誘導劑與抗氧化劑在化學合成培養基中對NS0細胞生長及重組蛋白生產的影響

本篇論文的主要目標為添加不同濃度誘導劑及抗氧化劑在適合骨髓瘤細胞（NS0）化學合成無血清培養基中，觀察對細胞生長與蛋白質表現的影響。我們先行馴化NS0細胞於自行配製的無血清培養基中，使其生長狀況及細胞型態保持穩定，並將細胞凍存。接著我們先利用六孔培養盤(six well plate)測試誘導劑(sodium propionate、 sodium butyrate、 DMSO )及抗氧化劑(vitamin C、N-acetyl L-cysteine、aurintricarboxylic acid)對NS0的毒性並測量蛋白質表現量，分別找出合適之添加濃度。在縮小濃度的範圍之下，我們以T型培養瓶（T-flask）為實驗系統，添加不同組合的誘導劑及抗氧化劑觀察其對細胞生長情形與蛋白質表現的影響。在初期多組交互作用實驗中發現誘導劑的丙酸(sodium propionate)對我們所使用的細胞茱具有非常獨特的效用，在增加重組蛋白質產量及對細胞的保護性方面比以往文獻中所常用的丁酸(sodium butyrate)更加出色。所以我們對丙酸及丁酸做一系列的探討。首先我們用T型培養瓶小範圍測試不同濃度的丙酸及丁酸，實驗結果顯示丁酸適合添加濃度為0.5mM以下，不至於造成細胞的死亡且蛋白質產量提高為原本的三分之一。而丙酸適合的添加濃度為2.0mM以下，蛋白質產量大幅度提高一倍的。但是兩者都有抑制細胞生長及防止細胞死亡的的趨勢。進一步採用搖瓶(Spinner flask)的系統進行測試，也具有一致的結果。基於丙酸及丁酸兩者都有提升蛋白質產量的效果，故嘗試同時將兩者添加於培養基中，研究是否具有加成效用。結果發現同時添加丙酸及丁酸對細胞的保護比單獨添加更具效果，蛋白質表現量與單獨添加丙酸結果相當。

關鍵字

骨髓瘤細胞；誘導劑；抗氧化劑；化學合成培養基

並列摘要

The aims of this thesis are to add different concentration inducer and antioxidant to a serum-free medium and to observe it’s influence on protein production and growing situation of recombinant NS0 cell. we used a serum-free medium adapted recombinant NS0 cell line as a model system to investigate the in teraction of various inducers and antioxidants and their effects on cell growth and recombinant prote in production. The serum-free medium, developed in house, is chemically defined and contains only one protein. We tested three inducer in six well plates, i.e., sodium butyrate, sodium propionate, and DMSO, and found that all three inducers were effective protein production enhancers, but sodium butyrate had high toxicity. We also tested three antioxidants, i.e., Vitamin C, N-acetyl L-cysteine, and ATA. Results show that these antioxidants could increase culture viability and extend culture life.Then we add suitable concentration inducer and antioxidant in T-flask together. In many interactive experiments found sodium propionate was better than sodium butyrate that be used Constantly.So we made a series of discussions for sodium propionate and sodium butyrate in T flask and spinner flask.The result indicated that the optimal induction concentration of sodium propionate for protein stimulation of NS0 cells was below 2.0mM and the expressions of fusion protein were elevated up 2.0–fold that of uninduced contro . The expression of fusion protein was similar to sodium propionate by the simultaneous addition of sodium propionate and sodium butyrate.

並列關鍵字

NS0 ； inducer ； antioxidant ； chemically defined medium

參考文獻

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誘導劑與抗氧化劑在化學合成培養基中對NS0細胞生長及重組蛋白生產的影響

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