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研究生:陳稼豫
研究生(外文):Chia-Yu Chen
論文名稱:藉由超音波結合微氣泡促進皮膚細胞藥物傳輸之機制探討
論文名稱(外文):Investigation of The Mechanism of Ultrasound Mediated Microbubbles Cavitation for Transdermal Drug Delivery Enhancement
指導教授:廖愛禾
指導教授(外文):Ai-Ho Liao
口試委員:江建平沈哲州莊賀喬王正康
口試委員(外文):Chien-Ping ChiangChe-Chou ShenHo-Chiao ChuangJehng-Kang Wang
口試日期:2019-07-16
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:65
中文關鍵詞:超音波白蛋白微氣泡皮膚穴蝕效應絲氨酸蛋白酶
外文關鍵詞:UltrasoundMicrobubbleSkinCavitationMatriptase
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藥物及保養品的導入方法較常見的為超音波,將超音波結合微氣泡增加藥物或保養品的滲透量也是近幾年熱門的經皮傳輸方法,不少研究指出,微氣泡結合超音波作用後能引發穴蝕效應,增加皮膚的通透度以此提升傳輸的藥量,本研究為探討此經皮傳輸於皮膚內的機制。
ㄧ開始先做了超音波的溫度檢測,為確保微氣泡是經由超音波施打後破裂而非熱效應,了解到1分鐘時其溫度上升幅度最小打破效率也最佳,再使用豬皮,找出微氣泡的使用濃度及超音波的施打次數,將豬皮以染劑模擬藥物滲透,超音波結合微氣泡施打後做染劑滲透,經過超音波的施打使微氣泡破裂,引發穴蝕效應,開啟皮膚的通透度,增加染劑的滲透量,無施打超音波其滲透深度為17.67 ± 1.56 μm,以超音波施打1分鐘後其染劑滲透深度為26.02 ± 1.11 μm,施打次數越多其染劑滲透深度越深,控制組與有施打超音波之染劑滲透深度皆有明顯的差異(p <0.001)。
經過豬皮的實驗證實超音波結合微氣泡的促滲效果,找出細胞實驗所使用的超音波參數,以細胞實驗了解經超音波施打後細胞的變化。matriptase為絲胺酸蛋白酶的ㄧ種,與表皮的分化、恆定與傷口癒合等生物調控起重要作用,細胞經過超音波結合微氣泡的作用後,使用西方墨點法觀察matriptase是否有被活化,將蛋白表現做影像強度量化後,可以知道M24的組別中其影像強度至少有74.32 ± 6.65 %的差異,M69的組別中其影像強度則至少有46.44 ± 4.12 %的差異,施打超音波後western蛋白表現所計算出的影像皆有40 %以上的差異,本研究從豬皮實驗及細胞實驗中可以觀察出超音波結合微氣泡的施打後可以開啟細胞間的通道,之後matriptase會修復所打開的通道,因而活化matriptase,同時細胞內合成matriptase的量增加,進而恢復細胞膜上matriptase的量,因此可以回復之前的狀態。
The most common method for introducing drugs and skin care products is ultrasound. Ultrasound mediated microbubbles to increase the penetration of drugs or skin care products is also a popular transdermal delivery method in recent years. Many studies have pointed out that ultrasound mediated microbubbles can induce cavitation effects and increase the permeability of the skin to increase the amount of drug delivered. This study was to investigate the mechanism of this transdermal delivery.
Starting from the temperature detection of the ultrasonic wave, it is ensured that the microbubbles are broken by the ultrasonic instead of the thermal effect. Understand that the temperature rises the smallest at 1 minute and destruction efficiency is also the best. Then use the pigskin to find out the concentration of microbubbles and the number of times the ultrasonic are applied. The pig skin is dyed with Evans blue to simulate drug penetration. After ultrasound mediated microbubbles is applied, the microbubbles are broken to cause cavitation effects, and the permeability of the skin is opened to increase the penetration amount of the dye. The group without ultrasound had a penetration depth of 17.67 ± 1.56 μm. After 1 minute of ultrasound application, the penetration depth of the dye was 26.02 ± 1.11 μm. There was a significant difference in the penetration depth between the Control group and the dyeing agent (p < 0.001).
