臺灣博碩士論文加值系統

摘要
副甲狀腺功能低下是目前難以治癒的內分泌系統疾病之一，臨床治療上使用副甲狀腺體同種異體移植的方式，但可能終身需要合併使用免疫抑制藥物，以避免移植排斥的問題。在本研究中，我們的目的希望針對此一臨床上細胞移植的需求，開發一個適合同種異體移植的細胞遞送技術，保護移植後的細胞有效阻隔宿主免疫細胞的侵入浸潤，但同時又能將移植細胞所分泌的內分泌激素釋放出來，以達到補償副甲狀腺功能缺損的目的。在本研究中，我們提出一創新製程技術，以多孔隙明膠載體載負副甲狀腺體細胞，進一步以海藻酸鈉包覆細胞載體，形成不溶性海藻酸明膠微粒，有效阻隔免疫細胞浸潤並提供3D環境，以提升細胞代謝與存活。實驗首先評估比較鈣與鋇離子製備之海藻酸微粒，進一步使用明膠載體預先載負細胞後，形成雙層結構微粒的機械性質與內分泌激素釋放、免疫細胞阻隔、活體動物移植相容性等生物功能。綜合評估鈣與鋇離子運用於海藻酸交聯與雙層細胞載體製備技術，在移植細胞存活度、增生比例、細胞激素分泌，以及載體物化性質的分析。從實驗結果發現，我們在雙層結構的微球體包覆技術條件下，在細胞增生、細胞存活率都有增加至少一倍以上的趨勢，且對環境鈣離子調節副甲狀腺素分泌比沒有明膠支架的海藻酸微粒更加快速反應，且分泌總量更多。綜合結果，透過不溶性海藻酸明膠微粒技術的細胞，可顯著提高存活率、增生並感應鈣離子濃度變化分泌副甲狀腺素、避免移植後產生外異體纖維化現象與免疫細胞攻擊。
關鍵字：副甲狀腺功能低下；海藻酸；微粒；細胞包覆；鈣反應

Abstract
Hypoparathyroidism is one of the endocrine system diseases that are hard to be treated. Hypoparathyroidism is caused by hereditary, surgical injury, autoimmune disease, and some unknown reason; it will make hypocalcemia, numbness, anxiety, and fatigue. The reason that causes hypoparathyroidism is neck operation that injury the parathyroid glands or removes the parathyroid glands. Currently, methods to treat hypoparathyroidism are oral calcium tablets and Vit D to balance the average blood calcium level and standard urine output. However, this way cannot provide the effective dose that maintains the blood calcium level immediately. Therefore, in clinical treatment, allotransplantation of parathyroid glands is used but restricted to immunologic rejection. Meanwhile, patients need suppression immunotherapies for their whole life. Therefore, we aim to develop a cell transplantation platform that protects the transplanted cells, impedes the immunological attack, and releases the parathyroid hormone. In this study, we developed alginate microbeads for the delivery of parathyroid cells, compared the microbeads crosslinked with calcium or barium ions, and also pre-loaded cells in a gelatin scaffold to manufacture the microbeads with double-layered structure. Data revealed that the microbeads prepared with double-layered structure possess optimal cells proliferation rate and parathyroid hormone (PTH) secretion capability, which shows no significant difference compared with a control group. Collectively, our data demonstrated parathyroid tissues and cells loaded in gelatin scaffolds and prepared as double-layered microbeads through alginate hydrogel encapsulation, possessing optimal cell proliferation rate and calcium-responsive PTH secretion capability. These results could be further used to transplant cells in the animal model evaluation.

Keywords: Hypoparathyroidism, alginate, microbeads, cell encapsulation, calcium-responsive

