臺灣博碩士論文加值系統

文獻中指出，移植後3-7天內常發現大量的巨噬細胞浸潤，其分泌的原發炎細胞激素（proinflammatory cytokine）：介白素-1（interleukin-1，IL-1）、腫瘤壞死因子-α（tumor necrosis factor-α，TNF-α）和干擾素-γ（interferon-γ，IFN-γ），進而啟動iNOS蛋白質的合成，使大量NO產生。NO能大量破壞植入的胰島β細胞，往往是造成原發性無功能（primary non-function）與移植失敗最主要的原因。
論文中將以AMT（HCl）作為iNOS inhibitor，並利用溶膠-凝膠法以及PLGA高分子進行包覆，藉以抑制IL-1β活化NO產生的途徑。結果顯示，以溶膠-凝膠法包覆AMT，至第三天仍能偵測到藥物的釋放。經由MTS assay分析細胞活性的結果也發現，silica-gel材料對RIN-m5F細胞並無細胞毒性，且AMT可自包覆顆粒中釋放並抑制IL-1β的毒害作用，達到保護體外培養的胰島癌細胞的生物活性。
最後也進一步的以糖尿病老鼠作為實驗model，將活體胰島細胞與AMT silica-gel共同移植進行測試，並探討其移植後原發性無功能排斥的影響。結果顯示連續監測血糖45天後，植入50顆islets與0.1 mg AMT silica-gel的糖尿病老鼠，其治癒率比單純只移植islets的效果來得更早呈現，似乎暗示由於AMT有效抑制發炎時產生的NO，進而達到保護植入物，使移植成功的目的。期望此種藥物控制釋放的系統對將來應用於臨床藥物治療上，能提供了另一個可行性。

In previous studies, it is indicated that macrophage infiltration in freshly transplanted islet grafts occurred within 3-7 days after transplantation. The infiltrated macrophages secreted a various of pro-inflammatory cytokines such as interleukin-1（IL-1）, tumor necrosis factor-α（TNF-α） and interferon-γ（IFN-γ） that induced nitric oxide (NO) overproduction via an inducible form of NO synthase (iNOS) synthesis. The destruction of pancreatic β-cells by NO might play an important role for primary non-function and result in transplantation failure.
In this study, AMT (HCl), an iNOS inhibitor, was used and encapsulated inside poly (lactide-co-glycolide) (PLGA) nanoparticles formulated by a single oil-in-water (O/W) and a double water-in-oil-in- water (W/O/W) emulsification methods or inside silica-gels by the sol-gel method to sustain released and inhibit NO production. The data suggested the sustained release of AMT silica-gel particles was still detectable in three days. It was also found that silica-gel powders had no cytotoxicity to RIN-m5F insulinoma cell line by MTS assay, and AMT could release from particles to inhibit the toxic effect of IL-1β to prevent the bioactivity of in-vitro cultured cells.
In further study, AMT encapsulation by the sol-gel method will be investigated by the local expression of AMT in animal models whether inhibition of NO at the grafted site could prevent initial damage to transplanted islets mediated inflammatory cytokines. 45 days posttransplantation, blood glucose levels in groups A (50 islets) and C (0.1 mg AMT) were significantly lower than groups B (1 mg blank) and D (1 mg AMT) after transplantation. Our data suggested that iNOS inhibitor: AMT was effective to enhance xenograft survival.

目 錄

指導教授推薦書…………………………………………………………...
口試委員議定書…………………………………………………………...
博碩士論文電子檔案上網授權書……………………………………...iii
長庚大學博碩士論文著作授權書…………………………….………..iv
致謝……………………………….………………………………………v
中文摘要...................................................................................................vi
英文摘要………………………………………….…………………….vii
目錄.........................................................................................................viii
圖表目錄……………………………………………………………….xiii
第一章 緒論...............................................................................................1
1.1 胰臟的生理功能...........................................................................1
1.2 糖尿病簡介...................................................................................1
1.3 原發性無功能（primary non-function, PNF）...............................2
1.4 一氧化氮（nitric oxide, NO）與一氧化氮合成酶
（nitric oxide synthase, NOS）.......................................................3
1.5 一氧化氮（nitric oxide, NO）與移植後發炎反應.........................5
1.6 AMT（hydrochloride）藥物............................................................6
1.7 藥物釋放系統...............................................................................7
1.7.1 溶膠-凝膠法（sol-gel methode）製備矽膠鹽......................8
1.7.1.1 以tetramethoxysilane（TMOS）矽烷氧化物製備藥物包覆載體..............................................................8
1.7.1.2 製備影響參數.........................................................10

