博碩士論文 106827021 詳細資訊




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姓名 洪翊玲(Yi-Ling Hong)  查詢紙本館藏   畢業系所 生物醫學工程研究所
論文名稱 針對糖尿病足潰瘍設計並製作一種抗菌且能促進傷口癒合的甲殼素複合式水凝膠之研究
(Design and Fabrication of an Antimicrobial-Wound Healing-Enhanced Chitosan Complex Hydrogel for Diabetic Foot Ulcer Treatment.)
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摘要(中) 糖尿病中最常見的是第二型糖尿病,屬於一種慢性疾病,它占糖尿病患者總
人口數的 90-95%。通常會有較長的潛伏期,第二型糖尿病患者主要原因是
自身的血液循環較差、新陳代謝異常和炎症時期較長等各種原因,導致傷口
癒合緩慢,嚴重者會影響到患者的生活品質或導致死亡。而本研究中開發一
種包含銀離子和包裹表皮生長因子 (EGF)之奈米粒子的殼聚醣水膠
(CPCHG),此水膠可以提供抗菌能力也因表皮生長因子的存在幫助傷口癒
合。本研究目前先以牛血清白蛋白(BSA)作為模擬蛋白代替表皮生長因子。
奈米載體的製備方式以聚陰離子三聚磷酸鹽作為交聯劑與殼聚醣進行交聯
製備包覆 BSA 的殼聚醣奈米載體(CTBNPs)。經過 DLS 分析,CTBNPs 的
粒徑大小與表面電荷分別為 265±26 nm 和 30.02±1.42 mV;CTBNPs 在 48
小時的釋放率為 89% 但在 CPCHG 的 BSA 釋放約為 70%。在銀離子釋放
分析中,銀離子在 48 小時中 4°C 和 37°C 的釋放量為 5.11 mg/L 及 6.53
mg/L。此外,在細菌抑制中,使用銀離子濃度為 24 mM 時,對於金黃色葡
萄球菌有顯著抑制的效果;在細胞毒性試驗中,細胞的存活率也可達到 90%。
生長曲線試驗中,將模擬蛋白(BSA)更改成表皮生長因子(EGF)製成的殼聚
醣水膠(CPEHG)。使用不同濃度表皮生長因子(EGF)去測試 NIH-3T3,結果
表明當 EGF 的濃度來到了 60 μg/mL 可讓細胞生長且不受銀離子的影響。
選定水膠(CPEHG)最終配方為 60μg/ mL EGF 和 24 mM 銀離子濃度。在動II
物實驗中,我們使用 Sprague Dawley(SD)大鼠並注射鏈脲佐菌素觀察 2 周
確定為糖尿病大鼠後在大鼠背上製造傷口並且使用敷料治療觀察傷口癒合
情況。結果表明在各組實驗組中 CPEHG 水膠的傷口癒合速度最快、炎症細
胞較少和有較多的膠原蛋白沉積,未來可用於治療糖尿病足傷口敷料。
摘要(英) Type II diabetes mellitus (T2DM) is the most common form of diabetes,
accounting for 90-95% of diabetics, and is a chronic disease often preceded by a
long asymptomatic period. Most of T2 diabetics suffer with delay wound healing
resulted from poor blood circulation, prolonged inflammation and lack of growth
signal due to disordered metabolism, and it may severely affect patients’ life
quality and even cause death. To overcome the aforementioned issues, a synthetic
chitosan-based hydrogel containing silver ions and growth factor-entrapped
nanoparticles that may provide both antibacterial and would healing-enhanced
functionalities were developed in this study. As being the preliminary
investigation, the bovine serum albumin (BSA) was employed as the model
protein molecule to substitute the practical growth factor protein in this study.
Furthermore, the BSA-encapsulated chitosan nanoparticles were fabricated by
using the polyanion tripolyphosphate as the coacervation crosslink agent.
Through the DLS analysis, the size and surface charge of the BSA-loaded
nanoparticles(CTBNPs) were 265 ± 26 nm and 30.02 ± 1.42 mV, respectively.
