The Influence of Rat Mesenchymal Stem Cell Cd44 Surface Markers on Cell Growth, Fibronectin Expression, and Cardiomyogenic Differentiation on Silk Fibroin - Hyaluronic Acid Cardiac Patches

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC ( rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5 -aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9 +/- 7.1 (%) or 48.4 +/- 6.0 (%) (n = 3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8 x 10(5)/cell, in fluorescent intensity) while cells with a CD 44-blockage treatment markedly diminished the expressions (e .g., 1.1 x 10 (4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P < 0.01 or better, n = 3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore , the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44- blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches

Silk fibroin-based patches on differentiation of mesenchymal stem cells into cardiomyogenic and regenerating myocardial

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。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

Sustained Release of Insulin from Poloxamer Gels Interpenetrated by Polyion Complexation of Chitosan-Hyaluronic Acid Network

This study investigates in vitro the protein drug delivery characteristics of new thermal sensitive gels, poloxamer P- chitosan CS/ hyaluronic acid HA gels P-CS/HA, in which a CS solution is interacting with various concentrations of HA that interpenetrates P gels. The polyion complexation occurs between CS and HA that can protect drugs from proteolysis. The results indicate that the swelling ratios of all P-CS/HA gels are markedly superior to those of nonswelling P and P- CS gels. For example, P-CS/HA 0.5% w/w gels have swelling ratios of 48.3 +/- 2.7% w/w, which are maintained for approximately 0.5 h in water at 37 degrees C. In vitro releases of insulin from P-CS/HA 0.5% w/w gels had significantly lowered initial burst release P < 0.01 and lasted much longer than those from gels without a CS network . The duration of insulin release was in a significantly sustained manner for up to 3.5 h, which was about two times or longer than the period of delivery using P or P-CS gels

「臺灣獎勵旅遊行程規劃競賽」企劃案

[[abstract]]臺灣目前獎勵旅遊面臨缺乏完善的配套，導致遇到大公司與大量員工來臺獎勵旅遊，無法完全給與貴賓級人士在臺灣最特殊最具有代表性的獎勵旅遊行程，因此本組認為舉辦「臺灣獎勵旅遊行程規劃競賽」是相當具有重大意義，若能提出優良的遊程規劃及創意性，便能夠吸引外國企業考慮臺灣成為獎勵旅遊的名單，因此將藉由本次的競賽，激發學生們規劃一份在臺灣舉辦的完善獎勵旅遊行程，且可透過舉辦競賽活動達到許多目的。 本次企劃競賽活動目的如下: 一、瞭解目前臺灣獎勵旅遊不足之處，提出改善的建議。 二、達到政府、企業、學校三方對此產業之重視，並相互交流，建立溝通管道。 三、期望業界與學校接軌，達到產學積極合作與時俱進。 四、發掘獎勵旅遊規劃人才，提早積極培育

「臺灣獎勵旅遊行程規劃競賽」企劃案

[[abstract]]臺灣目前獎勵旅遊面臨缺乏完善的配套，導致遇到大公司與大量員工來臺獎勵旅遊，無法完全給與貴賓級人士在臺灣最特殊最具有代表性的獎勵旅遊行程，因此本組認為舉辦「臺灣獎勵旅遊行程規劃競賽」是相當具有重大意義，若能提出優良的遊程規劃及創意性，便能夠吸引外國企業考慮臺灣成為獎勵旅遊的名單，因此將藉由本次的競賽，激發學生們規劃一份在臺灣舉辦的完善獎勵旅遊行程，且可透過舉辦競賽活動達到許多目的。 本次企劃競賽活動目的如下: 一、瞭解目前臺灣獎勵旅遊不足之處，提出改善的建議。 二、達到政府、企業、學校三方對此產業之重視，並相互交流，建立溝通管道。 三、期望業界與學校接軌，達到產學積極合作與時俱進。 四、發掘獎勵旅遊規劃人才，提早積極培育

CNC車床溝槽量測用夾治具

[[abstract]]近年來科技發展越來越快， 工具機是工業生產所必須的基礎設備。當此產業的能力強大時，不僅可使國內製造業不用採購國外昂貴的設備，並且有能力外銷機械賺取可觀的營業額。 數值控制概念，簡單來說，數值控制(Numerical Control 簡稱 NC)就是應用電腦的強大運算能力來幫我們處理計算一些複雜的運算程式或資料。像過去的銑、 車床都是用人腦或計算機來計算加工路徑以及加工位置，並應用雙手來調整加工機械的位置及定位，以達到預定的加工效果。 而當我們用CNC車床加工時，有時需要加工溝槽要量溝槽外徑的尺寸，量測該尺寸時，需要準備標準試棒配合量測，往往會花費較多的時間，而想出需要符合生產價值，且能達到一定經濟效益的量測用夾治具