝臟細胞是否能藉由同時表現胰臟轉錄因子Pdx1或 Ngn3重新編程成胰島素分泌細胞,並進一步開發能增進這些胰島素分泌細胞活性的培養因子。因此,我們測試正常個體中會調控貝他細胞生長、分化、胰島素製造及分泌的外在環境培養因子:葡萄糖濃度、菸鹼酸(Nicotinamide)、腸泌素 (Glucagon-like peptides 1) 、類腸泌素(Exendin-4)、血小板生長因子(platelet-derived growth factor),對肝細胞重新編程為胰島素分泌細胞的影響。結果發現這些因子會抑制肝細胞專一轉錄蛋白表現、細胞生長或造成細胞死亡。且在肝細胞中表達胰島分化必須轉錄因子Pdx1及Ngn3,並配合不同培養因子組合處理後,得知當葡萄糖、腸泌素、類腸泌素、血小板生長因子共同處理,對肝細胞轉變成胰島素生成細胞效率有加乘並顯著促進胰島素分泌的效果。因此我們藉由表現Pdx1及Ngn3配合特定培養因子提高肝細胞轉化效率,並提高胰島素分泌細胞活性,希望將來提供胰島移植應用。

探討HER2/EZH2訊號途徑調控glutamine代謝基因GOT2以影響胰臟癌細胞生長之作用

根據世界衛生組織的統計,胰臟癌高居全球癌症死亡人數第四位,亦為臺灣十大癌症死因之一,為最具侵略性、致死性及預後不佳的癌症。HER2為調控癌細胞增生重要致癌因子,在胰臟癌患者大量表現,其在乳癌細胞中可磷酸化並穩定負責調控組蛋白甲基化的EZH2蛋白表現;而GOT2已知在粒線體內調控麩醯胺酸(glutamine)代謝產生-ketoglutarate,並參與氧化磷酸化幫助胰臟癌生長。 本研究探討HER2/EZH2訊息傳遞途徑是否影響GOT2抑制對胰臟癌細胞之死亡作用,以及HER2/EZH2是否藉由甲基化GOT2調控glutamine代謝與malate-Aspartate循環,找尋出HER2是否藉由EZH2調控GOT2活性而參與癌細胞glutamine代謝反應,觀察HER2、EZH2、GOT2訊息傳遞途徑。 研究結果發現細胞生長作用與EZH2表現較HER2表現具有相關性,並證實EZH2與GOT2確實存在交互作用關係,透過EZH2與GOT2結合並甲基化GOT2而調控其作用,增加活性表現與抗藥效果,顯示EZH2與GOT2參與胰臟癌細胞glutamine代謝機制重要角色。

Cellular Effects of DNA Demethylation Enzyme TET2 Knockdown

近年來許多研究指出TET家族應是抑癌基因,然其許多抑癌機制卻尚未明瞭。根據本實驗室未發表的實驗結果,TET2表現量在大多乳癌檢體內較低,且TET2表現量低之病患存活率較差。本研究以Western Blot發現H3k4甲基化量在TET2 knockdown後減少。實驗室microarray結果更發現重要乳癌抑癌基因DKK1在TET2 knockdown後表現量下降。本研究以real-time PCR進一步證實DKK1表現量下降;Western Blot與ELISA結果也顯示DKK1蛋白質在細胞內及培養液中減少。細胞實驗更發現TET2 knockdown及DKK1 knockdown皆導致細胞有更顯著的移行(migration)與增生(proliferation)特性,因此推測DKK1可能為受TET2調控的下游基因。已知DKK1轉譯出之蛋白質能拮抗抑制Wnt/β-catenin傳遞路徑,因此本研究正在探討TET2是否也調控此Wnt路徑。如證實此假設,將可提出TET2操控DKK1,DKK1調控Wnt路徑,終而影響EMT、癌症轉移的路徑。