The encapsulation efficiency and the loading rate of the BSA in the nanoparticles
were 74± 8.62% and 1.23 ± 0.55%, respectively. Also, the release rate of BSA
from the nanoparticles was 89% within 4 h, while that from the synthetic hydrogel
was about 70% within the same time course. The nanoparticles were successful
insides the chitosan hydrogel. From hydrogel property test, BSA releases were
70% . When the silver ions released in 48 hours, the 4 and 37 degrees release were
5.11 and 6.53 mg/L. Moreover, the bacterial inhibit study, when used 24 mM
silver ions concentration the bacterial inhibit was significant. The hydrogel
toxicity for the cell. In the cell study, we used 24 mM silver ions concentration
the cell viability came to 90 %. In the growth curve study, we decide used
epidermal growth factor with different concentration. However, when EGF came
to 60 μg/mL cell weren′t affected by silver ions. We decided that the final
formulation of the hydrogel was 60 μg/mL EGF and 24 mM silver ion
concentration.In animal experiments, We used Sprague Dawley (SD) rats and
injected with streptozotocin to observe wound healing on rats′ backs after 2 weeks
of diagnosis as diabetic rats and to observe wound healing using dressings. The
results showed that the wound healing rate of CPEHG water gel in each group
was accelerated, inflammatory cell implantation and complication of collagen
deposition, which could be used to treat diabetic foot wound dressing in the future
關鍵字(中) ★ 第二型糖尿病
★ 糖尿病足潰瘍
★ 殼聚醣
★ 銀離子
★ 表皮生長因子
★ 傷口癒合
關鍵字(英) ★ Type 2 diabetes mellitus
★ Diabetic foot ulcer
★ Chitosan
★ Silver ions
★ Epidermal growth factor
★ Wound heling
論文目次 第一章緒論……...……..……………..…….………………………1
1.1 研究背景………………..………………..…...………………1
1.2 研究動機與目的……………..………………..………...……2
第二章文獻回顧………………………..…………………..…...…3
2.1 糖尿病介紹………………..……….….……………….……. 3
2.2 糖尿病主要併發症………………......….…...………..…...…5
2.3 糖尿病足潰瘍(Diabetic foot ulcers)……….....………. …..…7
2.4 糖尿病足潰瘍周邊醫療資源…………......…………..…….. 9
2.5 足部潰瘍等級……………………....……………………...…9
2.6 治療糖尿病足潰瘍方式…………....…………………….…10
2.6.1 足部完全接觸石膏(Total contact casts, TCCs)....…… 10
2.6.2 清創(Debridement)………………….…………………11
2.6.7 酶清創(Enzymatic Debridement)…………….………. 11
2.6.8 自溶性清創(Autolytic Debridement)…….…………....11
2.6.9 生物清創(Biologic Debridement)………………….…. 11
2.6.10 外科手術清創(Surgical Debridement)…….……...…12
2.6.7抗生素治療…………………….….…. ………...…......12
2.6.8 高壓氧治療(Hyperbaric oxygen therapy)…………..…12VI
2.6.9 負 壓 傷 口 治 療 (Negative Pressure Wound Therapy,
NPWT)..........................................................................................12
2.6.10 敷料治療………………………………………......…13
2.7 傷口癒合機制…………………………………………….…20
2.8 糖尿病足潰瘍常見細菌……………………………….....…22
第三章實驗部分………………………………………………….24
3.1 實驗藥品、材料、儀器設備……………..…………...………24
3.1.1藥品..……………………………………….…………. 24
3.1.2材料……..………………………………………….…. 25
3.1.3 儀器………..…………………………………………..26
3.2 檢量線……………….........…………………………………27
3.3 實驗整體流程………...……..……………..……………..…28
3.4 製備包覆模型蛋白 BSA 奈米載體……………………….. 28
3.5CTBNPs 物理特性分析…………………….…………....…29
3.6 製備銀離子及 CTBNPs 在殼聚醣水凝膠中(CPCHG)…..... 31
3.7 掃描式電子顯微鏡(SEM) 拍攝………………………….... 32
3.8 殼聚醣水膠釋放牛血清白蛋白(BSA)………………..…….32
3.9殼聚醣水膠釋放銀離子………………………………..…... 33
3.10抗菌試驗……………………………………..……………. 33VII
3.16 細胞培養…………………………...…………….……...... 36
3.17CPCHG 中的銀對 NIH-3T3 細胞毒性測試……..…………36
3.18CPCHG 對 NIH-3T3 生長試驗………………….…………37
3.14 製備表皮生長因子奈米載體及銀在殼聚醣水凝膠….......38
3.15CPCHG 對 NIH-3T3 生長試驗……….................................39
第四章結果與討論……………………………………………….43
4.1CTBNPs 物性及化性分析……………..…………………...43
4.2 CTBNPs 之表面形態分析………….………………….……43
4.3 CTBNPs 內 BSA 的包覆率、負載率分析……………..…..44
4.4 BSA 包覆 CTBNPs 之釋放試驗……..…..………..………. 44
4.5 CPCHG之表面分析…………………….....…... ………...…45
4.6CPCHG 釋放試驗……………………..……………………. 46
4.6.1 模擬蛋白(BSA)釋放………………………………..…46
4.6.2銀離子釋放………………………………….. ………..47
4.7CPCHG 中的銀離子對金黃色葡萄球菌抑菌試驗……..…. 48
4.7.1初步抑制圈試驗………………………………..…. ……...48
4.7.2抑菌試驗……………………..…………………………… 49
4.8CPCHG 細胞實驗分析…………………..…………………..51
4.8.1CPCHG 對小鼠胚胎成纖維細胞毒性試驗.. …………51VIII
4.8.2CPCHG 對小鼠胚胎成纖維細胞生長曲線試驗.......…52
4.9 CTENPs 內表皮生長因子包覆率、負載率分析…………...53
4.10CPEHG 體外細胞實驗分析……………………..…………53
4.11 機械性質試驗…………………………………. ………..…55
4.11.1CPCHG 和 CPEHG 延伸率及抗拉強度…………….. 55
4.11.2CPCHG 和 CPEHG 澎潤率及含水率….……………. 56
4.12動物試驗……………………………………..……………. 57
第五章結論……………………………….……………………….61
第六章未來展望………………………………………………….62
參考文獻…………………………………………………………….63
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指導教授 李宇翔(Yu-Hsiang Lee) 審核日期 2019-10-31
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