臺灣博碩士論文加值系統

中國倉鼠卵巢細胞經常被應用於生技工業上來生產重組蛋白。無血清培養的發展是相當有潛力的，其先決條件必須具備維持動物細胞的存活率以及降低細胞死亡。本實驗目的將嘗試尋找新的生長因子，在無血清條件下期望能維持細胞存活率並能促進細胞生長。研究中將使用可分泌巨噬細胞群落因子之中國倉鼠卵巢細胞進行探討。過程中，將評估多種效應物其中包含兩種生長因子是否對細胞生長有正面提升。在無血清條件下，將利用流式細胞儀及螢光顯微鏡來分析細胞週期與凋亡細胞之確認。測試結果發現，單獨添加胰島素與鹼性纖維母細胞生長因子可防止細胞死亡以及維持其存活率，但不能有效運作細胞週期而促進細胞增生。不過，於基礎培養基(DMEM/F12)培養條件下，同時添加胰島素與鹼性纖維母細胞生長因子之下，對於細胞生長有協同效應並能分泌巨噬細胞群落因子。本次的實驗結果，可以作為無血清培養基設計上的有效資訊並提供在無血清條件下抗細胞凋亡的研究訊息。關於間葉幹細胞無血清配方研究，利用單因子篩選實驗結果可以發現商業化血清取代物(lipumin)、ITS+1、bFGF、胰島素對於由WJ來源之間葉系幹細胞具有促進之能力，於之後的研究將利用哺乳類之phenotypic微陣列輔助觀察添加物對幹細胞增生之影響並進行新的取代物篩選。最後的目標仍是希望開發出一適合間葉幹細胞增生與分化的無血清培養基。

Chinese hamster ovary (CHO) cells are commonly used in the biotechnology industry to produce recombinant proteins. The development of serum-free media in order to maintain mammalian cell viability and reduce cell death in the absence of serum is highly desirable. Our aim was to find new growth factors that could maintain viability and further enhance the proliferation of CHO cells under serum-free conditions. Macrophage colony-stimulating factor (M-CSF)-secreting CHO cells were used as a model cell line in this study. The stimulatory effects of different compounds including two growth factors on the proliferation of CHO cells were evaluated. Flow cytometry and fluorescence microscopy were used to analyze the cell cycle and monitor apoptotic nuclei during the serum-free culture of CHO cells. Among the tested compounds, insulin and basic fibroblast growth factor (bFGF) each individually significantly prevented CHO cells from dying and helped maintain cell viability, but cells were unable to reenter the cell cycle and proliferate. Furthermore, simultaneous supplementation with insulin and bFGF synergistically promoted the growth of CHO cells and recombinant M-CSF synthesis in basal DMEM/F12 medium. The results presented here provide useful information for the design and development of serum-free media as well as for antiapoptotic studies of CHO cells under serum-free conditions. In preliminary results of mesenchymal stem cells (MSC), we found out the commercial serum substitute (lipumin), ITS, bFGF, insulin had the significantly effects on cell proliferation under serum-free conditions. After, we also used the commercial phenotype microarray to evaluate the effects of new serum substitutes. Finally, Our aim is to develop serum-free medium for MSC’s proliferation and differentiation.

目錄
指導教授推薦書
口試委員會審定書
授權書.......................................................................................................iii
誌謝 iv
中文摘要 v
英文摘要 vii
目錄 ix
圖目錄 xiii
表目錄 xv
第一章 緒論 - 1 -
1.1 研究動機與目的 - 1 -
1.2 研究架構 - 3 -
第二章 文獻回顧 - 5 -
2.1動物細胞與幹細胞培養概述 - 5 -
2.1.1 中國倉鼠卵巢細胞簡介 - 5 -
2.1.2 間葉幹細胞簡介 - 5 -
2.1.3 細胞培養基之簡介 - 7 -
2.1.3.1 血清培養基 - 8 -
2.1.3.2 無血清培養基 - 12 -
2.1.4 間葉幹細胞無血清培養 - 14 -
2.2 巨噬細胞群落刺激因子 (M-CSF) 之簡介 - 17 -
2.2 巨噬細胞群落刺激因子 (M-CSF) 之簡介 - 17 -
2.3 血清取代物對細胞生長之探討 - 19 -
2.3.1 胰島素對細胞增生之影響 - 21 -
2.3.2 鹼性纖維母細胞生長因子對細胞增生之影響 - 22 -
2.4 利用蛋白質體學技術探討CHO細胞表現重組蛋白 - 24 -
2.5 間葉幹細胞之蛋白質體學 - 28 -
2.6 細胞週期 - 30 -
2.6.1 細胞週期簡介 - 30 -
第三章、材料與儀器設備 - 32 -
3.1材料 - 32 -
3.2儀器 - 34 -
第四章 實驗方法 - 35 -
4.1 細胞株與培養繼代方式 - 35 -
4.2 細胞計數與存活率計算 - 36 -
4.3 細胞解凍 - 37 -
4.4 無血清與含血清細胞冷凍保存 - 38 -
4.5 化學物質製備 - 38 -
4.6 單一化學物之細胞試驗 - 39 -
4.7 酵素結合免疫分析法(ELISA) - 39 -
4.7.1 ELISA試劑製備 - 40 -
4.8 MTT assay - 42 -
4.9細胞週期分析 - 42 -
4.10 Hoechst stain - 43 -
4.11 微陣列分析 (Phenotype microarrays) - 43 -
第五章 結果與討論 - 44 -
第一部份 CHO細胞 - 44 -
5.1篩選血清替代物與確認 - 44 -
5.2胰島素劑量篩選 - 45 -
5.3鹼性纖維母細胞生長因子劑量篩選 - 46 -
5.4 細胞週期與細胞凋亡之影響 - 50 -
5.5 M-CSF產量之分析 - 59 -
5.6 篩選血清替代物與確認 - 62 -
5.7 無血清培養之生長趨勢 - 63 -
第六章 結論與展望 - 70 -
6.1 結論 - 70 -
6.2 未來展望 - 71 -
第七章 參考文獻 - 72 -
第八章 附錄 - 78 -
8.1 Phenotype MicroarraysTM panels PM-M3 to PM-M4 - 78 -
8.2 Phenotype MicroarraysTM panels PM-M7 to PM-M8 - 79 -
8.3 Microarray test of WJ-MSC on M7 and M8 - 80 -
8.4 Microarray test of WJ-MSC on M3 and M4 - 82 -
8.5 Microarray test of BM-MSC on M7 and M8 - 84 -
8.6 Microarray test of CHO on M3 and M4 - 86 -


