臺灣博碩士論文加值系統

以哺乳類動物細胞生產重組蛋白，獲得之蛋白結構與醣修飾與人體內的結構較相像，但產率遠比以微生物表現系統低，產率偏低的問題可藉由選殖較好的細胞株來改善。中國倉鼠卵巢細胞是最早用於生產重組醣蛋白藥物的細胞株，基因重組人類巨噬細胞刺激因子 (recombinant human macrophage colony-stimulating factor, M-CSF) 能刺激巨噬細胞增生與分化，是目前應用於血液疾病相當重要的蛋白質藥物。本實驗使用基因重組之中國倉鼠卵巢細胞株作為實驗用細胞株。
首先使用PLGA配合二次乳化法製備具有多孔洞之輕量化微載體，將細胞接種於含有微載體之培養基中，以旋轉角瓶配合低速攪拌器進行細胞培養，微載體之多孔洞結構可避免細胞受到攪拌所產生的剪切力影響，使細胞在懸浮的環境中增殖、分化及產生目標產物。
本實驗以乳化法所製備之實心微載體粒徑約為117.8±15.0 μm ，1 % NH4HCO3-PLGA微載體粒徑為375.8±78.5 μm，5 % NH4HCO3-PLGA粒徑為442.4±25.8μm，兩者孔洞大小均介於17~18μm。而發泡劑濃度越高，所製備之微載體粒徑越大，孔洞越多，相對密度較低。對照組為Ctyodex 3商品化微載體。商品化微載體細胞培養到達第四天即到最高密度，隨即引發大量細胞凋亡。以乳化法製備之實心微載體由於其表面平滑，細胞貼附在其表面，在spinner flask中生長的情況不佳。添加1 %發泡劑 NH4HCO3所製備之微載體所培養之細胞密度生長到達第12天才到最高，但其最終細胞數量仍可達到與商品化微載體相仿。添加5% 發泡劑之PLGA微載體細胞生長至第十六天才到達最高密度，其細胞生長速率及細胞密度均不如1% 發泡劑所製備之微載體良好。
以M-CSF ELISA kit 測定細胞所產生之M-CSF產量，1 % NH4HCO3-PLGA在細胞生長及M-CSF生產量上有較佳的表現，在100 ml spinner flask 中所測得的M-CSF含量為5.7 μg，約為12盤T-75細胞的產量。
本研究結果顯示，以1 % NH4HCO3-PLGA配合spinner flask在及時監控細胞密度和培養程序上都非常簡便，且確實可有效增加細胞密度及細胞產物的含量，具有發展為生產細胞產物系統的潛力。

Glycosylation and post-translational modification of recombinant proteins are the unique feature of mammalian cell system. However, the productivity of recombinant protein by mammalian cells is much lower than that of microorganisms. Chinese hamster ovary cell line is the most popular mammalian host for the commercial production of therapeutic proteins. We used Chinese hamster ovary cells (5/9 M alpha 3-18, BCRC 60185, CHO cells) to express macrophage colony-stimulating factor (M-CSF), which can stimulate macrophage proliferaction, differenciation and survival.
Cells were inoculated into culture system containing light weight porous microspheres, which were made of biodegradable polymer, PLGA. The support of PLGA microspheres reduced cell damage resulting from the shear force.
The particle size of dense PLGA particles was 117.8±15.0 μm；1% NH4HCO3-PLGA microcarrier was about 375.8±78.5 μm；5 % NH4HCO3-PLGA particle size was 442.4±25.8μm。The pore size of 1 % NH4HCO3-PLGA and 5 % NH4HCO3-PLGA was between 17~18μm。
Cytodex 3 is a commercial cell culture microcarrier, which was cross-linked dextran coated with denatured collagen. After cell culture with Cytodex 3 for 4 days, the cell density reached maximum, and the onset of cell death was observed. The cell culture with PLGA microcarriers, made of 1% NH4HCO3 and PLGA, the cells grew till day 13. The final density of cells was similar to that cultured with Cytodex 3. The 5 % NH4HCO3-PLGA microcarriers showed the massive cell death up to day 17, but the cell growth rate and cell density were both smaller to that with 1% PLGA microcarriers. The yield of M-CSF is higher for cell culture with 1 % NH4HCO3-PLGA microcarrier. The 1% NH4HCO3-PLGA microcarrier was the best in cell density and M-CSF production.

謝誌 i
中文摘要 ii
英文摘要 iv
目錄 vi
圖目錄 xi
表目錄 xiv
第一章 緒論 1
1.1前言 1
1.2研究動機與目的 4

第二章 文獻回顧 5
2.1 貼附型細胞培養 5
2.1.1 中國倉.鼠卵巢細胞 6
2.2 治療用蛋白 8
2.2.1 細胞週期 9
2.2.2巨噬細胞刺激因子 10
2.3 微載體培養貼附型細胞 11
2.3.1 微載體(microcarrier) 11
2.3.2 懸轉角瓶(spinner flask) 13
2.4 生物可降解性高分子 14
2.4.1聚乳酸-甘醇酸共聚物 15
2.5 聚乳酸-甘醇酸共聚物（Poly (lactide-co-glycolide), PLGA）製備微載體 16
2.5.1 乳化法製備PLGA微載體 16
2.5.2 發泡法製備多孔洞PLGA微載體 17
2.6 血清預處理 19

第三章 實驗藥品與方法 20
3.1 實驗流程 20
3.2 乳化法製備PLGA微載體 21
3.2.1 實驗藥品 21
3.2.2 實驗儀器 21
3.2.3 實驗方法 22
3.3 發泡法製備多孔洞PLGA微載體 24
3.3.1 實驗藥品 24
3.3.2 實驗儀器 24
3.3.3 實驗方法 25
3.4 微載體表面型態及性質測試 27
3.4.1 微載體表面型態觀察 27
3.4.1.1 使用儀器 27
3.4.1.2 實驗方法 27
3.5 細胞培養 28
3.5.1 實驗藥品 28
3.5.2 實驗儀器 28
3.5.3 實驗方法 30
3.5.3.1 冷凍細胞活化 30
3.5.3.2 細胞繼代培養 (Subculture) 31
3.6 微載體培養 32
3.6.1 實驗藥品 32
3.6.2 實驗儀器 33
3.6.3 實驗方法 34
3.6.3.1 微粒以血清預處理 34
3.6.3.2 微粒與細胞共培養 35
3.6.3.3 MTT 36
3.6.3.3.1 實驗原理 36
3.6.3.3.2實驗方法 37
3.6.3.4 EtBr / A.O. 螢光染色 39
3.7 M-CSF 濃度測定 40
3.7.1 實驗藥品 40
3.7.2 實驗儀器 41
3.7.3 前置作業 41
3.7.4 實驗方法 42

第四章 實驗結果與討論 43
4.1 微載體表面型態觀察及性質之測量 43
4.1.1 SEM觀察PLGA微載體表面型態 43
4.2 5/9 M alpha 3-18 細胞活化 46
4.3微載體培養M slpha 3-18 中國倉鼠卵巢細胞 47
4.3.1 血清前處理 47
4.3.2 懸浮培養 49
4.3.2.1 細胞貼附及生長 49
4.3.2.2 細胞生長曲線 54
4.3.2.3 轉速對細胞生長的影響 59
4.4 M-CSF 產量之比較 63

第五章 結論 67
參考文獻 71
附錄 77

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