第一章 緒論 1
一、 鈣離子功能 1
(一) 心血管系統 1
(二) 神經傳遞系統 2
(三) 內分泌系統 2
二、 副甲狀腺(Parathyroid gland) 3
三、 副甲狀腺素 (Parathyroid hormone, PTH) 4
四、 副甲狀腺功能低下 (Hypoparathyroidism) 4
五、 診斷 6
六、 現行副甲狀腺功能低下治療方式 6
七、 臨床副甲狀腺移植 8
八、 細胞包覆目前研究與挑戰 8
九、 海藻酸鹽微珠內的細胞包覆 9
十、 明膠支架(Gelatin Scaffold) 10
第二章 研究動機與目的 12
第三章 結果與討論 17
一、 組織 17
(一) 組織的存活度 17
(二) 組織培養在含有1 mM CaCl2培養基的存活率 18
二、 海藻酸微粒機械應力測試及孔隙觀察 19
(一) 海藻酸微粒機械應力測試 19
(二) 海藻酸微粒SEM孔隙觀察 24
三、 組織包覆 25
(一) 包覆組織存活度 26
(二) 組織副甲狀腺素釋放測試 27
四、 副甲狀腺細胞包覆 28
(一) 細胞分解/包覆 29
(二) 細胞副甲狀腺素釋放測試 29
(三) 細胞包覆存活率 32
(四) 細胞增殖試驗 36
(五) 細胞品質 38
五、 動物實驗 40
(一) 解剖顯微鏡觀察 40
(二) 組織切片染色 40
六、 討論 44
第四章 結論 47
第五章 實驗部分 48
一、 藥品 48
二、 儀器設備及實驗器具 49
三、 副甲狀腺組織存活測試 50
四、 包覆材料建構與製備 51
(一) 海藻酸鈣微粒(Ca beads) 51
(二) 海藻酸鈣明膠微粒(Sca-Ca beads) 52
(三) 海藻酸鋇微粒(Ba beads) 52
(四) 海藻酸鋇明膠微粒 53
五、 海藻酸微粒機械應力測試 54
六、 海藻酸微粒孔徑及表面觀察 55
七、 副甲狀腺組織製備 55
八、 副甲狀腺組織的微囊化 56
(一) 海藻酸鈣微粒(Ca beads) 56
(二) 海藻酸鋇微粒(Ba beads) 56
九、 組織/細胞存活試驗染色 57
十、 組織副甲狀腺素分泌試驗 58
十一、 副甲狀腺細胞分離製備 59
十二、 副甲狀腺細胞的微囊化 60
(一) 海藻酸鈣微粒(Ca beads) 60
(二) 海藻酸鈣明膠微粒 61
(三) 海藻酸鋇微粒(Ba beads) 62
(四) 海藻酸鋇明膠微粒 62
十三、 細胞副甲狀腺素分泌試驗 63
十四、 副甲狀腺素(PTH) ELISA測定 66
十五、 細胞增殖試驗 67
(一) 蛋白萃取 67
(二) 總蛋白試驗 67
十六、 蛋白表現量分析 68
(一) 西方墨點法Western Blot 68
十七、 實驗動物 72
(一) 手術分組 72
(二) 腹腔手術 73
(三) 皮下手術 73
(四) 檢體外觀拍攝 74
(五) 冷凍切片製備 74
(六) H&E染色 75
參考文獻 76

圖目錄
圖 1副甲狀腺調控血鈣濃度示意圖 4
圖2 實驗方法示意圖 13
圖3實驗流程圖 15
圖4 兩種包覆方式示意圖 16
圖5以完全的培養基培養的組織存活圖。 18
圖6 含1mM CaCl2 培養基培養的組織存活度。 19
圖7海藻酸鋇微粒機械應力測試結果。 21
圖8海藻酸鈣微粒機械應力測試結果。 22
圖9海藻酸鋇明膠微粒機械應力測試結果。 23
圖10海藻酸鈣明膠微粒機械應力測試結果。 24
圖11海藻酸微粒SEM圖。 25
圖12海藻酸鋇微粒(Ba beads)包覆圖。 26
圖13包覆完後五天 組織存活圖。 27
圖14不同鈣離子濃度測試海藻酸鋇微粒(Ba beads)內的組織PTH釋放。 28
圖15(A)海藻酸鋇微粒(Ba beads)包覆示意圖 (B) 海藻酸鋇明膠微粒(Sca-beads) 包覆示意圖。 29
圖16包覆細胞不同鈣離子濃度測試PTH釋放。 31
圖17海藻酸鋇微粒(Ba-beads)包覆細胞存活率。 33
圖18海藻酸鋇明膠微粒(Sca-beads)包覆細胞存活率。 34
圖19(A)海藻酸鋇微粒(Ba beads)螢光量化結果(B)海藻酸鋇明膠微粒(Sca-beads)螢光量化結果。 35
圖20(A) 海藻酸鋇微粒總蛋白測試(B)海藻酸鋇明膠微粒總蛋白測試 37
圖21(A) 海藻酸鋇微粒總蛋白測試(B)海藻酸鋇明膠微粒總蛋白測試。 39
圖22將Sca-Ba beads由皮下取出相比無明顯纖維化現象。 41
圖23將Sca-Ba beads由腹腔取出相比無明顯纖維化現象。 42
圖24 皮下移植進行H&E染色觀察相比無明顯纖維化現象。 43
圖25腹腔移植進行H&E染色觀察相比無明顯纖維化現象。 44
圖26 副甲狀腺組織切開分成1 x 1-2 x 2 mm碎片。 55
圖27為海藻酸微粒結構示意圖。 57
圖28組織PTH釋放檢測示意圖。 59
圖29海藻酸微粒結構示意圖。 63
圖30細胞PTH釋放檢測示意圖。 65

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