圖目錄
Fig. 2-1 dihydrofolate reductase (DHFR) reaction pathway - 21 -
Fig. 2-2 Effect of insulin on glucose uptake and metabolism - 22 -
Fig. 2-3 Functional distribution of identified proteins of UCB-MSC. - 29 -
Fig. 5-1 Seven materials: Olive oil(10 mg/L), Soybean oil(10 mg/L), Estasan(10 mg/L), Insulin(10 mg/L), BSA(10 mg/L), Ser EX(1 %) and bFGF (40 ng/ml)screen results - 48 -
Fig. 5-2 Dosage effect of insulin on CHO cells in DMEM/F12 medium………………………………………………………….- 48 -
Fig. 5-3 Dosage effect of bFGF on CHO cells with 80 μg/ml insulin in DMEM/F12 medium. - 49 -
Fig. 5-4. Compare viable cell density in three different condition (insulin alone、bFGF alone、insulin with bFGF) and HyQ serum-free medium………………………………………………………….- 49 -
Fig. 5-5 The cell of CHO cell after culture for 72 h. (a) HyQ serum-free medium, (b) insulin and bFGF in DMEM/F12, (c) insulin alone, (d) bFGF alone, (e) DMEM/F12. - 54 -
Fig. 5-6 Percentages of CHO cell in the cell cycle of Sub, G0/G1, S, G2/M phase. (a) The CHO cells were synchronized by culturing in DMEM/F12 supplemented with insulin, bFGF (b) HyQ serum-free medium. - 55 -
Fig. 5-7 Percentages of CHO cell in the cell cycle of Sub, G0/G1, S, G2/M phase. (a) The CHO cells were synchronized by culturing in DMEM/F12 supplemented with insulin (b) bFGF. - 56 -
Fig. 5-8 The nuclear morphologies of CHO cell after culture for 72 h…… - 58 -
Fig. 5-9 Effect of insulin and bFGF addition days on M-CSF production… - 61 -
Fig. 5-10 Effect of insulin and bFGF addition days on cell growth. - 61 -
Fig. 5-11 Four materials: Lipumin, bFGF, Serotonin, insulin screen results - 65 -
Fig. 5-12 Dosage effect of insulin on MSC in 5 % lipumin alone in α-MEM medium. - 65 -
Fig. 5-13 Dosage effect of bFGF on MSC in 5 % lipumin alone in α-MEM medium. - 66 -
Fig. 5-14 Dosage effect of ITS+1 on MSC in 5 % lipumin alone in α-MEM medium. - 66 -
Fig. 5-15 Dosage effect of ITS+1 on MSC in 5 % lipumin alone in α-MEM medium. - 67 -
Fig. 5-16 Dosage effect of ITS+1 on MSC in 5 % lipumin alone in α-MEM medium. - 67 -
Fig. 5-17 Dosage effect of ITS+1 on MSC in 5 % lipumin alone in α-MEM medium. - 68 -
Fig. 5-18 Effect of lipumin, ITS+1, bFGF addition days on cell growth in α-MEM medium - 69 -




表目錄
Table 1. Major protein components of plasma - 10 -
Table 2. Major functions of serum in mammalian cell culture - 10 -
Table 3. Selecting a suitable medium - 11 -
Table 4. Many of the functions of serum are listed, along with potential replacements to be used in serum-free media - 13 -
Table 5. Phenotype characterization and expression of growth factors by human mesenchymal stromal culture - 16 -
Table 6. Summary of proteome analysis in recombinant CHO cell production - 27 -
Table 5.1 Gating ratio of four type culture medium in different culture time - 57 -

劉繼賢 動物細胞培養與基因重組蛋白質生產之研究，清華大學化學工程研究所博士論文，2001

鄭琮霖 鹼性纖維母細胞生長因子對子宮上皮細胞死亡之影響，成功大學生理學研究所碩士論文，2001

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