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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 謝銘鈞 | |
| dc.contributor.author | Ming-Chia Yang | en |
| dc.contributor.author | 楊明嘉 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:10:04Z | - |
| dc.date.available | 2013-02-11 | |
| dc.date.copyright | 2010-02-11 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-02-01 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45234 | - |
| dc.description.abstract | 生物系統中，蛋白質及聚醣類對於細胞活性及分化扮演很重要的角色，尤其對於細胞間質的合成細胞分化。本研究使用新的方法將蠶絲蛋白(SF)與幾丁聚糖(CS)和玻尿酸(HA)使用噴霧乾燥的方式製備成微粒再以壓錠的製程製備薄膜，以再生醫學觀念將間葉幹細胞(MSC)誘導分化後植入心肌壞死部位。並探討MSC表面抗原CD44與材料表面HA對rMSCs分化及生長影響。在材料特性方面，以ATR-FTIR來評估材料表面官能基，發現在SF掺合CS後會有ㄧ級胺平移的現象發生，SF的1654cm-1與CS的1634cm-1平移至1645cm-1，但HA許多特性波峰與CS相似，之後會使用靜態接觸角及Toluidine Blue O (TBO)來分析SF薄膜上的HA。靜態接觸角及TBO的結果可證明SF薄膜表面含有大量的HA。以上的結果證明我們成功製備SF/CS、SF/HA及SF/CS-HA三種材料。之後大鼠間葉幹細胞(rMSCs)培養在經不同改質蠶絲蛋白薄膜上，MTT細胞活性測試中發現在第七天SF/CS patch以及SF/CS-HA patch都比SF patch MTT數值來的高。之後使用CD44-blockage將rMSCs表面CD44 block後探討SF表面HA是會為影響rMSCs增生，與未blockage rMSCs相比其細胞生長有意義降低甚至比培養在SF patch的數值都來的低。在fibronectin免疫染色中也發現rMSC在SF/HA 未經CD44-blockage其fibronectin的分泌也是最多的。證明rMSCs表面CD44會與HA作用增加生長活性且CS也可以促進rMSCs的生長。使用5aza分化rMSCs使其產生cardiomyogenic，利用real time PCR及免疫螢光染色來分析分化後心肌特殊基因及蛋白的表現，在SF/CS-HA patch與其他兩種材料SF及SF/CS patch比較，其心肌基因的表現最高，而免疫螢光染色也有相似的結果。因此我們近一步探討是否材料表面的HA會與rMSCs的CD44作用來影響其分化，我們比較CD44-blockage及未blockage rMSCs表面CD44培養於SF/HA表面上來探討心肌特殊基因表現。研究結果顯示rMSCs 未用CD44-blockage培養於SF/HA patch其Gata4, Nkx2.5, Tnnt2 及 Actc1心肌特殊基因表現都比SF及經CD44-blockage施用來的高。免疫螢光染色分析結果顯示，未blockage rMSCs表面CD44培養於SF/HA表面其TroponinT、Cardiotin及connexin 43蛋白質表現也是rMSCs 未施用CD44-blockage培養於SF/HA patch最高。在動物實驗中，先將大鼠左心室壁以cryoinjuried的方式造成心肌壞死後，分別植入SF/CS-HA及SF/HA patch。經兩個月後以超音波心電圖觀察，植入patch組其左心室終端收縮及舒張距離都比cryoinjured組有意義的低。在心肌短縮分率上有植入patch都比cryoinjured組有意義的高。綜合上述研究結果，SF/HA上的HA會與rMSCs表面抗原CD44作用加速rMSCs細胞生長及分化為心肌細胞，最適合用來做為心肌重建的材料。在初步動物研究結果中也發現植入SF/CS-HA及SF/HA可以防止心肌擴張及改善心肌功能。 | zh_TW |
| dc.description.abstract | Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated into or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS), SF/hyaluronic acid (HA) or SF/CS-HA microparticles that were further processed by mechanical pressing and genipin crosslinking to produce hybrid cardiac patches. We examined the influence of a CD44-blockage treatment of rMSCs on the aforementioned issues on new SF-based hybrid cardiac patches after they were cultivated. The ATR-FTIR spectra and the contact angle confirmed the co-existence of CS, HA or CS-HA and SF in microparticles and patches. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD44 and CD31, respectively, by a flow cytometric technique. To examine the growth of rMSCs on the patches, MTT viability assays were performed, and the results demonstrated that the growth of rMSCs on SF hybrid patches significantly exceeded (P<0.001) that on cultural wells after seven days of cultivation. This was also observed by adding vimentin to the cells. RMSCs cultured on cultural wells and SF/HA patches with a CD44-blockage treatment were 100%, 208.9 ± 7.1 (%) and 48.4 ± 6.0 (%) (n=3, for all), respectively, after five days of cultivation. Moreover, rMSCs grown on SF/HA patches highly promoted fibronectin expressions of the cells, while those with a CD44-blockage treatment markedly diminished the expression. To investigate the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real time quantitative polymerase chain reaction (real-time PCR) analysis. The results showed that cardiomyogenic differentiation of induced rMSCs on SF/CS-HA hybrid patches significantly improved the expressions of cardiac genes Gata4, Nkx2.5, Tnnt2 and Actc1 (all, P<0.01 or better, n=3) compared to those on SF and SF/CS patches and cultural wells. To investigate the interaction between CD44 of rMSCs and HA of SF/HA patches possibly modulated cardiomyogenic differentiation. Cardiomyogenic differentiation significantly promoted the expressions of cardiac genes Gata4, Nkx2.5, Tnnt2 and Actc1 (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions for the cells with CD44-blockage treatment. