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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻 | |
dc.contributor.author | Tsung-Wei Huang | en |
dc.contributor.author | 黃琮瑋 | zh_TW |
dc.date.accessioned | 2021-06-15T04:59:33Z | - |
dc.date.available | 2010-08-06 | |
dc.date.copyright | 2010-08-06 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46241 | - |
dc.description.abstract | 呼吸上皮是一種黏液纖毛組織,是呼吸道對抗外來毒素,病菌的第一道防線,臨床上,鼻部呼吸上皮的缺損容易產生一些症狀,例如鼻腦脊髓液漏的患者會有頭痛、腦部感染等症狀;鼻中膈缺損的患者常患有發炎、結痂增生、出血、甚至出現呼吸哨音的問題,而當其症狀明顯時即需要接受修補手術。到目前為止,常用的修補術式包括黏膜皮瓣、側邊鼻皮瓣、及顳骨筋膜自體皮瓣,然而這些術式常會遭遇到一些問題,包括移植部位病變,組織攣縮,或是殘存原來的組織特性等。因此,近年來發展出以生體相容性的材料來當作移植物,目前大部分的植入物是膠原蛋白製品主要是因為其具有促進呼吸上皮細胞生長分化的能力,但是卻有容易降解、機械強度不足、以及價格昂貴的問題,限制其臨床應用價值。幾丁聚醣(chitosan)是一種類似細胞外基質的醣胺多醣,具有成本低、生體降解性、無抗原性、以及易塑性等特點,在臨床上被廣泛使用在各種領域,包括藥物攜帶載體、外科縫線、以及促進傷口癒合的敷料等,然而幾丁聚醣是否適合被用來當作鼻部上皮組織的移植物以及它是否對呼吸上皮的黏液纖毛分化產生影響,並未被探討,因此本研究首先以幾丁聚醣基質膜來培養人類呼吸上皮細胞並評估其對黏液纖毛分化的影響。
首先,從人類下鼻甲分離鼻上皮細胞培養在三種材料上:幾丁聚醣膜,膠原蛋白,以及幾丁聚醣膜−膠原蛋白膜。以光學及電子顯微鏡觀察細胞型態,並以雷射共軛焦顯微鏡計算纖毛面積比率。黏液基因表現則以反轉錄聚合鏈反應評估。細胞植入後三天發現鼻上皮細胞可貼附於膠原蛋白及幾丁聚醣−膠原蛋白膜,但是並無法貼附於幾丁聚醣膜,進一步分析在膠原蛋白上,纖毛分化比率與纖毛擺動頻率相較於幾丁聚醣− 膠原蛋白並無統計學差異。三種黏液基因(MUC5AC,MUC5B,MUC2)於膠原蛋白及幾丁聚醣−膠原蛋白膜上則均有表現,且表現量並無差異。根據本研究的結果發現微量的膠原蛋白混合幾丁聚醣即可改善幾丁聚醣的生體相容性並促進鼻上皮細胞的黏液纖毛分化,與純膠原蛋白相比更可以大幅的降低成本,因此本研究首先確立幾丁聚醣−膠原蛋白膜適合當作鼻上皮細胞培養的支架,未來更可以進一步應用臨床鼻黏膜組織的修補上。 此外,臨床上,在廣泛的呼吸道缺損如氣管、支氣管腫瘤切除手術後,或是結核菌造成支氣管攣縮經手術切除,往往產生氣管支氣管組織缺損的問題,如何修補則是一大挑戰。目前新的治療策略是發展呼吸道氣管、支氣管組織工程,然而具備促進呼吸上皮細胞黏液纖毛分化的生醫材料支架,是呼吸道組織工程成功的關鍵。透明質酸是細胞外基質的主要成分具有促進細胞增生、遷移、及分化的能力,在呼吸系統則具有促進纖毛振動頻率的效果,因此透明質酸衍生物膜可能可以被應用在呼吸上皮組織工程的支架上藉以促進黏液纖毛分化。 由於透明質酸只有膠態,並不適合用來當作培養細胞的支架,因此需要在結構上改變,藉以提高其強度以及抵抗降解的能力。在過去,透明質酸衍生物膜被認為不是培養呼吸上皮細胞適當的生醫材料,然而,本研究以透明質酸苯酯化合物(HYAFFR)製成的膜顯示出其可以提供呼吸上皮細胞一個比傳統膠原蛋白支架更好的環境。呼吸上皮細胞的生長與黏液纖毛分化的程度以MTT 分析法、掃瞄式電子顯微鏡、免疫螢光染色、西方點墨法以及反轉錄聚合鏈反應檢測。雖然MTT 分析顯示呼吸上皮細胞在膠原蛋白上比在透明質酸衍生物膜上具有較快的增生能力,但是透明質酸衍生物膜卻具有比較高的促進黏液纖毛分化的能力,纖毛細胞的比率在膠原蛋白支架上為12.4%,在透明質酸衍生物膜上則提高為20.4%,並且具有假多層構造與活體上皮組織更加相近。黏液蛋白基因MUC5AC 與MUC5B 在透明質酸衍生物膜上的表現量則比在膠原蛋白上高。免疫螢光染色則顯示出培養的呼吸上皮細胞具有透明質酸受體,CD44 與透明質酸介導細胞移動受體(RHAMM)。因此,透明質酸衍生物膜具有促進黏液纖毛分化的能力並適用於未來呼吸上皮細胞組織工程的應用。 然而,有關透明質酸衍生物膜調控纖毛分化的機轉卻不清楚,透明質酸在體內經由兩個主要的受體,CD44 以及RHAMM,來影響細胞的增生與分化,其中RHAMM所扮演的角色至今仍然不清楚,雖然RHAMM 也是一種細胞表面受體但是也存在細胞質、細胞骨架、以及細胞核中執行與透明質酸相關或與透明質酸無關的功能。更重要的是,RHAMM 也是一種與微小管相關的蛋白質(microtubule-associatedprotein),跟微小管有交互作用,而微小管則是構成呼吸道纖毛的主成分。