全國中小學科展

醫學與健康科學

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。

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。

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。

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。

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。

Development of a neurointerface glove with tactile feedback

Research Question or Engineering Problem A stroke continues to be the most important medical and social problem of the modern world. Stroke is a type of acute cerebrovascular accident (ACVA) and is characterised by a sudden (within minutes, less often - hours) appearance of focal neurological symptoms (motor, speech, sensory, coordinating, visual and other disorders) and / or general brain disorders (depression of consciousness, headache, vomiting, etc.) that persist for more than 24 hours or lead to death of the patient in a short period of time due to a cause of cerebrovascular origin. There are two clinical and pathogenetic forms of stroke: ishemic stroke (cerebral infarction) is caused by acute focal cerebral ischemia, leading to infarction (zone of ischemic necrosis) of the brain; hemorrhagic stroke (non-traumatic intracerebral hemorrhage) is caused by rupture of an intracerebral vessel and blood penetration into the brain parenchyma or rupture of an arterial aneurysm with subarachnoid hemorrhage (SAH). ACVA also includes transient disorders of cerebral circulation, characterised by the sudden occurrence of focal neurological symptoms that develop in a patient with cardiovascular disease (arterial hypertension, atherosclerosis, atrial fibrillation, vasculitis, etc.), last for several minutes, less often hours, but no more than 24 hours, and ends with a full restoration of the impaired brain functions. Transient disorders of cerebral circulation include: transient ischemic attack (TIA), which develops as a result of short-term local cerebral ischemia and is characterised by sudden transient neurological disorders with focal symptoms; hypertensive cerebral emergency, which is a condition associated with an acute (usually significant) rise in blood pressure (BP) and accompanied by the appearance of general cerebral (less often focal) neurological symptoms secondary to hypertension. The most severe form of hypertensive crisis is acute hypertensive encephalopathy, the basis of pathogenesis of which is cerebral edema. Cerebral infarction generally is the result of the interaction of many etiopathogenetic factors, which can be subdivided into local and systemic ones. Local factors include: morphological changes in the brachiocephalic or intracerebral arteries (pathological tortuosity, etc.), atherosclerotic lesions of the vessels of the aortic arch and cerebral arteries, cardiac lesions as a source of thromboembolic cerebral infarctions, fibromuscular dysplasias of the walls of the brachiocephalic and cerebral arteries, brachiocephalic artery dissection, vasculitis (arteritis), changes in the cervical spine with the formation of extravasal compression of the vertebral arteries, anomalies in the structure of the vessels of the neck and brain (hypoplasia of the vertebral artery, trifurcation of the internal carotid artery), etc. Systemic factors include: disorders of central and cerebral hemodynamics (a sharp change in BP or a decrease in cardiac output, etc.), hereditary and acquired coagulopathies, polycythemia, certain forms of leukemia, hypovolemia, psychoemotional stress / distress, etc., hypercoagulative / hyperaggregatory side effects of a number of medications (oral contraceptives, etc.). In the 俄羅斯n Federation, more than 500 thousand people have a stroke every year. About 25,000 new cases of stroke occur in St. Petersburg every year. The incidence of stroke in the 俄羅斯n Federation is 3.48 ± 0.21 cases per 1000 people. The incidence of various types of ACVA varies widely, in particular, cerebral infarctions account for 65–75%, hemorrhages (including subarachnoid hemorrhages) – 15–20%, transient cerebral circulation disorders account for 10–15%. The frequency of cerebral strokes in the population over 50–55 years old increases by 1.8–2 times in each subsequent decade of life. Medical and socio-economic consequences of ACVA are very significant, in particular, death in the acute period of stroke occurs in 34.6% cases, during the first year after the end of the acute period in 13.4% cases; severe disability with the need for constant care is present in 20.0% of stroke patients; 56.0% have limited working capacity and only 8.0% return to their previous work activity. Disability due to stroke (the national average is 56–81%) in our country ranks first among all causes of primary disability, amounting to 3.2 per 10 thousand people. Stroke mortality among working-age population has increased in the 俄羅斯n Federation by more than 30% over the past 10 years. The annual death rate from stroke in our country is 175 per 100 thousand people. Stroke annually becomes the main cause of disability: 85% of victims experience a decrease in strength or a complete lack of ability to control the muscles of half of the body and only half of them recover limb functions partially or completely; the rest of those who have suffered a stroke remain paralysed and require care, since they are not able to completely independent existence. In this regard, recently, in the process of rehabilitation, the technology of brain-computer interfaces (BCI) has begun to be actively used. on the basis of this technology exercise machines are created. These exercise machines are controlled directly by the patient himself. This feature of the technology increases the effect of the procedure by providing a direct connection between the patient's desire and effort with the work of the simulator. The greatest development of this technology is observed in the field of medicine, where BCIs are used as a means of communication or as one of the tools of neurorehabilitation. In this regard, it seems very promising to develop the most optimal brain-computer interfaces. The goal of our project was to create an automated training complex in the form of a neuro-controlled glove with tactile feedback, designed to simplify access to rehabilitation means.

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。

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.

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。