Studies On The Applied Technologies Of Major Royal Jelly Proteins (MRJPs) In Culturing Of Human Cells To Replace Fetal Bovine Serum (FBS)

Royal jelly (RJ) is a kind of viscous liquid that is secreted by the hypopharyngeal gland, the mandibular gland and the postcerebral glands of the honeybee worker aging5-15days. RJ is the lifelong food for the queen, and has many nutritional functions for human beings. Fresh RJ contains60%-70%water,12%-15%protein,10%-16%carbohydrate,3%-6%fat and a few vitamins, minerals and free amino acids. Major RJ proteins (MRJPs), the whole water-soluble proteins in RJ, accounts for82%-90%of all proteins. Recent studies have demonstrated that MRJPs is the key factor which decides whether honeybee larvae develop into queen or worker. Studies on the proliferative activities of MRJPs on five human cell lines and the preliminary mechanism of action, and optimization with mixture of MRJPs and fetal bovine serum (FBS) culturing human cell lines were implemented. Furthermore, the effects on cell proliferation of several cytokines added in mixture of MRJPs and FBS were investigated.1. The proliferative effect of MRJPs on Chang Liver cell line and mechanism ofits actionThe effects of FBS, MRJPs and the mixture of MRJPs and FBS (M/F) with different ratios on the proliferation of Chang Liver cell line were assayed by MTT. The effects of different treatments on cell cycle distribution of Chang Liver cell line were detected by flow cytometry. Results showed that MRJPs alone could not promote proliferation of Chang Liver, and the best concentration of MRJPs in a complete medium (10%FBS) was0.5mg/mL. The mixture with M/F60/40exhibited best proliferative activity on Chang Liver. The effect of the mixture on the cell was not different significantly with that of FBS (p<0.05). It was shown by flow cytometry that the S phase and G0/G1phase of the cell in the medium with M/F60/40at5th day were not significantly different from the cell in FBS (p<0.05), indicating that MRJPs might promote DNA synthesizing in S phase or RNA and protein synthesizing in G0/G1phase. Therefore, MRJPs mixed with FBS could partially replace FBS to culture Chang liver cell line in practice.2. The proliferative effects of the mixture of MRJPs and FBS on four human cell linesFBS was replaced by MRJPs (5mg/mL) with different percentages (30%,60%,90%) to culture293T, HCT116,231and HFL-I cell lines. The10%FBS and10%PBS were set as positive control and negative control, respectively. The relative cell viabilities of different treatments were detected by MTT assay after5-8days of culturing. It was shown that the best M/F ratio for293T, HCT116, HFL-I cell lines was60/40as well. Cells cultured with this M/F ratio showed the highest cell viabilities, which was not significantly different from the cells cultured with FBS, even higher than that of the latter. However, the mixed M/F serum had no significant proliferative activity on231cell line. The cell viability on the5th day was only34.4%of the cell cultured with FBS, indicating M/F has selectivity for cells.3. The proliferative effects of the mixed M/F serum with cytokines on five human cell linesEpidermal growth factor (EGF), Insulin-Transferrin-Selenium (ITS) and dexamethasone (DMSM) in full combinations were added into the mixed M/F serum(60/40) to culture Chang Liver,293T, HCT116,231and HFL-I cell lines.10%FBS and the mixed M/F serum (no cytokines) were set as positive control and negative control, respectively. The relative cell viabilities of each treatment were detected by MTT assay after3-8days of culturing. Results showed that the mixed M/F serum, together with EGF and ITS could significantly promote proliferation of Chang Liver,293T, HCT116and HFL-I. The relative viabilities of those cell lines cultured with mixed M/F serum containing EGF and ITS were significantly higher than those cell lines in the positive control. The addition of dexamethasone showed no significant proliferative activities to all cell lines. Besides, the addition of cytokines showed no proliferative effect on231cell line, and231cells could not adhere to the bottom properly.These studies demonstrated that MRJPs which could promote prolication of cells could partially replace FBS to culture many human cell lines. Four cell lines fitted cultured with MRJPs were selected, and the formulation of M/F medium and cytokines were optimized. All those provide new evidences to develop new usages of RJ in future.