因此吾人預期RHAMM 在透明質酸衍生物膜促進呼吸上皮纖毛分化的調控上扮演一個重要的角色。此外,而根據過去的文獻,維生素A 酸是體外培養呼吸上皮細胞重要的添加物,它可以促進呼吸上皮細胞產生纖毛分化。因此,本研究將進一步探討透明質酸衍生物膜及膠原蛋白在維生素A 酸添加與否的環境中對呼吸上皮細胞纖毛分化的影響,並進一步釐清RHAMM 在促進呼吸上皮細胞纖毛分化所扮演的角色。 研究結果顯示在缺乏維生素A 酸的環境之下,只有透明質酸衍生物膜可以促進纖毛細胞分化,在電子顯微鏡顯示出聚集的纖毛構造,而在膠原蛋白上的呼吸上皮細胞則轉變為扁平且不規則的細胞。反轉錄鏈聚合反應顯示在缺乏維生素A酸之下,RHAMM 的訊息核糖核酸表現量在透明質酸衍生物膜上的組別較在膠原蛋白上增加許多;而在膠原蛋白的組別中,添加維生素A 酸的組別比缺乏維生素A 酸則有明顯的RHAMM 訊息核糖核酸表現量。因此,為了釐清RHAMM 所扮演的角色,本研究進一步以慢病毒為載體利用抗RHAMM 的shRNA 來降低RHAMM 的基因表現,結果顯示在添加維生素A 酸的膠原蛋白組別及有/無添加維生素A 酸的透明質酸衍生物膜組別,纖毛細胞分化均被明顯抑制。本研究的結果顯示透明質酸生醫材料可以經由RHAMM 的作用部分取代了維生素A 酸在呼吸上皮細胞纖毛分化上所扮演的角色,另一方面也證實了維生素A 酸對於呼吸上皮細胞纖毛分化會受到RHAMM的調控。 | zh_TW |
dc.description.abstract | Comprising mucociliary epithelium, respiratory epithelium serves as an important defense mechanism against inhaled toxins, pathogens, and particles. Discontinuity of the epithelium, e.g., CSF rhinorrhea, may cause headache and central nervous system infection. Septal perforation may cause inflammation, nasal crusting, bleeding, and whistling while breathing. To date, various surgical repairs for the septal perforation are proposed, such as advancement mucosal flaps, lateral nasal wall flaps, and autografts using temporal fascia. However, several drawbacks are encountered, i.e., donor site morbidity, tissue shortage, and retention of the original characteristics of the donor tissue. Therefore, several biocompatible biomaterials are developed to serve as grafts. Most graft materials are composed mainly of collagen, because collagen can promote epithelial growth and mucociliary differentiation. However, several disadvantages of collagen, e.g., fast biodegrading rate, low mechanical strength, and extremely high cost, restrict its clinical usage. Chitosan, like glycosaminoglycan in extracellular matrix, is characterized by its low cost, biodegradable, nonantigenic, and tailorable properties. Nevertheless, whether chitosan is exploited as a scaffold for growth and differentiation of the nasal epithelium remains unclear. The aim of this study is first to evaluate whether chitosan-based membranes can be used for culturing respiratory epithelial cells (RECs) and investigate their effect on mucociliary differentiation.
