複合材料性質對細胞交互作用影響之研究

The Study of Interaction Between Novel Bio-materials and Human Cells

施乃嘉(Nai-Chia Shih)

指導教授 ： 林欣杰

國立交通大學/工學院/奈米科技研究所/碩士(2018年)

摘要

儘管生物醫學技術蓬勃的發展，但許多國家的慢性病罹患率仍居高不下。在許多國家的醫療健保系統中，慢性疾病不僅造成巨大的經濟負擔，情緒壓力並影響患者及家人的生活質量。因此目前需要並進一步改良目前慢性病的治療方法。本文研究了開發性的複合材料應用，希望將之應用於慢性病症上之治療。
第一個研究題目主要是開發一種新型的PLA-Gelatin貼片藥劑，用於對抗壓瘡之病症，這種疾病是對於臥床不起或被限制在輪椅上的患者來說是非常常見的。因此為了促進褥瘡病症傷口的癒合，我們開發了一種溫度敏感性的凝膠貼劑系統，該系統可以裝載微脂體脂質體，並在體溫下釋放它們。由於氧化壓力是壓力型潰瘍進展的主要原因。Tempamine（TMN）是一種抗氧化劑，用來被包覆於脂質體內部之藥物（Lipo-TMN）。隨後將Lipo-TMN包埋在PLA-明膠系統中，其中脂質體藥物能在33℃下成功自明膠內釋放。由於皮膚傷口的閉合需要上皮形成及血管再生成，因此在此我們使用過氧化氫（H2O2）處理的過的人類上皮細胞（HaCaT）與人類臍帶靜脈內皮細胞（HUVEC）作為釋放含有Lipo-TMN的明膠治療組別，與接受含有TMN的PLA-Gelatin中系統組別相比，接受含有Lipo-TMN的PLA-Gelatin細胞別中出現了更好的細胞活力，這也表明脂質體維持了TMN抗氧化之活性。在添加含有Lipo-TMN的PLA-明膠系統所處理的細胞內，細胞在表達凋亡基因中的蛋白質有顯著下降的現象。以所有實驗數據表明PLA-明膠系統在33℃釋放Lipo-TMN是可有效減輕人類上皮細胞（HaCaT）與人類臍帶靜脈內皮細胞（HUVEC）內的氧化損傷。
論文的第二部分是開發一種新型3D列印墨水配方，可應用於需要使用血液透析的終末期腎臟病（ESRD）患者，可客製化製制及設計動靜脈移植管（AVG）。由於血小板粘附是導致AVG功能失調的主要因素，如果不立即解決則會導致死亡，因此重要的是新開發一種含有抗沾粘特性的3D列印墨水。因此本實驗篩選了幾種以有研究作為抗沾粘材料的3D列印墨水，故為了確保材料確實可以有效防止血漿蛋白吸附和血小板粘附，我們將新型開發的材料以3D列印方式列印出，並將之浸沒在富含人血小板的血漿（PRP）中，並對血纖維蛋白原和血小板的進行免疫染色，或是將材料長時間浸泡於胎牛血清蛋白 ( FBS )中，對人類臍帶靜脈內皮細胞 ( HUVEC ) 進行細胞貼附實驗，相信這種新穎的配方將有助提升及促進ESRD患者在AVG的使用上有更好的生活質量，因為他們不必再因為管徑狹窄而不斷更換植入之AVG。

關鍵字

微脂體 ； 氧化壓力 ； 雙層貼片釋放系統 ； 積層製造 ； 抗沾黏 ； 溫感型釋放貼片

並列摘要

Despite of recent advances in the development of biomedical technology, prevalence of chronic diseases remains high in many countries. Not only it is a huge financial burden in many countries’ healthcare systems, chronic diseases also create emotional toll and affect quality of life of patients and their family. Therefore, there is an urgent need to continue to build on the current understanding and further improve current treatments for chronic diseases. Two different chronic diseases were investigated in this thesis, and that two different novel technologies were developed.
The first chronic disease that was investigated was pressure ulcer. The disease is common to patients that are bedridden or confined to a wheelchair, in which localized damage occur in the skin as a result of pressure. If the wound is not closed, the disease can result in serious infections and kidney failure. Therefore, to promote wound closure, a temperature sensitive gel-patch system in harboring drug-loaded liposomes was developed and released them at body temperature. Since oxidative stress is a major cause of progression of pressure ulcer, tempamine (TMN), an antioxidant, was formulated into a liposomal drug (Lipo-TMN). Subsequently, Lipo-TMN were embedded in PLA-Gelatin system, in which the liposomal drugs could only be successfully released at 37C. Since epithelialization and angiogenesis are the two major events that need to happen for a successful skin wound closure, the therapeutic efficacy of the released Lipo-TMN were investigated in human epithelial cells (HaCaT) and human endothelial cells (HUVECs) that were treated with hydrogen peroxide (H2O2). Better cell viability was noticed in cells that received Lipo-TMN that embedded in PLA-Gelatin compared to the cells received TMN embedded in PLA-Gelatin, suggesting liposomes were required to sustain the anti-oxidant activity of TMN.. Expressions of apopotic genes and proteins were significantly downregulated in cells treated with Lipo-TMN embedded in PLA-Gelatin. Collectively, the data indicated the PLA-Gelatin system was effective in releasing Lipo-TMN at 37C and the released Lipo-TMN were able to alleviate the oxidative damages in human epithelial and endothelial cells.
The second part of the thesis was to develop a novel 3D printing ink formulation that could be used to fabricate custom-designed arteriovenous graft (AVG) for patients with end renal stage disease (ESRD) that requires hemodialysis. Since platelet adhesion is a major factor that resulted in dysfunctional AVG, which can cause mortality if not address immediately, therefore it is important the newly developed 3D printing ink formulation contain anti-adhesive properties. Several anti-adhesive materials were screened for their ability to be 3D printed. To ensure the materials could indeed prevent plasma protein adsorption and platelet adhesion, the 3D printed AVGs were submerged in human platelet rich plasma (PRP), and the presence of fibrinogen and platelets were immunostained. It is believe this novel formulation will be useful in the future development of a custom-designed AVG for ESRD patients, which will promote a better quality of life as they don’t have to have their implanted AVGs constantly replace because of restenosis.

並列關鍵字

Liposome ； Tempamine ； 3D printing ； Anti-fouling ； oxidative stress ； Thermal patch

參考文獻

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