After confirming the effect of ultrasonic wave combined with the promotion of microbubbles, find out the ultrasonic parameters used in the cell experiment. After confirming the effect of ultrasound mediated microbubble penetration, find out the ultrasonic parameters used in cell experiments. Cellular experiments were used to understand the changes in cells after supersonic application. Matriptase is an enzyme of serine protease, which plays an important role in the biological regulation of epidermal differentiation, constant and wound healing. After the cells were subjected to ultrasound mediated microbubbles effects, western blotting was used to observe whether matriptase was activated. After the protein relative expression was quantified, it was found that the image intensity of the M24 group was at least 74.32 ± 6.65 %, and the M69 group had at least 46.44 ± 4.12 % of the image intensity. After the western protein expression, the calculated images all have more than 40% difference. This study observed that after the application of ultrasound mediated microbubbles, it will activate matriptase. After ultrasound mediated microbubbles, it will affect the expression of proteins in the cells, and then open the permeability of the skin. Ultrasound mediated microbubble can open the channel between cells and activate matriptase.
中文摘要 i
ABSTRACT ii
目 錄 iv
圖目錄 vii
表目錄 ix
第1章、緒論 1
1.1皮膚結構生理學 1
1.1.1表皮層(Epidermis) 2
1.1.2真皮層(Dermis) 2
1.1.3皮下組織(Subcutaneous tissue) 2
1.1.4皮膚屏障-表皮層之分層 3
1.2超音波簡介(Ultrasound) 4
1.2.1診斷型超音波(Diagnostic medical ultrasound) 5
1.2.2治療型超音波(Therapeutic ultrasound) 5
1.3超音波微氣泡對比劑 6
1.4穴蝕效應(Cavitation) 7
1.4.1穩態穴蝕效應(Stable cavitation) 8
1.4.2慣性穴蝕效應(Inertial cavitation) 8
1.5超音波結合微氣泡藥物傳輸機制 9
1.6 matriptase 10
1.6.1絲胺酸蛋白酶(serine protease) 10
1.6.2 matriptase的發現 11
1.6.3 matriptase的結構 12
1.6.4 matriptase的活化機轉與調控 13
1.6.5 matriptase生理功能 15
1.7研究動機 16
第2章、材料與方法 17
2.1研究架構 17
2.2藥品與設備 17
2.2.1藥品 17
2.2.2設備 19
2.3無菌白蛋白微氣泡製作 19
2.4超音波導入系統 20
2.5超音波導入系統參數測試 21
2.5.1溫度檢測 21
2.5.2 仿體之打破效率 22
2.5.3 24 well內之打破效率 22
2.5.4打破效率結果量化 23
2.6豬皮滲透實驗 24
2.6.1不同稀釋倍率白蛋白微氣泡之豬皮滲透 24
2.6.2超音波施打不同次數之豬皮滲透 25
2.7活豬皮實驗 26
2.7.1 以超音波施打豬皮表面 26
2.7.2蘇木紫與伊紅染色(Hematoxylin & Eosin stain, HE stain) 26
2.7.3掃描式電子顯微鏡(SEM) 27
2.8細胞實驗 28
2.8.1細胞株及繼代培養 28
2.8.2培養液配製 28
2.8.3冷凍細胞及解凍細胞 29
2.8.4細胞計數 29
2.9超音波結合微氣泡體外細胞實驗 30
2.9.1細胞施打超音波結合微氣泡存活率測試 30
2.9.2西方墨點轉漬法(western blotting) 32
2.9.3免疫細胞化學染色(ICC) 33
2.10影像處理 34
第3章、實驗結果 35
3.1超音波導入系統參數 35
3.1.1溫度檢測 35
3.1.2仿體之高頻超音波影像系統打破效率 35
3.2豬皮滲透實驗 37
3.2.1不同稀釋倍率白蛋白微氣泡之豬皮滲透 37
3.2.2超音波施打不同次數之豬皮滲透 38
3.3活豬皮實驗 41
3.3.1蘇木紫與伊紅染色(Hematoxylin & Eosin stain, HE stain) 41
3.3.2掃描式電子顯微鏡(SEM) 42
3.4無菌白蛋白微氣泡對比劑 43
3.5 24 well之高頻超音波影像系統打破效率 44
3.6超音波結合微氣泡體外細胞實驗 47
3.6.1細胞施打超音波結合微氣泡存活率 47
3.6.2西方墨點轉漬法(western blotting) 48
3.6.3免疫細胞化學染色(ICC) 54
第4章、討論 56
第5章、結論 59
參考文獻 60
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