RECs are cultured on three various substrates, e.g., chitosan membranes, collagen, and chitosan-collagen membranes. Morphology of RECs is examined via light and electron microscopy, the area of ciliated cells is measured by confocal microscopy. Expression of mucin genes is investigated with reverse-transcription polymerase chain reaction. RECs are found to be successfully adhesive with collagen and chitosan-collagen membranes at day 3 after seeding, but not with chitosan membranes. The cilia area on collagen is non-significantly different from that on chitosan-collagen membranes. The expression levels of mucin genes, namely, MUC5AC, MUC5B, and MUC2, in RECs on both collagen and chitosan-collagen membranes do not differ significantly. This study demonstrates that a small amount collagen mixed with chitosan substrate may improve the biocompatibility and promote the mucociliary differentiation in RECs. It appears that chitosan-collagen membrane is a promising scaffold for culture of the nasal epithelium and can be applied for repairing nasal mucosa defect in the future. In addition, how to repair extensive respiratory tract defect after resecting trachea or bronchus due to tumor or tuberculosis remains a challenge. The new therapeutic strategy has been suggested using tissue engineered airway replacement. A key issue for this strategy is the selection of biomaterials which can facilitate growth and differentiation of RECs. Hyaluronan, one of the chief components of the extracellular matrix, contributes significantly to cell proliferation, migration, and differentiation. In the respiratory system, hyaluronan has been proposed to serve a pivotal role in mucosal host defense by stimulating ciliary beating of RECs. Therefore, we hypothesize that hyaluronan may serve as the ideal scaffold for tissue engineering of respiratory epithelium. However, unmodified hyaluronan is found only in gel form and possesses a very short degradation time. For it to be used in tissue engineering, it would need to be chemically modified to improve its structural properties and increase its resistance to degradation. Previously, hyaluronan derivatives were considered as unsuitable biomaterials for culture of respiratory epithelium. In contrast, this study demonstrates that the membranous scaffolds made from benzyl esters of hyaluronic acids (HYAFFR) are capable of providing a more preferential environment for human RECs than conventionally used collagen-based scaffolds. The proliferation and mucociliary differentiation of RECs were examined by MTT assays, scanning electron microscopy, immunofluorescence, immunoblotting and gene expression. Although MTT reveals that RECs on collagen have higher proliferation rates than that on HYAFF, HYAFF promotes more ciliary differentiation of RECs than collagen. The percentage of ciliated cells in cultured RECs increases from 12.4% on collagen to 20.4% on HYAFF with a pseudostratified polarized layer that closely resembles the composition of the native epithelium. The expression levels of MUC5AC and MUC5B mRNA are higher on HYAFF than those on collagen. The presence of a hyaluronan-binding domain, CD44 and the receptor for hyaluronan-mediated motility (RHAMM) of RECs are also demonstrated. Accordingly, the mucociliary differentiation-promoting effect of hyaluronan-derivative membranes indicates that it may be applied to the tissue engineering of respiratory epithelium. However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. Hyaluronan influences proliferation and differentiation of various cells through two main receptors, CD44 and RHAMM. RHAMM remains the most poorly understood hyaluronan receptor as it can act as a cell surface receptor but it can also be localized in the cytoplasm, cytoskeleton, or in the cell nucleus, displaying both hyaluronan dependent and independent functions. Additionally, RHAMM also functions as a microtubule-associated protein interacting with microtubules, which are the components of cilia. Therefore, it is anticipated that RHAMM may play a role in regulating