研究生: |
王湘華 Hsiang-Hua Wang |
---|---|
論文名稱: |
小分子量絲膠蛋白在幹細胞分子生物學及免疫學上的影響 Effects of Low Molecular Weight Sericin on Stem Cell Molecular Biology and Macrophage Immunology |
指導教授: |
洪伯達
Po-Da Hong |
口試委員: |
程君弘
Juin-Hong Cherng 劉正哲 Chen-Che Liu 蔡協致 Hsieh-Chih Tsai |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 材料科學與工程系 Department of Materials Science and Engineering |
論文出版年: | 2018 |
畢業學年度: | 106 |
語文別: | 英文 |
論文頁數: | 54 |
中文關鍵詞: | 蠶絲 、絲膠 、小分子量 、高溫高壓脫膠 、幹細胞 、巨噬細胞 、免疫反應 、人類細胞激素 、斑點黏合 、即時聚合酶鏈鎖反應 |
外文關鍵詞: | Silk, Sericin, Low molecular weight, HTHP degumming, Stem cell, Macrophage, Immune response, Human cytokine, Focal adhesion, qPCR |
相關次數: | 點閱:314 下載:2 |
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絲膠蛋白(Sericin)為蠶絲蛋白的一種,許多文獻已經證實了絲膠蛋白擁有良好的生物降解性、生物相容性及抗菌、抗紫外線等特性,是胺基酸組成相當豐富的天然高分子。並且,從文獻中發現水溶性的絲膠蛋白對於細胞貼附和組織工程之發展能提供相當的助益,驅使在細胞生物學應用上產生本研究主題與方向。另外,為了避免大分子量蛋白可能造成人體的過敏反應,以及提升材料進入皮膚表層的效率,此研究著重在於提取分子量小於10 kDa的絲膠蛋白進行細胞生物實驗。
本研究目的在於了解小分子量絲膠蛋白作用於幹細胞貼附及巨噬細胞產生的免疫反應情形。利用高溫高壓反應(HTHP)將蠶繭中的絲膠與絲素進行分離,分離的過程稱為脫膠(Degum);相較於其他添加物(酵素和化學物)的脫膠方法得到的絲膠蛋白斷裂的點與分子量固定,而利用高溫高壓的方式,絲膠藉由熱能得以全面性斷鍵,不僅可以簡化脫膠程序,還能避免添加物汙染,得到純度較高的小分子量絲膠蛋白。脫膠過後的絲膠蛋白,會利用高效能過濾膜(Ultrafiltration Membrane System),針對鎖定的分子量範圍進行篩選,同時,利用傅立葉轉換紅外光譜(FTIR)對高溫高壓脫膠後的絲膠產物進行二級結構鑑定,確保溫度及壓力的作用下,不會影響蠶絲蛋白結構。分子量篩選過的絲膠蛋白應用不同的分子量鑑定技術確認其分子量,包括十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (SDS-PAGE)、膠體滲透層析儀(GPC)、液相層析質譜儀(LC-MS)和基質輔助雷射脫附游離飛行質譜儀(MALDI-TOF),結果比對之後,可以得到在預期10 kDa以下,接著將絲膠材料與細胞培養進行實驗。
小分子量絲膠蛋白首先經過人體皮膚纖維母細胞(WS1)共同培養的生物相容性測試,相較於尚未分離分子量的絲膠和大分子量的絲膠結果,都顯示小分子量的絲膠明顯提升細胞存活率。而材料的免疫反應亦是未來實際應用在生物體上的一大課題,本研究是利用人類巨噬細胞株(U937)與小分子量絲膠進行共同培養,觀察細胞內部之基因表達,發現絲膠對於巨噬細胞免疫反應的調節作用,其結果與巨噬細胞分泌的功能性蛋白表現一致,得到了小分子量絲膠不會引發巨噬細胞活化,甚至可以調控輕微發炎反應的重要結論。另外,將人體口腔脂肪幹細胞植入到含有小分子量絲膠塗層的培養盤中,以正常的培養條件下進行培養,觀察絲膠蛋白是否影響細胞存活率,並且利用即時聚合酶鏈式反應(qPCR)和細胞激素蛋白質濃度分析(Cytokine Assay),同時查看幹細胞內的基因表現和釋放的功能性蛋白表現,推測出絲膠蛋白影響幹細胞的訊息傳導路徑,希望未來能夠由此發展絲膠蛋白在幹細胞的多元應用。
Sericin is a kind of globular protein and it is a natural polymer with biodegradable, biocompatible, and anti-bacterial characteristics. Due to the multiple amino acid and water-soluble properties, it provides more conducive to use in cell adhesion for tissue engineering. And low molecular molecules can easily permeate into human skin and to avoid allergen.
This research aimed to harvest the lower molecular weight of sericin (LMw-sericin) and its use to study the influence of the stem cell focal adhesion and macrophage immune responses. In order to harvest the low molecular weight of sericin, firstly, the silkworm Bombyx mori cocoon have been degummed and hydrolysed by using the condition of high-temperature and high-pressure (HTHP). The degumming material has filtered with ultrafiltration membrane system in order to gather low molecular weight of sericin. The low molecular weight of obtained sericin was determined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel permeation chromatography (GPC), liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization- time of flight (MALDI-TOF). The results showed that the molecular weight of the harvested sericin was consistently below 10 kDa.
In the stem cell boost and induction system, the low molecular weight of sericin was coated on plates before cells injected into maintained culture medium. Further, this study focused on the gene expression of focal adhesion, human cytokines and chemokines was analyzed by quantitative real-time polymerase chain reaction (qPCR) and cytokine assay. Then, the toxicity of sericin and gene expression were compared. The gene expression of stem cell has been shown that the pathways and it leads to motility, survival and proliferation. Furthermore, sericin-induction macrophages indicate that the regulation of the immune responses and appear without violent activation. Overall, based on the findings, it is believed that the low molecular weight of sericin can regulate cellular inflammatory responses and have potential applications in stem cell biology.
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