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探討生醫材料對間皮細胞培養的影響以應用於腹膜缺損修復

為了解決kuga st的問題，作者高皓璽 這樣論述：

Table of ContentsRecommendation letter from thesis advisorThesis/Dissertation Oral Defense Committee CertificationChinese Abstract ....................................................................................................... - i -English Abstract ..........................................

............................................................ - iv -Table of Contents .................................................................................................. - viii -List of Figures ............................................................................................

............. - xi -List of Tables ..........................................................................................................- xiv -Abbreviations ..........................................................................................................- xv -Chapter 1 Introduction

- 1 -1.1 Background - 1 -1.2 Mesothelial cells - 1 -1.3 Peritoneal fibrosis - 2 -1.4 Biomaterials - 5 -1.5 Interaction of substrate and mesothelial cells - 5 -1.6 Experiences (History) in Culture of Mesothelial Cell - 6 -1.7 Marker of mesothelial cells - 6 -1.7.1 E-cadherin -

6 -1.7.2 ICAM-1 - 7 -1.7.3 Calretinin - 7 -1.7.4 Desmin - 8 -1.7.5 Cytokeratin - 8 -1.7.6 Vimentin - 9 -1.8 Materials - 10 -1.9 Instruments - 13 -Chapter 2 Gelatin/ hyaluronic acid cryogel scaffolds - 15 -2.1 Introduction - 16 -2.2 Materials and Methods - 18 -2.2.1 Mat

erials - 18 -2.2.2 Preparation of Gelatin (G) and Gelatin-Hyaluronic Acid (GH) Scaffold by Cryogelation - 19 -2.2.3 Characteristics of Cryogel - 20 -2.2.4 Culture of Mesothelial Cells in Cryogel Scaffolds - 22 -3.2.4.1 Isolation and Harvest of Mesothelial Cells - 22 -2.2.4.2 In Vitro

Cell Culture - 22 -2.2.4.3 SEM Analysis - 23 -2.2.4.4 DNA Quantification - 23 -2.2.4.5 Live/Dead Staining - 23 -2.2.4.6 Cell Cytoskeleton Staining - 24 -2.2.4.7 Quantitative Real-Time Polymerase Chain Reaction (qPCR) - 24 -2.2.4.8 Immunofluorescence (IF) Staining - 25 -2.2.5 In

Vivo Studies - 26 -2.2.6 Statistical Analysis - 28 -2.3 Results - 28 -2.3.1 Synthesis and Characterization of Gelatin (G) and Gelatin/Hyaluronic Acid (HA) Cryogels - 28 -2.3.2 In Vitro Cell Culture - 31 -2.3.3 In Vivo Studies - 35 -2.4 Discussion - 37 -2.5 Conclusions - 47 -C

hapter 3 Chitosan/polycaprolactone blends application on mesothelial cells - 49 -3.1 Introduction - 49 -3.2 Materials and methods - 51 -3.2.1 Preparation of chitosan /PCL solution and blend membranes formation - 51 -3.2.2 Evaluation of cell Viability (LIVE/DEAD) - 52 -3.2.3 Evaluation

of Cell proliferation - 52 -3.2.4 SEM - 52 -3.2.5 Immunofluorescent study - 53 -3.2.6 Western blotting - 53 -3.2.7 quantitative polymerase chain reaction (qPCR) - 54 -3.2.8 Statistical methods - 54 -3.3 Rresults - 55 -3.3.1 The transparency of chitosan/polycaprolactone blend me

mbranes - 55 -3.3.2 Proliferation of mesothelial cell line (Met-5A) - 56 -3.3.3 SEM - 57 -3.3.4 The phenotype of mesothelial cells - 58 -3.4 Disscusion - 61 -Chapter 4 Chitosan/polycaprolactone electrospun web - 65 -4.1 Introduction - 66 -4.2 Materials and Methods: - 67 -4.2.

1 Materials - 67 -4.2.2 Preparation of the Electrospinning Emulsion and web fabrication - 67 -4.2.3 Characteristics of electrospun web - 68 -4.2.4 Culture of Mesothelial Cells in electrospun web - 68 -4.2.4.1 Isolation and Harvest of Mesothelial Cells - 68 -4.2.4.2 In Vitro Cell Cultu

re - 69 -4.2.4.3 SEM Analysis - 69 -4.2.4.4 DNA Quantification - 70 -4.2.4.5 Quantitative Real-Time Polymerase Chain Reaction (qPCR) - 70 -4.2.5 In Vivo Studies - 71 -4.2.6 Statistical Analysis - 73 -4.3 Results - 73 -4.3.1 Synthesis and characterization of the PCL and PCL/ chit

osan electrospun webs - 74 -4.3.2 In vitro cell culture - 76 -4.3.3 In Vivo Studies - 80 -4.4. Discussion - 82 -Chapter 5 Conclusion and Outlooks - 86 -5.1 Conclusion - 86 -5.2 Future works - 88 -References - 89 - List of FiguresFigure 2.1 The SEM micrographs (A) and porosity

(B) of gelatin (G) and gelatin/hyaluronic acid (GH) cryogels. Bar = 100 μm. ………………………………………. - 29-Figure 2.2 The water uptake kinetics in phosphate buffered saline (PBS) (A) and degradation kinetics in collagenase (B) of G and GH cryogels. …………….…………. - 30-Figure 2.3 The typical compressive s

tress–stain curves of the G and GH cryogels. The lines are fitted curves from Equation (5). ………………………………………………………..…. - 30-Figure 2.4 The cell morphology from SEM observation (A) and cell proliferation from DNA assays (B) of mesothelial cells cultured in G and GH cryogels. Bar = 50 m. …………………………