the ciliary differentiation of RECs on hyaluronan-based biomaterials. Additionally, based on the literature, retinoic acid (RA) is commonly used as a constant medium supplementation to promote ciliary differentiation of RECs in vitro. The aim of the study is to investigate the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA and further to elucidate the role of RHAMM in promoting ciliary differentiation of RECs. Analytical results of culturing RECs on collagen and HYAFF indicate that only HYAFF can increase the ciliary differentiation of RECs under RA-free conditions. The expression level of RHAMM mRNA of RECs more significantly decreases on collagen than that on HYAFF without RA. RECs on collagen with RA also express higher level of RHAMM mRNA than those without RA. Therefore, by using lentiviral vector-based short hairpin RNA targeting RHAMM, the study further reveals that knockdown of RHAMM obviously inhibits the ciliary differentiation of RECs on collagen with RA and on HYAFF with/without RA. In addition to demonstrating that hyaluronan-based biomaterials partially “replace” RA in the ciliary differentiation of RECs, which is regulated by RHAMM, this study establishes that RHAMM regulates the ciliary differentiation-promoting effect of RA on RECs. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:59:33Z (GMT). No. of bitstreams: 1 ntu-99-D96548018-1.pdf: 7831494 bytes, checksum: 7cdc58ef8e49e2a8caa4b9c38e2ec103 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目 錄
口試委員會審定書……………………………….… i 誌謝…………………………………………………… ii 中文摘要…………………………………………….. iii 英文摘要………………………………….……...... vi 縮寫表…..................................... xi Chapter 1: Background and Introduction……... 1 Chapter 2: Culture of respiratory epithelial cells using chitosan-based membranes 2.1 Materials and Methods…………….. 7 2.1.1 Preparation of membranes………………………. 7 2.1.2. Isolation and culture of human respiratory cells 7 2.1.3. Morphological examination…………………… 8 2.1.4. Immunocytochemistry ………..……………. 9 2.1.5. Examination of mucin genes…….………….. 9 2.2. Results………………….….………………. 11 2.2.1. Attachment and proliferation of RECs……….. 11 2.2.2. Morphology and ciliogenesis of RECs…… 11 2.2.3. Genetic expression of mucin of RECs…… 12 2.3. Discussion…………………………………… 13 Chapter 3: Increased mucociliary differentiation of human respiratory epithelial cells on hyaluronan derivative membranes 3.1. Materials and Methods………………………… 20 3.1.1 Preparation of membranes…………………... 20 3.1.2. Isolation and culture of human RECs …… 20 3.1.3. MTT assay………………………………… 22 3.1.4. Morphological examination……………… 22 3.1.5. Immunocytochemistry ……….………... 23 3.1.6. Western blot analysis…………….. 24 3.1.7. Reverse transcription polymerase chain reaction for MUC5AC and MUC5B.. 24 3.1.8. Assessment of hyaluronan binding domain of RECs… 25 3.1.9. Statistical analysis……………………… 25 3.2. Results……………………….……………. 27 3.2.1. Attachment and proliferation of RECs on collagen vs. HYAFF… 27 3.2.2. Morphology and ciliogenesis of RECs on collagen vs. HYAFF …. 28 3.2.3. Expression of mucin genes of RECs on collagen and HYAFF….. 29 3.2.4. Expression of hyaluronan binding domain of RECs on HYAFF… 30 3.3. Discussion………………………………….………… 32 Chapter 4: Regulation of ciliary differentiation of human respiratory epithelial cells by the receptor for hyaluronan-mediated motility on hyaluronan-based biomaterials 4.1. Materials and Methods………………………………… 49 4.1.1 Preparation of membranes…………………………... 49 4.1.2. Isolation and culture of human RECs …………… 49 4.1.3. Morphological examination………….…………… 51 4.1.4. Immunocytochemistry ………………..………... 51 4.1.5. Reverse transcription polymerase chain reaction (RT-PCR)…. 52 4.1.6. Western blot analysis…………………………….. 52 4.1.7. Transduction of lentiviral vector-based short hairpin RNA (shRNA) against RHAMM……………………. 53 4.1.8. Statistical analysis……………………………… 54 4.2. Results……..………………….………………….. 55 4.2.1. Morphology and ciliogenesis of RECs on HYAFF vs. collagen 55 4.2.2. Genetic expression of RHAMM and CD44 of RECs on HYAFF vs. collagen.. 