1.7.2 聚乳酸與聚甘醇酸共聚物（poly(lactide-co-glycolid), PLGA）..............................................................................13
1.7.2.1 PLGA物理化學特性簡介.......................................13
1.7.2.2 以PLGA製備微小球.............................................15
1.7.2.3 微小球的降解與藥物釋放.....................................17
1.7.2.4 製備過程.................................................................18
1.7.2.5 製備影響參數.........................................................19
1.8 研究動機與目的……………………………….………………21
第二章 材料與方法.................................................................................22
2.1 實驗設備及試劑.........................................................................22
2.1.1 實驗設備...........................................................................22
2.1.2 實驗試劑...........................................................................24
2.1.2.1 AMT 藥物包覆.......................................................24
2.1.2.1.1 以PLGA製備AMT微小球..........................24
2.1.2.1.2 以溶膠-凝膠法包覆AMT............................25
2.1.2.2 RIN-m5F insulinoma cell line細胞培養.................25
2.1.2.2.1 細胞株...........................................................26
2.1.2.2.2 細胞培養試劑…………………………...…26
2.1.2.3 細胞活性分析（MTS assay）………….……...…...26
3.1.2.4 動物實驗……………………..…………...………26
2.2 實驗方法…………….............…………………...…………….27
2.2.1 AMT藥物包覆……………………………………….….27
2.2.1.1 以溶膠-凝膠法包覆AMT………………..………27
2.2.1.2 以PLGA製備AMT微小球………………………28
2.2.1.2.1 油/水 乳化法（O/W emulsion）…….……28
2.2.1.2.2 水/油/水 乳化法（W/O/W emulsion）……..28
2.2.2 粒徑分析儀（photon correlation spectroscopy, PCS）量測…………………….......................................................29
2.2.3 掃瞄式電子顯微鏡（scanning electromicroscopy, SEM）觀察…………………………………………...…………30
2.2.4 體外藥物釋放實驗（in-vitro release experiment）…….30
2.2.4.1 以溶膠-凝膠法包覆藥物AMT………………..…30
2.2.4.2 以PLGA製備AMT微小球………….……...……31
2.2.5 體外AMT釋放濃度測試……………….……...……….31
2.2.6 RIN-m5F insulinoma cell line細胞培養………….……..32
2.2.6.1 解凍細胞…………………...……………………..32
2.2.6.2 繼代培養…………………...……………....……..33
2.2.6.3 細胞計數…………………………...……....……..33
2.2.6.4 冷凍細胞……………………..….................……..33
2.2.7 細胞活性分析（proliferation assay, MTS assay）....…..34
2.2.8 葡萄糖刺激胰島素分泌試驗
（glucose-stimulation of insulin secretion，GSIS）..…….…35
2.2.9 動物實驗…………………….……......…………………36
2.2.9.1 胰島細胞的純化（purification of islets）………..36
2.2.9.2 糖尿病老鼠的誘導…………………...…………..37
2.2.9.3 腎包膜下（sub-renal capsule）移植……………..38
2.2.9.4 監測移植後體重、血糖值……………………...…38
2.2.9.5 亞硝酸鹽分析（nitrite assay）……………..………38
2.2.10 統計分析……………………...………………………..39

第三章 結果與討論…………………………….………………………40
3.1 AMT（HCl）藥物濃度的測定…………………………………..40
3.1.1 測定AMT（HCl）藥物吸收峰…………...…..…………..40
3.1.2 AMT（HCl）藥物濃度減量線…………………...……..40
3.2 AMT藥物包覆…………………………………………...…….41
3.2.1 以溶膠-凝膠法包覆AMT…………………..…………..41
3.2.1.1 探討製備過程中添加鹼性催化劑對silica-gel之影響………………………………………………….41
3.2.1.2 探討冷凍乾燥時間對silica-gel釋放曲線之影響.43
3.2.1.3 於hood中自然揮發溶劑與水分的silica-gel….....44
3.2.2 以PLGA製備AMT微小球……………….…...……….45
3.2.2.1 探討O/W與W/O/W兩種乳化法對AMT微小球的影響………………..……………………………...45
3.2.2.2 探討不同的PLA：PGA聚合比例對AMT微小球的影響……………………………………......…...46
3.3 微小球表面型態的電子顯微鏡影像（SEM image）…….…..47
3.3.1 以溶膠-凝膠法包覆AMT…………….……………..….47
3.3.1.1冷凍乾燥與hood自然揮發製備而成的silica-gel影像…………………………………………………..47
3.3.2 以PLGA製備AMT微小球………………….……...….48
3.3.2.1 以O/W及W/O/W乳化法製備PLGA（50/50）微小球…….……………………………………...….48
3.3.2.2 以O/W及W/O/W乳化法製備PLGA（75/25）微小球….………………………………………....….48
3.4 細胞活性分析（proliferation assay, MTS assay）.......................49
3.4.1 Silica-gel材料對MTS assay之O.D值的影響…….….49
3.4.2 Silica-gel材料對RIN-m5F insulinoma cells的毒性測試49
3.4.3 AMT silica-gel抑制IL-1β效果之探討.............................50
3.5 葡萄糖刺激胰島素分泌試驗
（glucose-stimulation of insulin secretion, GSIS）.....................51
3.6 動物實驗（animal experiment）..................................................52
3.6.1 移植前後的血糖值變化...................................................52
3.6.2 移植前後的體重變化.......................................................53
3.6.3 亞硝酸鹽分析（nitrite assay）...........................................54
3.6.3.1 製作亞硝酸鹽標準減量線.....................................54
3.6.3.2 血清中NO2於移植後的變化……………….........54
3.6.4 治癒（存活）率分析...........................................................56
第四章 結論.............................................................................................57
參考文獻...................................................................................................98