…………………………………………………………………….………………..…. - 32-Figure 2.5 Confocal microscopy observation of mesothelial cells cultured in G and GH by live/dead (A) (bar = 150 m) and nucleus/cytoskeleton staining (B) (bar = 30 m). The live cells were stained green and the dead cells were stained red in (A), while

the cell nuclei were stained blue by Hoechst 33342 and the actin cytoskeleton was stained red by rhodamine-phalloidin in (B). Both the merged top-view image and cross-sectional-view image are included in (A). ……………………………………………..…. - 33-Figure 2.6 Gene expression of the mesothelial cells cultured

in G and GH from a quantitative real-time polymerase chain reaction (qRT-PCR). * p < 0.05 compared with G. ………………………………………………………………………………………………………………. - 34-Figure 2.7 The immunofluorescence (IF) staining of calretinin and E-cadherin of the mesothelial cells cultured in G and GH for seven days. Th

e protein was stained green by a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, while the nuclei were stained blue by Hoechst 33342. Bar = 30 m. ……………………………..…. - 35-Figure 2.8 Gross view of the initial mesothelium wound and the transplanted cell/scaffold constructs at differen

t time points post-implantation. ……………………. - 36-Figure 2.9 Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining of E-cadherin and calretinin of the cell/cryogel constructs 7- and 21-days post-implantation (bar = 20 m). Native peritoneum tissue was used for comparison. The

inserts are enlarged views on the surface of the specimen (bar = 10 m). ……. - 37-Figure 3.1 The lines of graph paper is clearly visible through the chitosan because of its transparency. The lines on the graph paper could hardly be traced when the PCL content in the chitosan was greater than 50%.

……………………………………………….…. - 55-Figure 3.2 Cell proliferation as measured by the MTT assay. A marked increase in the number of Met-5A cell line according to the proportion of MTT value for PCL 50 and PCL 75 in the day 7 ………………………………………………………………………….…………….…. - 56-Figure 3.3 The attachment of Met-5A

cell line is also prominently increased with the increase of PCL. PCL 75 is the best. ………………………………………………………………..…. - 57-Figure 3.4 The cell infiltration and migration within the scaffolds were noted after 7 days of incubation especially in the right side of this figure. …………………..….…..…. - 58-

Figure 3.5 Red: F-Actin, Green: Expressed protein, Blue: Nuclei, DAPI. The cell infiltration and migration increased also noted after 7 days of incubation on the membrane of PCL 75. ………………………………………………………………………………………. - 58-Figure 3.6 The mRNA expression of mesothelial cell markers (cytokeratin and

desmin). There was not significant difference in gene expression at day 7. But prominent increased gene expression of desmin and cytokeratin were noted in PCL 75 at day 14. ………………………………………………………………………………………………………..…. - 61-Figure 4.1 The SEM micrographs of PCL/chitosan (A) and PCL (B) webs. ……….

- 74-Figure 4.2 Water contact angle measurement of PCL/chitosan and PCL webs. As the chitosan blended to PCL the webs became more wet (contact angle decreased from 113.6° to 57.6°). ………………………………………………………………………………………………. - 75-Figure 4.3 The typical tensile stress–stain curves of the PCL and PCL

/C electrospun webs. …………………………………………………………………………………………………………………. - 75-Figure 4.4 Comparing day one and day seven, the cells became more elongated, but the general phenotype remained. More cells, together with their secreted ECM, were also found to attach to the surface on day seven. ………………………………

…………..…. - 76-Figure 4.5 Cell proliferation from DNA assays. During the cell culture process, the number of cells in PCL/chitosan is significantly higher than that in PCL. ……..…. - 77-Figure 4.6 The immunofluorescence (IF) staining of calretinin and E-cadherin of the mesothelial cells cultured

in PCL and PCL+C for seven days. The protein was stained green by a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, while the nuclei were stained blue by Hoechst 33342. ……………………………………………………..…. - 78-Figure 4.7 Confocal microscopy observation of mesothelial cells cultured in PCL

and PCL+C by nucleus/cytoskeleton staining (bar = 75 m). The cell nuclei were stained blue by Hoechst 33342 and the actin cytoskeleton was stained red by rhodamine-phalloidin. …………………………………………………………………………………….…. - 79-Figure 4.8 Gene expression of the mesothelial cells cultured in PCL/chitosan an

d PCL from a quantitative real-time polymerase chain reaction (qRT-PCR). * p < 0.05 compared with PCL. ……………………………………………………………………………………..…. - 80-Figure 4.9 Gross view of the initial mesothelium wound and the transplanted cell/web constructs. We covered the surface of the web with cells on the wou

nd. …………..…. - 81-Figure 4.10 Three whole slide images (H&E staining, ×20 scanned) were taken out of rats with three different conditions on the seventh day of implantation. ………………………………………………………………………………..………………..… - 81-Figure 4.11 Hematoxylin and eosin (H&E) staining and immunohistochemical

(IHC) staining of E-cadherin and calretinin of the empty, PCL+C and PCL+C groups 7-days post-implantation (bar = 50 m). The inserts are enlarged views on the surface of the specimen (bar = 25 m). …………………………………………………………………………….…..…. - 82- List of tablesTable 1. Mechanical properties of G and GH

cryogels. Values are the mean ± standard deviation (SD) of five independent measurements. ………………………………………..…. - 31-Table 2. Mechanical properties of PCL and PCL/C electrospun webs. Values are the mean ± standard deviation (SD) of three independent measurements. …………….…. - 76-