Differential Protein Spot Expression In Mouse Thymocyte Treated With The Third Polysaccharides From Radix Hedysari

Objective:1.To verify whether the third polysaccharides from Hedysarum polybotys saccharide (HPS-3) can regulate the immunity of mouse thymus organizations.2. To choose the best method for preparing the protein of the mouse thymus tissue treated with HPS-3through comparison the two-dimensional gel electrophoresis (2-DE) maps and give others some reference. It is also a strong basis for the identification of differential protein spots and the possible target-related.3. To find out the differential protein spots expression in mouse thymus organizations treated with HPS-3, and discuss the molecular biology mechanism of enhancing the immunity with HPS-3.Methods:l.The mice were treated with different doses of HPS-3(50mg/kg/day,100mg/kg/day) for fourteen days, the thymus gland index, spleen index and proliferation index of T lymphocyte were detected and the ultrastructure of mouse thymocyte through transmission electron microscope (TEM) was observed at the same time.2. Thymus totally proteins from the mice, which have given HPS-3in100mg/kg/d for fourteen days, were extracted using standard tissue lysis method, then trichloroacetic acid (TCA)-acetone precipitation method and tri-n-butylphosphate:acetone:methanol protein purification method were used respectively to purify the thymus protein. In the meantime, the tri-n-butylphosphate:acetone:methanol protein purification method was utilized to precipitate the protein from A549cell. The proteins were separated by means of immobilized pH gradient based on two-dimensional gel electrophoresis, and the condition of isoelectric focusing (IEF) was also adjusted and optimized, then the gels were stained by Beyotime Fast Sliver Stain kit, digitized images of2D gels that were generated using Image Scanner were analyzed by PD Quest software.3. The protein of mouse thymus organizations were separated utilizing2-DE which have been chose from the second method, analyzed the digitized images of2D gels with PD Quest software and found out the protein spots which differentially expressed between the two groups.Results:1.Compared with control group, thymus gland index and spleen index were raised obviously, T lymphocyte proliferation was promoted in HPS-3dosed with100mg/kg/day, but which treated with50mg/kg/day didn’t cause observable change. The ultrastructure characteristics of mice thymocyte showed that the mice which were given HPS-3with100mg/kg/day could promote the thymocyte disintegrating and proliferating.2. Tri-n-butylphosphate:acetone: methanol protein purification method not only decreased protein decomposition, but also increased protein solubility. There were1165±12spots expressed in the2-DE mapping which the protein has been precipitated through the tri-n-butylphosphate:acetone:methanol protein purification method, and better expression in acidic protein and low abundance than the TCA-acetone precipitation method. Besides, the focus voltage for gel electrophoresis was optimized on the traditional focus conditions, and a two-dimensional electrophoresis system with a clear background and good protein separation was successfully established for thymus tissue. Horizontal and vertical tail was improved significantly by the optimized focusing condition, and a high-resolution two-dimensional electrophoresis map with clearer protein spots and more complete separation was obtained easily, the tri-n-butylphosphate:acetone:methanol protein purification method was also suitable for the protein from A549cells.3. Analytic results of the2-DE mapping with PD Quest software is that:there were1106±37spots expressed in control group, and1165±12spots displayed in HPS-3group, there were192protein spots which were uifferentially expressed with more than twofold increased or decreased volume between control and HPS-3group.Conclusion:1. HPS-3can promote the thymocyte proliferating and elevate the immune responses of the mouse.2. The method of extracting thymus tissue protein was established. The protein spots in2-DE map were significantly increased using tri-n-butylphosphate:acetone: methanol protein purification method; in addition, by using the optimized method described above, satisfactory2-DE maps of thymus tissue has been obtained, which lays a foundation for the further study of the proteome of thymus tissue treated with HPS-3.3. HPS-3significant impact the expression of the mouse thymus organizations proteins.