抑制Hippo途徑做為體外擴增受接觸性抑制之人類角膜內皮細胞以用於移植之方法

探討經由抑制Hippo途徑,誘發受接觸性抑制之人類角膜內皮細胞(HCEC)增生及其機轉之研究。 研究過程 以攜帶YAP基因之質體轉染培養之受接觸性抑制之HCEC,或添加溶血磷脂酸(LPA)後,觀察HCEC之YAP蛋白核轉移、細胞分化及增生。再利用專一性激酶抑制劑探討可能之訊息途徑。 研究結果 轉染之YAP能顯著誘發HCEC細胞增生,同時保有其功能。LPA能誘發HCEC的YAP蛋白核轉移,進而增進HCEC細胞增生。而PI3K與ROCK抑制劑能顯著抑制HCEC的YAP蛋白核轉移及細胞增生。 結論 LPA能誘發YAP蛋白核轉移與受接觸性抑制之HCEC細胞增生,此現象應與PI3K及ROCK途徑之活化有關。 應用 此研究提供了一種創新的誘發細胞增生策略以用於移植或細胞療法。

Potential Diagnosis of Cancerous Cells Through Utilising Optical Spectroscopy

Cancer is responsible for an estimated 9.6 million deaths in 2018. Deaths from cancer worldwide are projected to reach over 13 million in 2030. Thus, developing a device that has the capability to solve today’s toughest global challenge is crucial by utilizing a simple yet robust approach - “SEEING THE UNSEEABLE” through bold innovation. Although removing cancer is much more effective than either radiation or chemotherapy, when unseen residual cancer cells remain, they could grow back into tumour overtime. The reoccurrence of cancer contributes to a greater risk of death. Hence, launching a system that is able to distinguish between the cancerous cell and normal cell is ultimately essential to make sure no cancer is left behind during surgery. This robust optical system is established with quantitative approach by exploring the integration of an algorithm into the developed software. The end result of this device has the capability to provide users an accurate numerical pH value. The developed system is integrated with the smart IoT gateway capability whereby this powerful analytical device is incorporated with the real-time monitoring, data transformation and data analyzer. Harnessing the power of technology lets us fight cancer better. Each time a pathologist analyzes tissue after operation, it can take up 2 to 3 days because the tissue has to be frozen, thinly sliced, and stained so it can be viewed under the microscope during the process of biopsy. Thus, it is crucial to invent this Surgeons’ VisionMetric device which has an IoT-based microcontroller that is capable of providing real-time numerical value on-site.

以小鼠腎小管IMCD3細胞株探討纖毛生成和上皮細胞極化的關係

纖毛(cilia)病變是許多疾病的成因,像是多囊性腎臟病、巴德-畢德氏症候群等,但目前仍不清楚纖毛生成的機制。我們的研究初步證實纖毛生成和閉鎖小帶(Tight junction)可能有密切關聯性,且閉鎖小帶完整的細胞,纖毛基部到細胞底部的平均距離較長。因基體上的遠端附器(Distal appendage)能辨認細胞頂端膜(Apical membrane)上的特殊構造,有完整閉鎖小帶的細胞纖毛生長在細胞頂端,閉鎖小帶不完整時,細胞的極性未表現,纖毛無法生成在細胞頂端。 本研究以IMCD3(小鼠腎小管)細胞株,在去血清(serum starvation)的刺激誘導下,使IMCD3細胞長出纖毛,再以免疫螢光染色分析閉鎖小帶與生成纖毛的關係。並利用克隆形成試驗(Colony formation assay),將IMCD3細胞株分為四類型態,也進一步發現在三維培養中,四種細胞型態皆無法排列成空腔構造。最後我們建立載體DNA:pLAS2w.Ppuro-EGFP-Arl13B,以綠色螢光標示纖毛蛋白,在活細胞中追蹤基體的移動及纖毛生成的過程。期望藉由研究纖毛形成的過程機轉,能探討致病的作用機制,找出治療纖毛病變的療法。

後轉譯化學修飾對Programmed Cell Death 5(PDCD5)功能之影響

PDCD5,簡稱PD5,屬於 PDCD (programmed cell death)家族成員,本身具有125個胺基酸,本身能引起細胞凋亡,也有報導細胞自噬,並且在作用時會從細胞質進入細胞核,除此之外,PD5在人類許多腫瘤表達下降,跟p53也有協同促進細胞凋亡的作用。PD5被推測可能可以誘使癌細胞進行細胞凋亡,雖然目前與這個蛋白質有關的研究並沒有很多,作用機制也尚不明確,但我們相信PD5有值得我們研究的價值,於是我們進一步探討PDCD5的結構,以及各種蛋白質修飾後可能的結果,還有相關的反應機制。

