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Title: | 活細胞分析式微流道晶片之開發及其應用於癌症轉移潛能診斷之評估暨
SIK2藉由磷酸化CAPS2調控胰島素分泌之探討 Study I: A portable microfluidic device for the rapid diagnosis of cancer metastatic potential Study II: Identification of CAPS2 as a downstream target of SIK2 in regulating insulin secretion |
Authors: | Li-Yu Chen 陳立瑜 |
Advisor: | 周涵怡 |
Keyword: | 微流道,生物晶片,肺癌,癌症轉移,胰島素,糖尿病,磷酸化, Microfluidics,Biochip,Lung cancer,Metastasis,Insulin,Diabetes,Phosphorylation, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 研究一 活細胞分析式微流道晶片之開發及其應用於癌症轉移潛能診斷之評估
摘要 肺癌的轉移若及早發現並加以治療,便能顯著提升患者改善病情的機會。然而,現行臨床癌症檢驗之方式,如胸腔攝影、斷層掃描及組織切片等,均未能有效判斷肺癌細胞轉移之可能性,以致患者無法獲得適當的治療。本研究中,吾人設計開發出一新型微流道晶片系統,以期作為快速評估癌症轉移潛能之臨床檢驗。本系統設計係以恆定環境溫度及酸鹼度為基礎,再以甲殼素附著之微流道表面偵測目標細胞之貼附性,作為判斷細胞遷移特性之依據。於實際測試中,吾人將不同轉移潛能之肺癌細胞株,經標定後以本系統進行細胞貼附性測試。結果顯示本系統確能有效區分不同轉移潛能之肺癌細胞株。因此,透過本系本研究所開發之微流道系統,僅需少量自患者經活體採樣分離之細胞樣本,便能迅速取得評估癌細胞轉移潛能之資訊。此微流道系統未來若能應用於臨床診斷,便能為肺癌及其他癌症患者,於早期研判適合之治療模式。 研究二 SIK2藉由磷酸化CAPS2調控胰島素分泌之探討 摘要 動物體的各種器官之協同運作,需仰賴內分泌系統對細胞之調節作用。胰島素即係一種負責控制血糖濃度之內泌素。當胰島素的生成或分泌發生異常,抑或是體內細胞出現胰島素失敏之現象,便可能引起糖尿病。佔總糖尿病患數90 – 95%之第二型糖尿病,其起因爲體細胞逐漸產生胰島素抗性,進而造成胰島素過量分泌之代償作用,終而導致β 細胞劇烈死亡。因此,改善其病徵之方法可為刺激胰島素分泌及抑制β 細胞凋亡。Salt inducible kinase 2 (SIK2) 係一屬AMPK族群之酵素,於β 細胞中調控胰島素之分泌,並藉由抑制CREB-mediated IRS-2基因表現,促使β 細胞凋亡。本研究室先前研究指出,SIK2可透過其磷酸酶活性控制β 細胞中胰島素小泡之遷移及分泌,而抑制SIK2磷酸酶活性可增加胰島素之分泌。為進一步探討SIK2對胰島素分泌調控之機轉,吾人以Scansite網站資料庫預測,發現calcium-dependent activator protein for secretion 2 (CAPS2) 蛋白具有SIK2之磷酸化目標序列,可能為SIK2之下游調控因子。欲了解SIK2是否會透過CAPS2影響胰島素分泌,本研究首先確認SIK2及CAPS2均於β 細胞中表現,再以免疫沈澱法驗證其交互作用,並以in vitro kinase assay證明CAPS2確為SIK2之磷酸化目標。然而,吾人實驗結果亦顯示於SIK2及CAPS2共同過量之小鼠β 細胞中,SIK2可透過CAPS2強化細胞對葡萄糖刺激之反應。綜合上述,本研究結果指出SIK2可藉磷酸化CAPS2調控β細胞中胰島素之分泌,然其中詳細之分子機制,尚待未來進一步研究發掘。 Study I: A portable microfluidic device for the rapid diagnosis of cancer metastatic potential which is programmable for temperature and CO2 Abstract If metastasis of lung cancer can be found and treated early, a patient might have an improved chance to prevail over it, but routine examinations such as chest radiography, computed tomography and biopsy cannot characterize the metastatic potential of lung cancer cells; critical diagnoses to define optimal therapeutic strategies are thus lost. In this study, we designed a portable microfluidic device for the rapid diagnosis of cancer metastatic potential. Featuring a micro system to control temperature and a bicarbonate buffered environment, our device discriminates a rate of surface detachment as an index of the migratory ability of cells cultured on pH-responsive chitosan. We labeled metastatic subpopulations of lung cancer cell lines, and verified that our device is capable of separating cells according to their metastatic ability. As only few cells are needed, a patient's specimen from biopsies, e.g. from fine-needle aspiration, can be processed on site to offer immediate information to physicians. We expect that our design will provide valuable information in pre-operative evaluations to assist the definition of therapeutic plans for lung cancer, as well as for metastatic tumors of other types. Study II: Identification of CAPS2 as a downstream target of SIK2 in regulating insulin secretion Abstract Through the endocrine system, communications between cells in various tissues in our body are well coordinated to drive a variety of physiological events properly. Insulin, one of endocrines, is secreted in response to elevated blood concentration of glucose, thereby keeps blood sugar level within certain range. Either failures of production and secretion of insulin, or cellular insulin insensitivity could result in diabetes mellitus. Type 2 diabetes (T2D) accounting for 90–95% of those with diabetes, is caused by the cellular resistance to insulin stimulation which leads to progressive insulin secretion defect in order to compensate for the less responsive reactions, and finally massive death of β cells. Ways to treat T2D are to stimulate more insulin secretion and to inhibit β cell death. Salt inducible kinase 2 (SIK2), a member of AMPK family, plays important roles in both β cell survival as well as insulin secretion. SIK2 has been reported to suppress β cell survival by repressing CREB-mediated IRS-2 gene expression. Our group found that SIK2 is expressed in insulin-producing β cells and regulates dynamics of insulin secretion via its kinase activity. Inhibition of SIK2 kinase activity resulted in vesicle mobilization to the membrane and increased insulin release. To further understand the machinery of the regulation of insulin secretion by SIK2, we applied Scansite database search program and found that CAPS2 (calcium-dependent activator protein for secretion 2), the vesicle related protein containing SIK2 phosphorylation consensus motif, was considered as a putative downstream substrate of SIK2. Herein, we firstly confirmed the physiological protein interaction between CAPS2 and SIK2, and found the interaction was correlated to SIK2 kinase activity. In addition, our in vitro kinase assay results provide clear evidences to show that CAPS2 is the direct substrate of SIK2. However, in SIK2/CAPS2 overexpressing mouse primary β cells, SIK2 might regulate insulin secretion via CAPS2 by sensitizing the responses to glucose in β cells. Taken together, our findings suggest that the function of CAPS2 is involved in insulin secretion and is regulated by protein kinase SIK2. Future studies are needed to reveal the mechanisms of the SIK2/CASP2 pathway in controlling insulin secretion. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4512 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 口腔生物科學研究所 |
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