請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88022
標題: | 層粘連蛋白改質褐藻酸微米球包覆人參皂苷及脂肪衍生幹細胞應用在乳房腫瘤切除術後乳房重建技術 Synthesis, Characterization, and Evaluation of Laminin-Alginate Microspheres Encapsulated with Ginsenoside Rg1 and ADSCs for Breast Reconstruction after Lumpectomy |
作者: | 楊易軒 I-Hsuan Yang |
指導教授: | 林峯輝 Feng-Huei Lin |
共同指導教授: | 陳右昇 Yo-Shen Chen |
關鍵字: | 層粘連蛋白改質褐藻酸微米球,脂肪衍生幹細胞,人參皂苷Rg1,乳房重建手術,幹細胞療法, laminin-alginate microspheres,adipose-derived stem cells,ginsenoside Rg1,breast reconstruction,stem cell therapy, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 乳房重建手術是讓女性罹患乳癌患者在進行手術切除乳房後的一種重建方式,儘管目前市面上已經有很多被開發的技術,但仍存在很多的問題,例如:義乳植入產生的異物反應造成莢膜攣縮、皮瓣手術所需的自體組織不足以及植入物易發生再吸收導致頻繁的手術需求等等。另一方面,組織工程已越來越被廣泛於研究中,利用組織工程來重建各個損傷部位已經成為了趨勢。本論文假說為使用幹細胞和可促進血管新生的小分子,同時包覆於生物材料之中形成微米球,與自體的脂肪細胞共混形成可注射的組織工程化脂肪組織,經過體內注射植入和數周的分化,幹細胞可以分化成脂肪細胞,促血管新生的小分子使組織間新生許多血管,可提高注射進去的細胞存活率以減緩植入物再吸收的情形。
本研究中,將透過低濃度的高碘酸鈉對褐藻酸進行改質,並將層粘連蛋白修飾於褐藻酸上,與培養基中的脂肪衍生幹細胞和人參皂苷Rg1共混,透過生物電噴灑技術,噴入氯化鈣溶液中,交聯形成層粘連蛋白改質褐藻酸微米球(ADSC-G-LAMS)。包覆脂肪衍生幹細胞和人參皂苷Rg1的微米球會與自體的脂肪細胞共混形成可注射的工程化脂肪組織,自體細胞釋放出的旁分泌訊號可以使脂肪衍生幹細胞走向脂肪新生分化。本實驗開發的材料備製成可注射的形式並用於乳房重建。利用傅立葉變換紅外分光光度計、光學顯微鏡、掃描式電子顯微鏡分別對ADSC-G-LAMS進行官能基鑑定、球尺寸量測和微結構的觀察;高效液相層析儀評估了人參皂苷Rg1從微米球中釋放情形;用HUVEC細胞體外管形成試驗評估從微米球釋放之人參皂苷Rg1的成血管能力;細胞活性、細胞毒性、基因毒性分別透過WST-1,活/死染色和染色體結構異常分析進行評估,確認設計的G-LAMS的安全性;通過定量即時聚合酶鏈鎖反應確定PI3K,Akt和eNOs的基因表現量評估G-LAMS微米球的血管生成能力,將脂肪細胞和微米球結合以注射形式用於大鼠體內研究。用組織學染色和免疫螢光染色評估動物實驗結果;全血分析和血清學分析評估已開發的脂肪細胞和ADSC-G-LAMS微米球組合的安全性。 結果表明ADSC-G-LAMS微米球的大小約為232.42 μm,提供一個很好的環境供細胞存活、生長,並不具任何的細胞毒性、基因毒性和全身性毒性。ADSC-G-LAMS微米球擁有兩階段的藥物釋放,人參皂苷Rg1可以透過PI3K/AKT/eNOs路徑影響HUVEC細胞,並提升血管細胞35.9%的血管形成能力。動物實驗部分,本研究開發的ADSC-G-LAMS微米球可以很好地與宿主脂肪組織整合,持續釋放的人參皂苷Rg1造成組織具有適當血管生成能力,可應用於乳房重建手術。 Numerous advancements have been made in the field of breast reconstruction following lumpectomy. While these technologies have shown promising results in clinical settings, there are still several challenges that researchers must address. Tissue engineering has emerged as a potential solution for breast reconstruction after lumpectomy, offering hope to patients. In this study, a new approach utilizing laminin-modified alginate (abbreviated as ADSC-G-LAMS) was developed using a low concentration of sodium periodate. This modified alginate was mixed with ADSCs and Rg1 in a medium and then transformed into microspheres (ADSC-G-LAMS) using a bio-electrospray technique with a power syringe. The ADSC-G-LAMS microspheres were collected and combined with adipocytes to stimulate the production of necessary growth factors, guiding the ADSCs towards the adipogenic pathway. The resulting combination of ADSC-G-LAMS microspheres and adipocytes was prepared in an injectable form for breast reconstruction. The synthesized laminin-modified alginate microspheres loaded with Rg1 (G-LAMS) were characterized using various techniques such as Fourier transform infrared spectrophotometry (FTIR), optical microscopy, and scanning electron microscopy (SEM) to identify functional groups, measure sphere size, and examine microstructure, respectively. The release profile of Rg1 from the G-LAMS microspheres was evaluated using high-performance liquid chromatography (HPLC). The ability of the developed G-LAMS microspheres to support vascularization was assessed through the HUVEC tube formation assay using MatrigelTM. Safety evaluations, including cell viability, cytotoxicity, and genotoxicity, were conducted using WST-1, live/dead staining, and chromosome aberration tests, respectively. The angiogenic potential of the G-LAMS microspheres was assessed by analyzing gene expression levels of PI3K, Akt, and eNOs using real-time quantitative PCR (Q-PCR). An in vivo study using SD rats involved combining the adipocytes and microspheres in an injectable form. The efficacy of the treatment was evaluated through hematoxylin & eosin (H&E) and immunofluorescence staining. Additionally, blood element analysis and serological analysis were conducted to assess the safety of the combined treatment of adipocytes and ADSC-G-LAMS microspheres. The results demonstrated that ADSC-G-LAMS microspheres successfully integrated with the host adipose tissue, promoting angiogenesis through the sustained release of Rg1 for breast reconstruction. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88022 |
DOI: | 10.6342/NTU202301268 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 醫學工程學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-111-2.pdf | 3.5 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。