標題: 葡萄糖與金精三羧酸(ATA)對中國倉鼠卵巢細胞 生長、貼附與重組蛋白表現之影響
The effects of glucose and aurintricarboxylic acid on Chinese hamster ovary cell growth, adhesion and recombinant protein expression
作者: 李宗益
Lee, Tsung-Yih
曾慶平
Tseng, Ching-Ping
生物科技系所
關鍵字: 中國倉鼠卵巢細胞;葡萄糖;金精三羧酸;Br-cAMP;P19 細胞;小鼠胚胎海馬迴神經細胞;Chnese hamster ovary cell;glucose;aurintricarboxylic acid;Br-cAMP;P19 cell;mouce embryonic hippocampal neuron
公開日期: 2012
摘要: 由美國食品藥物管理局的資料顯示,目前大部分核准之藥用蛋白生產細胞株,皆是透過中國倉鼠卵巢(Chinese hamster ovary; CHO)細胞來生產,因此我們使用該細胞株為研究對象,進行以下各項研究與其應用的探討。 本論文分成兩個部份進行研究:第一部分,葡萄糖對CHO細胞的生長抑制效應、機制與應用。第二部分,金精三羧酸(aurintricarboxylic acid;ATA)於無血清培養狀態下,對CHO細胞、P19細胞與小鼠胚胎海馬迴細胞的貼附現象。 在第一部份研究中,由於葡萄糖提供細胞生長所需碳源,但在高葡萄糖濃度培養基中,卻會導致CHO細胞生長抑制現象,在我們的研究中顯示,高葡萄糖培養條件下(10 g/L),CHO細胞比生長速率從1.29 d-1下降到0.91 d-1,培養基乳酸堆積增加一倍,細胞內cAMP濃度提高四倍,此一情況導致Extracellular signal-regulated kinases (ERK) 及細胞內p27kip 的磷酸化上升,造成細胞週期G0/G1遲滯(G0/G1 arrest)的情況。 從文獻探討發現,當CHO細胞呈現G0/G1 遲滯的情況時,會促進細胞重組蛋白的產生,因此我們利用會表現重組蛋白β-galactosidase (β-Gal) 的CHO細胞株(CHO-β-Gal)來探討此一抑制現象對重組蛋白表現之影響,從我們的實驗結果顯示,透過增加葡萄糖培養濃度並無法提高重組蛋白β-Gal產量,但是利用cAMP 類似物(Br-cAMP)的添加,卻可以提升β-Gal的表現量,當提高Br-cAMP濃度至250 μM時可提升1.6倍,比較目前被利用來促進蛋白質表現的誘導物質,發現Br-cAMP結合其他促進蛋白質表現的誘導物質對重組蛋白β-Gal的表現具有累加效果(最高提升4.6倍)。 第二部份,我們提供了一項細胞培養的替代技術,藉由添加非生肽化合物ATA來促進神經細胞的貼附生長無須透過培養材質表面前處理。我們先證明了ATA可以在無血清培養基中促進CHO細胞的附著和增生。接著我們發現,ATA可增強經retinoic acid (RA)分化的小鼠胚胎癌P19神經細胞的貼附生長(最佳ATA濃度30 mg/mL)。而運用到小鼠胚胎海馬迴細胞的培養中,也可觀察到類似的結果,最佳的ATA濃度為15 mg/mL。進一步的細胞型態觀察分析顯示,比較使用經poly-L-lysine (PLL)前處理的培養方法,兩者平均神經突觸長度和神經細胞的極化效果幾乎是相同的。
According to the U.S. Food and Drug Administration information, it was show that the majority of approved therapeutic proteins were produced by Chinese hamster ovary (CHO) cells culture. In this study, we use it as a model cell line for the various studies and their application. The dissertation is divided into two sections for my study: First, the mechanism and application of the glucose inhibitory effect on CHO cell growth. Second, the adhesion phenomena of CHO cells, P19 cells and mouse embryonic hippocampal cell growth by using aurintricarboxylic acid (ATA) in serum-free culture. In the first part of this study, glucose is an essential carbon source for cell growth. However, the high glucose concentration culture resulted in CHO cell growth inhibition. Our studies showed that in high glucose culture (10 g/L) condition, the cell specific growth rate of CHO decreased from 1.29 d-1 to 0.91 d-1, the culture medium of lactate accumulation increased 2-fold, and the concentration of intracellular cAMP increased by four times. This situation induced the extracellular signal-regulated kinases (ERK) activation and p27kip phosphorylation, and resulted these in G0/G1 arrest. From the literature review, G0/G1 arrest is one of useful strategy to promote recombinant protein production. In this study, we use CHO-β-Gal cells which is overexpress recombinant protein β-galactosidase (β-Gal) for further application. Our results show that the production of recombinant β-Gal can’t be enhanced by increasing glucose concentration. However, we found that the usage of cAMP analogs (Br-cAMP) enhanced the expression of β-Gal to 1.6 fold, when Br-cAMP increased to 250 μM. Next, we test the protein expression effect by combination of Br-cAMP with some well-known compounds of protein expression enhancer. Our finding indicated the compound of protein expression enhancer combining with Br-cAMP can promote more recombinant β-Gal production up to 4.6-fold. On the second part of this study, we provide an alternative technique to promote the adhesions of the neuron cell growth using ATA, a non-peptide compound, without surface manipulations. We first demonstrated that ATA can promote CHO cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid (RA) differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 μg/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 μg/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine (PLL) surface.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079029510
http://hdl.handle.net/11536/74217
顯示於類別:畢業論文