圖表目錄

圖一、非特異性發炎反應示意圖……………………………………….58
圖二、一氧化氮（nitric oxide）…………………………………………..59
圖三、NOS催化NO生合成反應……………………………………….59
圖四、Inducible nitric oxide synthase (iNOS) pathway…………………60
圖五、AMT（hydrochloride）的分子式………………………………….61
圖六、不同劑型之血液中藥物濃度曲線圖…………………………….62
圖七、PLA、PGA與PLGA分子式……………………………………...62
圖八、（a）油包水乳化液（W/O）；（b）( O/W ) 水包油乳化液………....62
圖九、複合乳化法流程示意圖………………………………………….63
圖十、微小球的降解與藥物釋放關係………………………………….64
圖十一、以溶膠-凝膠法包覆AMT流程……………………………….65
圖十二、以PLGA製備AMT微小球流程圖…………………………66
圖十三、AMT（HCl）藥物全波長掃瞄………………………………….67
圖十四、AMT（HCl）藥物濃度檢量線………………………………….67
圖十五、三種製備方式的silica-gel之單一時間點的AMT釋放量…68
圖十六、三種製備方式的silica-gel之AMT累積的釋放圖…………...68
圖十七、延長冷凍乾燥時間後AMT單一時間釋放圖………………69
圖十八、延長冷凍乾燥時間後之AMT累積釋放圖…………………...69
圖十九、silica-gel之單一時間點的AMT釋放量……………………...70
圖二十、Hood中自然揮發的silica-gel之AMT累積的釋放…………..70
圖二十一、PLGA（75/25）單一時間點的AMT釋放量………………...71
圖二十二、PLGA（75/25）的AMT累積釋放量………………………...71
圖二十三、PLGA（50/50）單一時間點的AMT釋放量………………...72
圖二十四、PLGA（50/50）的AMT累積釋放量………………………72
圖二十五、Hood揮發一個禮拜製成的silica-gel SEM影像圖………..73
圖二十六、冷凍乾燥製成的silica-gel SEM影像圖……………………74
圖二十七、PLGA（50/50）微小球電子顯微鏡影像…………………….75
圖二十八、PLGA（75/25）微小球電子顯微鏡影像…………………….76
圖二十九、細胞毒性測試長條圖……………………………………….77
圖三十、AMT silica-gel抑制IL-1β毒殺細胞的存活率直條圖……….78
圖三十一、移植前後的血糖變化曲線………………………………….79
圖三十二、移植前後的體重變化曲線………………………………….80
圖三十三、亞硝酸鹽（nitrite）標準減量線……………………………...81
圖三十四、Kaplan-Meier curve（存活率分析）…………………………82
表一、民國93、94年台灣國人十大死因排名表………………………..83
表二、免疫抑制藥物對胰島細胞的影響……………………………….84
表三、三種不同製備方式的silica-gel之粒徑大小…………………….85
表四、三種silica-gel分別於單一時間點及累積的AMT釋放比例（％）…………………………………………………..………….85
表五、延長冷凍乾燥時間後的silica-gel分別於單一時間點及累積的AMT釋放比例（％）…………………………………………….86
表六、Hood中自然揮發的silica-gel之粒徑大小……………………...86
表七、單一時間點及累積的AMT釋放比例（％）……………………87
表八、分別以不同比例的PLGA及乳化法製備之AMT微小球粒徑表..……………………………………………………………….87
表九、PLGA（75/25，50/50）微小球單一時間的AMT釋放比例（％）…..………………………………………………………….88
表十、PLGA（75/25，50/50）微小球的AMT累積釋放比例（％）………88
表十一、Blank、pure AMT和sol-gel AMT之MTS assay…………...…89
表十二、Blank、pure AMT和sol-gel AMT的細胞毒性測試…………89
表十三、AMT silica-gel抑制IL-1β毒殺細胞的MTS assay…………90
表十四、葡萄糖刺激胰島素分泌試驗（GSIS）…………………………91
表十五、移植前後的血糖紀錄表（one-way ANOVA）………………...92
表十六、移植前後的血糖紀錄表（paired-t test）………………………..93
表十七、移植前後的體重紀錄表（one-way ANOVA）………………...94
表十八、移植前後的體重紀錄表（paired-t test）………………………..95
表十九、亞硝酸鹽分析（nitrite assay）（one-way ANOVA）…………..96
表二十、亞硝酸鹽分析（nitrite assay）（paired-t test）…………………96
表二十一、Kaplan-Meier風險統計分析………………………………..97

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