MafF對LRH-1調控代謝機制之探討

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)。LRH-1則是僅與具有完整basic region (BR)以及leucine zipper (LZ) 兩個domain的MafF產生交互作用,單獨的BR以及LZ無法與LRH-1結合。另外,在肝臟細胞株HepG2中過度表現Myc-MafF,Western blot結果發現GCK有減少的趨勢,SHP則是有增加的現象,而LRH-1亦呈現增多的趨勢。Real-time PCR結果,比對Myc以及Myc-MafF,發現MafF存在時會使LRH-1以及GCK mRNA含量較控制組Myc有減少的趨勢,對SHP mRNA 則是沒有顯著影響。此外,我們在肝臟細胞株HepG2中加入胰島素,發現二者表現量皆有明顯的增加,說明胰島素刺激之下,MafF以及LRH-1蛋白質增加能夠增強肝臟能量代謝。

Fabrication and Characterization of Biological Electrospinning Nanofiber Scaffold Based on Cellulose Diacetate-Gelatin-Green Tea for Tissue Engineering Applications

Tissue engineering has developed novel therapies such as many types of wound dressings, bio-pads, scaffolds and bandages, in order to reduce the effects of deep and extensive skin wounds. Here, we have produced an electrospun nanofiber scaffold, based on biodegradable materials such as gelatin (as a natural and hydrophilic polymer) and cellulose diacetate (with optimal biodegradability), in order to increase wound healing using nanotechnology. We also used green tea extract for its anti-oxidant and anti-bacterial effect, to improve the biological properties of the scaffold. In the fabrication process, two polymer solutions: 1. Gelatin (with acetic acid solvent) and 2. Cellulose Diacetate (with acetone solvent) mixed with green tea extract, were prepared. Then they were spun using a two-nozzle electrospinner to produce a hybrid nanofiber scaffold. SEM images showed enough finesse and uniformity of the produced scaffold to simulate the extracellular matrix. Further, measuring the contact angle of water droplet and the web surface, indicated optimal hydrophilicity of the nanofiber scaffold, which controls the level of scaffold degradability and cell adhesion. Also, the results of antibacterial tests for two bacterial strains (E. coli and S. aureus) showed the antibacterial characteristics of the extract-containing scaffold. In addition to previous tests, evaluation of fibroblast morphology on the nanofiber scaffolds, indicated appropriate cell adhesion and expansion, that confirms the biocompatibility of this produced scaffold.

Lighting Up The Brain

Alzheimer’s disease (AD) is a neurodegenerative disease in which current diagnostic tools are invasive and lack the ability to diagnose early-onset dementia. Current antibody-based diagnostic tests for neurodegenerative diseases require invasive measures such as a lumbar puncture, and lack specificity to biomarkers that are found in both healthy individuals and patients with AD. In this project, a design for a carbon dot(CD)-bound bispecific antibody is developed for the minimally- invasive diagnosis of AD. The molecular probe can be easily synthesized with a specificity to amyloid- beta (Aβ) oligomers as it distribution and abundance in the brain suggest they are better predictors of disease progression and are present in the early-onset of the dementia. The bispecific antibody conjugated to the CD displays a low affinity to transferrin receptors (TfRs) which allows the probe to cross the blood-brain barrier via receptor mediated transcytosis leading to a minimally invasive diagnosis. A synthesis technique was developed to conjugate the bispecific antibody to the CD. As a proof of concept, this technique was used to couple bovine serum albumin (BSA) to CDs. The structural and optical properties of the CDs were observed. By synthesizing a novel carbon dot conjugated specific antibody that emits light at a specific wavelength in the near-infra red region, the molecular probe displays optical properties suitable for the minimally-invasive diagnosis using fNIR- spectroscopy.