| Summary: | 碩士 === 文化大學 === 生物科技研究所 === 82 === HepG2, a human hepatoma cell line, was capable of secreting twenty six major proteins in human plasma. The results of initial work done in this lab indicated that HepG2 cell can be long-term and large scale cultivated in serum-free medium using a hollow fiber bioreactor and HepG2 crude conditioned medium can substitute fetal calf serum in supporting the growth of several cell lines in vitro. The goal of this study is to further purify and characterize the growth factors from HepG2 crude conditioned medium which could be used as serum substituents.
A bioassay using propidium iodide staining and flow cytometer (FACSort) was established to analyze the viability and proliferation of cells in vitro. The application of HepG2 conditioned medium as serum subsitituents was studied using 16 cell lines and CEM and H9 cells were chosen as the target cells thoughout the entire purification process.
HepG2 growth factors were purified from HepG2 conditioned medium by the following methods: ammonium sulfate precipitation, DEAE Sepharose fast flow ion exchange liquid chromatography. Blue Sepharose CL6B affinity chromatography and FPLC MonoP chromatofocusing. The results indicated that the specific bioactivity was increased one to 1.8 fold, 12 to 17 fold, 5 to 9 fold and 13 to 32 fold in the fraction of 60-80% ammonium sulfate precipitation, DEAE 0-0.05M NaCl, Blue 0-20 mM and 20-100 mM Tris, respectively.
The N-terminal 22 amino acids sequence of Frac-Ⅰ, Frac-Ⅱand Frac-Ⅲ following FPLC chromatofocusing were sequenced. As compared to peptide data base (FASTA), the similarities of 72%, 57% and 93% were found between Frac-Ⅰ and human transferrin, Frac-Ⅱ and bacterial alcohol dehydrogenase, and Frac- Ⅲ and human liver mangnase-superoxide dismutase, respectively.
To improve the effect of HepG2 growth factor as serum substituent, BSA, transferrin and insulin were supplemented as medium additives. The results indicated some enhanced effects with additives; however, the effects vary with cell types tested.
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