| Summary: | 博士 === 國立交通大學 === 生物科技系所 === 101 === 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.
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