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標題: | 調控胰島細胞群集行為以探討再生胰島建構之條件 Modulating cellular clustering behaviour for regenerative islet formation |
作者: | Yun-Hsuan Chang 張芸瑄 |
指導教授: | 周涵怡(Han-Yi Chou) |
關鍵字: | 胰島細胞,Pannexin1,血清素,胰島細胞構型,胰島素分泌, Pancreatic islet cell,Pannexin1,Serotonin,Islet cell constitution,Insulin secretion, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 在現今社會中,隨著生活型態的改變以及高糖高脂食物攝取量增加,罹患糖尿病的人口也逐年遞增。根據世界衛生組織的統計,每11人中就有一人罹患糖尿病,且預估到了2045年,全球糖尿病人口將會暴增至629百萬人之多。針對糖尿病患者,多以藥物以及胰島素注射的方式控制血糖濃度。然而,藉由外在藥物控制,無法給予病患及時的血糖調控。
幹細胞誘導胰島細胞移植是一種不僅能從根本解決病患及時血糖控制的問題,也大幅度降低移植器官來源不足以及排斥的問題。本所以研究口腔生物科學為導向,也包含口腔幹細胞相關的研究。近年來,由於口腔幹細胞取得較其他幹細胞容易,因此許多研究著重在口腔幹細胞的分化以及如何應用在幹細胞治療。目前大多數的研究多著重在幹細胞誘導胰島素分泌細胞再生的相關研究。然而,胰島細胞是由五種細胞所組成,並藉由連接蛋白互相在細胞間傳遞訊號,建立協調系統,進而調節不同激素分泌。若僅以單一種細胞作為再生胰島的來源,可能因失去不同細胞間緊密的連結以及調控系統,致使單一種細胞的再生胰島在移植後無法達到較好的功能以及移植體存活率較低的問題發生。 因此,本研究希望觀察胰島細胞在重組過程中,細胞排列以及細胞間蛋白的表現。並探討若是在重組過程中加入神經傳導物質,是否會提升細胞間蛋白的表現,藉以提升移植後的功能性以及存活率。 首先,以平面培養打散後的胰島細胞,發現在第四天,胰島細胞會聚集成一定大小的群落,並以免疫螢光染色法觀察到細胞群落中,與原生胰島相似,不同型態的細胞會有互相接觸的現象 。然而,這些細胞群落在葡萄糖刺激下,胰島素分泌的量與原生胰島有極大的差距。 由於在自然的情況下,胰島細胞是橢圓形立體結構,有可能重組胰島的型態會影響到功能的表現。因此採取懸滴式培養的方式,建立具有立體構型的重組胰島。在懸滴式培養的第三天,可以看到有與原生胰島型態相似的類胰島細胞成型。然而,這些類胰島在葡萄糖刺激下,胰島素分泌的量與胰島細胞群落是相似的。因此我們藉由免疫螢光染色, 發現與原生胰島相比,這些重組後的類胰島的pannexin1蛋白(一種調控細胞與細胞溝通的蛋白,其表現會影響到胰島細胞中激素的分泌)幾乎沒有表現。 有鑒於血清素可以刺激pannexin1蛋白的表現,因此,我們在胰島細胞重組的過程中,加入血清素,觀察到在血清素的濃度為10µL的培養條件下,pannexin1蛋白的表現有顯著的增加,同時,對於葡萄糖刺激下胰島素的分泌量也較未受到血清素刺激的類胰島佳。 此外,我們將未受血清素刺激的類胰島以及受到血清素刺激的類胰島,移植到小鼠體內,並進行組織切片,觀察到受到血清素刺激的類胰島,在移植後兩週存活的細胞量多於未受血清素刺激的類胰島。 本研究藉由觀察胰島細胞重組的過程,可以了解到建立胰島的立體構型,並利用血清素提升重組胰島的GSIS能力,以及移植後細胞的存活率,對於未來在建構再生胰島方面的研究是不可或缺的條件。 Islet auto-transplantation is a thoroughly treatment to achieve blood sugar homeostasis, that would potentially overcome the limitation of scarcity of donor and immune-rejection. However, without a proper guidance to achieve correct architectural conformation of natural islets, the regenerated islet cell would have lower survival rate and inefficient Glucose-Stimulated Insulin Secretion (GSIS) response after transplantation. Hence, we aim to design a reconstitution assay to observe the behavior of islets cell reconstitution, and improve the function and survival of reconstituted islet cells. First of all, we observed the islet single were able to forming the islet-like cluster with insulin expression in culture plate. Moreover, being similar with primary islet cell, these islet-like cluster were heterotropism cell clusters with both homotropism cell contacts and heterologous cell contacts. However, the result of Glucose Stimulated Insulin Secretion(GSIS) indicated these islet-like cluster had poor ability of insulin secretion. In view that primary islet cell has three-dimension structure. We supposed that the difference of morphology between primary islet cell and reconstituted islet cell would influence the insulin secretion. To construct reconstituted islet cell with similar morphology with primary islet cell, we cultured islet single cells by hanging-drop method. The pseudo-islet cell would be constructed after 60 hours of culture. However, these pseudo-islet cells had poor ability of insulin secretion as same as islet-like cluster. Furthermore, we found that in pseudo-islet cell, pannexin1, a kind of protein, regulating intercellular communication and hormone secretion in islet cell, had mere expression. In light that serotonin could promote pannexin1 expression, we reasoned that supplementation of serotonin would improve pannexin1 expression in reconstituted islet cell, promoting intercellular communication and insulin secretion. With 10 µM serotonin stimulation, it obviously increased pannexin1 expression in pseudo-islet cells. Moreover, the outcome of GSIS was better than the pseudo-islet cell without serotonin treatment. In addition, we transplanted these pseudo-islet cells into wildtype C57BL/6J mice. After 14 days of transplantation, the cell survival of pseudo-islet cell treated with serotonin was better than the pseudo-islet cell without serotonin treatment. Hence, we can conclude that serotonin intervention would improve pannexin1 expression, promoting GSIS function and cell survival after transplantation in reconstituted islet cell. By modulating islet cell reconstitution, this study demonstrates that achieving proper cell architecture, improving GSIS and cell survival after transplantation are indispensable factors when constructing regenerative islet cell in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21269 |
DOI: | 10.6342/NTU201903521 |
全文授權: | 未授權 |
顯示於系所單位: | 口腔生物科學研究所 |
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