56 4.2.3. Inhibition of ciliogenesis for RECs on HYAFF and collagen by RNA interference……..…………………….... 57 4.3. Discussion…………………………………… 60 Chapter 5: Conclusion and future prospects……… 75 Chapter 6: References………………………...... 79 Chapter 7: Clinical and biomechanical analyses to select a suture material for uvulopalatopharyngeal surgery ……. 91 Chapter 8: List of publications ……….………….. 116 圖表目錄 Chapter 2 Figure 1 RECs cultured on chitosan, collagen and chitosan-collagen membranes at day 3 after seeding and day 21 after confluence…..…. 16 Figure 2 Scanning electron microscopy of human RECs on collagen and chitosan-collagen membranes………………. 17 Figure 3 Confocal images of RECs cultured at day 21 after confluence… 18 Figure 4 RT-PCR analyses of mucin genes expression in cultured RECs on collagen and chitosan-collagen membranes express MUC5AC, MUC5B and MUC2… 19 Chapter 3 Table 1 Oligonucleotide primer sequences utilized in the RT-PCR…… 40 Figure 1 Human respiratory epithelial cells cultured on collagen and HYAFF on day 3 after seeding.…………. 41 Figure 2 MTT assays of RECs cultured on collagen and HYAFF…… 42 Figure 3 Scanning electron micrographs of RECs on collagen and HYAFF… 43 Figure 4 Typical appearance of RECs cultured on collagen and HYAFF on day 21 after confluence. Shown are fluorescent microscopy images of cilia on collagen compared with HYAFF)………………….……. 44 Figure 5 Western blot analysis of acetylated tubulin and actin proteins in RECs…… 45 Figure 6 Representative confocal images are shown of RECs cultured on day 21 after confluence……..…… 46 Figure 7 RT-PCR analysis for mucin genes expression in RECs…… 47 Figure 8 Confocal images of immunofluorescent staining of RECs cultured on HYAFF on day 21 after confluence with anti-CD44 and anti-RHAMM antibodies…….… 48 Chapter 4 Table 1 Oligonucleotide primer sequences utilized in the RT-PCR…… 65 Figure 1 Human respiratory epithelial cells cultured on HYAFF and collagen with/without retinoic acid on ALI day 21…66 Figure 2 Scanning electron micrographs of RECs on HYAFF and collagen with/without retinoic acid on ALI day 21……………….. 67 Figure 3 Western blot analysis of acetylated tubulin and actin proteins in RECs on ALI day 21………………………………………. 68 Figure 4 RT-PCR analysis of RHAMM and CD44 genes expression in RECs on ALI day 21………………………………………….. 69 Figure 5 Lentiviral-based shRNA against RHAMM used to knock down RHAMM in RECs……………………………………..……… 71 Figure 6 Typical appearance of RECs cultured on collagen under RA-supplemented conditions on ALI day 21 after transduction of anti-RHAMM shRNA and controls 72 Figure 7 RECs cultured on HYAFF under RA-supplemented conditions on ALI day 21 after transduction of anti-RHAMM shRNA and controls……. 73 Figure 8 RECs cultured on HYAFF under RA-free conditions on ALI day 21 after transduction of anti-RHAMM shRNA and controls…………… 74 Chapter 5 Figure 1 Microporous membranes of various synthetic biomaterials under SEM… 77 Figure 2 Wound healing assay of RECs on various synthetic biomaterials……78 Chapter 7 Figure 1 CONSORT flow diagram………………….…. 111 Figure 2 Wound healing on postoperative day 7 using different sutures…… 112 Figure 3 Percentage change in Young’s modulus of four sutures over postoperative time…………………………....… 113 Figure 4 Scanning electron micrographs of four sutures on postoperative days. 114 Figure 5 Percentage of saliva absorbed by various sutures as a function of time 115 | |
dc.language.iso | en | |
dc.title | 生醫材料於呼吸上皮組織工程之應用 | zh_TW |
dc.title | The applications of biomaterials in tissue engineering of respiratory epithelium | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 楊泮池,楊怡和,侯勝博,宋信文,孫一明,鄧文炳 | |
dc.subject.keyword | 幾丁聚醣,膠原蛋白,透明質酸,呼吸上皮細胞,黏液纖毛分化,維生素A酸,透明質酸介導細胞移動受體, | zh_TW |
dc.subject.keyword | Chitosan,Collagen,Hyaluronan,Respiratory epithelial cells,Mucociliary differentiation,Retinoic acid,Receptor for hyaluronan-mediated motility (RHAMM), | en |
dc.relation.page | 116 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-29 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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