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS, SF/HA and SF/CS-HA hybrid patches were much more pronounced compared with SF patches, indicating the improvement of cardiomyogenic differentiation on the hybrid patches. In echocardiographic examinations, the SF/CS-HA and SF/CS patch groups effectively reduced progressive LV dilatation and preserved LV systolic function as compared to the cryoinjured group. The SF/CS-HA and SF/HA groups showed a significantly higher LVFS than the cryoinjured group. The results of this study demonstrate that the novel SF/HA and SF/CS-HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues. By examining the results of CD44-blockage treatment on rMSCs, we found that CD44 of rMSCs modulated the growth, fibronectin expression and cardiomyogenic differentiation of rMSCs cultured on a new cardiac SF/HA patch that may have great potential for regenerating cardiac tissue. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:10:04Z (GMT). No. of bitstreams: 1 ntu-99-D95548015-1.pdf: 4180833 bytes, checksum: 9d1971d4165504095f29dfdf31e26add (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | Table of Contents
口試委員會審定書 i 誌謝 ii Abstract iv Table of Contents v List of Figures vii List of Tables ix Chapter 1: Introduction - 1 - 1.1 Foreword - 1 - 1.2 Myocardial Infarction - 4 - 1.3 Myocardial tissue engineering - 5 - 1.4 Mesenchymal stem cells - 10 - 1.5 Silk Fibroin (SF) - 14 - 1.6 Chitosan (CS) - 17 - 1.7 Hyaluronic Acid (HA) - 18 - 1.8 Purpose of Study - 21 - Chapter 2: Materials and Methods - 24 - 2-1 List of materials - 24 - 2-2 List of machines - 25 - 2.3Experimental setup - 26 - 2.4 Preparation of SF-based patches - 27 - 2.5 Surface characterization of SF hybrid patches - 28 - 2.6 Flow cytometric analysis of rMSCs - 29 - 2.7 Viability of rMSCs on SF-based hybrid patches - 30 - 2.8 rMSCs culture and differentiation of cardiomyocyte-like cells on various SF-based hybrid patches - 31 - 2.9Total RNA isolation and real-time PCR - 32 - 2.10 Immunofluorescence staining of morphology and cardiomyogenic differentiation of 5-aza inducing rMSCs - 34 - 2.11 Rat cryoinjured model and scaffold implantation - 35 - 2.12 Histology, histochemistry and morphometry - 36 - 2.13 Functional evaluation of cryoinjured myocardium - 37 - 2.14 Statistical analysis - 37 - Chapter 3: Results and Discussion - 39 - 3.1 Stage I — rMSCs on silk fibroin–polysaccharide cardiac patches in vitro - 39 - 3.1.1 Characterizing SF/CS and SF/CS–HA microparticles of the hybrid patches - 40 - 3.1.2 The growth rates and morphologies of rMSCs on SF, SF/CS and SF/CS–HA patches after culturing for seven days - 44 - 3.1.3 Real-time PCR analysis for the cardiac-specific gene expressions of cardiomyogenesis of rMSCs - 50 - 3.1.4 Immunofluorescence staining for the expressions of cardiacspecific proteins of cardiomyogenesis of rMSCs - 55 - 3.2 Stage II — rMSCs CD44 surface markers on silk fibroin – Hyaluronic acid cardiac patches - 63 - 3.2.1 Growth of rMSCs on SF and SF/HA patches with or without CD44-blockage treatment - 64 - 3.2.2 Fibronectin expression of rMSCs on SF and SF/HA patches with or without CD44-blockage treatment - 67 - 3.2.3 Real-time PCR analysis for the cardiac-specific gene of cardiomyogenic differentiation of rMSCs with or without CD44- blockage treatment - 74 - 3.2.4 Immunofluorescence staining for the expressions of cardiacspecific proteins of cardiomyogenesis of rMSCs - 78 - Chapter 4: Stage III —Animal study - 83 - 4.1 Gross and histological examination of rat animal study - 83 - 4.2 Left ventricular function assessment - 88 - Chapter 5: Conclusions - 91 - References - 93 - List of Figures Figure 1.1. Plaque in a coronary arteries - 4 - Figure 1.2. Histological stages of myocardial infarction - 6 - Figure 1.3. Tissue engineering: it is the use of a combination of cells, scaffold and materials methods, and suitable biochemical and physio-chemical factors - 8 - Figure 1.4. Protocol for the clinical trial using mesenchymal stem cells (MSC) for the treatment of heart failure - 12 - Figure 1.5. Hematopoietic and mesenchymal stem cell differentiation - 13 - Figure 1.6. The structure of silk fibroin (SF) - 14 - Figure 1.7. The structure of chitosan - 18 - Figure 1.8. The structure of hyaluronic acid - 19 - Figure 2.1. Schematic illustration the experimental setup - 26 - Figure 3.1.1 The SEM micrograph of surface morphology of SF/CS–HA patch - 40 - Figure 3.1.2. The contact angles of SF-based hybrid patches - 41 - Figure 3.1.3. ATR–FTIR transmission spectra of SF, CS, HA, SF/CS and SF/CS–HA microparticles - 42 - Figure 3.1.4. ATR-FTIR transmission spectra of SF, HA and SF/HA microparticles - 43 - Figure 3.1.5. Flow cytometric spectra for examining the expressions of various surface markers of rMSCs - 46 - Figure 3.1.6. Relative growth rates of rMSCs, determined by MTT assay - 47 - Figure 3.1.7. Immunofluorescence images of the obtained rMSCs on SF hybrid patches after 7days cultured. - 49 - Figure 3.1.8 Real-time PCR analysis for the relative quantities of gene expressions of specific proteins of cardiac muscles for rMSCs induced by 5-aza and then cultured on various SF and SF-hybrid patches for six days - 54 - Figure 3.1.9. Immunofluorescent stained differentiation rMSCs by cardiomyocytes specific protein - 61 - Figure 3.10 Relative growth rates, determined by MTT assay, for rMSCs were cultured on cell culture wells, SF, SF/HA patches compared with rMSCs with CD44-blockage treatment that cultured on SF/HA patches after five days of cultivation - 65 - Figure 3.11 Vimentin (green color) and nuclei (blue color) staining for the rMSCs without (a) or with CD44-blockage (b) treatment cultured on SF/HA patches after five days - 66 - Figure 3.12 Fibronectin (green color) and nuclei (blue color) staining for rMSCs without (a) or with CD44-blockage treatments (b) for five days of culturing on SF patches, and rMSCs without (c) or with treatment CD44-blockage (d) for five days of culturing on SF/HA patches - 70 - Figure 3.13 The CD44 (in green) and nuclei (in blue) staining of CD44＋ rMSCs on SF (a), SF/HA (b), and CD44-blockage treated rMSCs on SF/HA (c) patches for one day of cell seeding - 73 - Figure 3.14 Real-time PCR analysis of the gene expressions for the specific proteins of cardiac muscles of rMSCs were without or with CD44-blockage treatment - 77 - Figure 3.15 Cardiomyocytes specific proteins of troponin T, cardiotin and connexin 43 staining for rMSCs or with CD44-blockage treatment, the 5-aza inducing and cultured on SF/HA patches for six days - 82 - Figure 4.1 Gross appearance, hematoxylin–eosin, and Masson’s trichrome staining of sham group (a)-(b), cryoinjured heart (c)-(d), SF/CS-HA patch (e)-(f) and SF/HA patch (g)-(h). All specimens shown in panels (a–h) were after 8 weeks cryoinjured. - 85 - Figure 4.2 Photomicrographs of the sham-operated ((a)-(b)), cryoinjured ((c)-(d)), SF/CS-HA patch ((e)-(f)) and SF/HA patch ((g)-(h)) retrieved at 8 weeks after the patch implantation stained with hematoxylin & eosin stain ( (a), (c), (e) and (g)) and Masson's trichrome ( (b), (d), (f) and (h)). - 87 - Figure 4.3 Fractional shortening examination at 8 weeks after myocardial infarction. - 90 - List of Tables Table 1. Selected members of cardiomyocytes primer-probe to use real-time PCR - 34 - Table 2 Left ventricular characteristics assessed by M-mode in infarcted animals pre- and post-treatment as well as in noninfarcted animals - 90 - | |
| dc.language.iso | en | |
| dc.subject | 心肌分化 | zh_TW |
| dc.subject | 蠶絲蛋白摻和材料 | zh_TW |
| dc.subject | 間葉幹細胞 | zh_TW |
| dc.subject | CD44 | zh_TW |
| dc.subject | mesenchymal stem cell | en |
| dc.subject | cardiomyogenic | en |
| dc.subject | CD44 of mesenchymal stem cells | en |
| dc.subject | Silk fibroin hybrid patches | en |
| dc.title | 探討以蠶絲蛋白為基質摻和多醣類對間葉幹細胞生長及分化為心肌應用於心肌再生 | zh_TW |
| dc.title | Silk fibroin-based patches on differentiation of mesenchymal stem cells into cardiomyogenic and regenerating myocardial | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 黃義侑,鍾次文,王水深,宋信文,劉得任 | |
| dc.subject.keyword | 蠶絲蛋白摻和材料,間葉幹細胞,CD44,心肌分化, | zh_TW |
| dc.subject.keyword | Silk fibroin hybrid patches,CD44 of mesenchymal stem cells,cardiomyogenic,mesenchymal stem cell, | en |
| dc.relation.page | 97 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-02-01 | |
| 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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