ABSTRACT
AIM: To develop a novel hepatocyte serum-free medium based on sericin, and to explore the effect of sericin on the hepatocyte transcriptome. METHODS: A controlled trial comparing novel serum-free medium and other media: C3A cells were cultured in our novel serum-free medium, HepatoZYME, complete medium (DMEM/F12 with 100 mL/L FBS), and DMEM/F12, and then cell attachment, proliferation, and function as well as the biocompatibility of the media were assessed. A comparative study of serum-free media with or without 2 mg/mL sericin: the effect of sericin on C3A growth was assessed by cell viability and proliferation, the effect of sericin on C3A cell cycle distribution was determined by flow cytometry, and the effect of sericin on the C3A transcriptome was assessed by gene-chip array and RT-qPCR. RESULTS: More C3A cells attached to the plate containing our serum-free medium than to those containing HepatoZYME and DMEM/F12 at 24 h post-seeding. Both the viability and proliferation rate of C3A cells in sericin-based serum-free medium were superior to those of cells in HepatoZYME and DMEM/F12 (P < 0.001). The content of albumin and urea in our serum-free medium was significantly higher than that in HepatoZYME and DMEM/F12 throughout the whole culture period (P < 0.001) and was similar to that in complete medium at day 3, 4, and 5. In part 2, cell viability and proliferation were greater in the presence of 2 mg/mL sericin (P < 0.001), as was the proportion of cells in S phase (16.21% ± 0.98% vs 12.61% ± 0.90%, P < 0.01). Gene-chip array analysis indicated that the expression of CCR6, EGFR, and FOS were up-regulated by 2 mg/mL sericin, and RT-qPCR revealed that the expression of CCR6, EGFR, FOS, AKT1, JNK1, NFkB1, MMP-9, MEK2, ERK1/2 and MYC was up-regulated by 2 mg/mL sericin (P < 0.05). CONCLUSION: We developed a novel hepatocyte serum-free medium. Sericin probably enhances cell attachment through the CCR6-Akt-JNK-NF-κB pathway and promotes cell proliferation through CCR6-mediated activation of the ERK1/2-MAPK pathway.
Subject(s)
Cell Culture Techniques/methods , Hepatocytes/drug effects , Sericins/pharmacology , Transcriptome/drug effects , Cell Adhesion/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Culture Media, Serum-Free/chemistry , Culture Media, Serum-Free/pharmacology , Gene Expression Profiling/methods , Hepatocytes/metabolism , Humans , Oligonucleotide Array Sequence Analysis/methodsABSTRACT
Inactivation effect of pulsed electric field (PEF) on polyphenol oxidase (PPO) and lipoxygenase (LOX) was investigated using a laboratory PEF system with a coaxial treatment chamber. Circular dichroism (CD) and fluorescence analysis were used to study the conformation change of the protein. The experimental results show that PPO and LOX can be effectively inactivated by the PEF treatment. Inactivation effect of PPO and LOX increases with the increase in the applied electric strength and the treatment time. Activity of PPO and LOX can be reduced by 60.3% and 21.7% at 20 kV x cm(-1) after being treated for 320 micros respectively. The decrease of the negative peaks (208 and 215 nm in PPO spectra, 208 nm and 218 nm in LOX spectra) in CD spectra of PPO and LOX shows that PEF treatment caused a loss of alpha-helix and increase in beta-sheet content, indicating that conformation changes occur in the secondary structure of PPO and LOX enzyme. This effect was strengthened as the applied electric field increased: alpha-helical content of PPO and LOX was 56% and 29% after being treated at 8 kV x cm(-1), however, when the electric field was increased up to 20 kV x cm(-1), alpha-helical content of PPO and LOX decreased to 21% and 16% respectively. The decrease rate of alpha-helix and increase rate of beta-sheet in PPO are higher than LOX, indicating that the second conformation of PPO is less resistant to PEF treatment than LOX. The fluorescence intensity of LOX increases after PEF treatment. At the same time, increasing the applied pulsed electric field increases the fluorescence intensity emitted. Fluorescence measurements confirm that tertiary conformation changes occur in the local structure of LOX. However the possible mechanism of the conformation change induced by the PEF treatment is beyond the scope of the present investigation.
