Experimental Study On The Regulating Effects Of Hedysari Radix Polysaccharide On Immune Function Of Lymphocytes In Mice

Objective:To study the regulating effects of Hedysari Radix polysaccharide(HPS) on immune function of lymphocytes in mice and analyze the differentially expressed proteins after treating mouse spleen lymphocytes with HPS.Methods:1. Effects of HPS on proliferation of spleen lymphocytes of mouse were tested by MTT test, percentage of CD3T lymphocyte and CD4+/CD8+T lymphocyte was tested by cytometric method, and the level of cytokines (IL-2/IL-4/INF-y) in the cultural supernatants was tested by enzyme-linked immuno sorbent assay (ELISA).2. To separate the spleen lymphocytes of mouse into HPS group and control group, the total proteins of mouse spleen lymphocytes which have treated with HPS in 100μg/ml for seventy-two hours were extracted by using standard pyrolysis, and then were purifyed by using acetone precipitation.we can verify whether the acetone precipitation is suitable method for protein extraction of mouse spleen lymphocytes, meanwhile we optimized the condition of the isoelectric focusing electrophoresis (IEF), and compared the 2-DE patterns which were stained by using Beyotime Sliver Stain kit in two groups. 3. The protein of mice spleen lymphocytes were separated by using the two-dimensional electrophoresis which have been optimized, and the differentially expressed proteins of were detected on the 2-DE maps by image analysis software named PDQuest 8.0.Results:1.MTT outcome showed that the ability of spleen lymphocyte proliferation increases significantly when the HPS concentration were 40/80/100/200μg/ml(P<0.05), and under the same culture conditions, the ability of spleen T lymphocyte proliferation in HPS group treated with 100μg/ml of HPS-3 concentration was significantly higher than the other drug group; in HPS group, the percentage of CD3+ and CD3+CD8+ T lymphocyte was increased, the ELISA tested on cell culture supernatant showed that the level of IL-2(P<0.01) and INF-ywas improved(P<0.05), the level of IL-4 was resuced(P0.05).2. Aeetone Precipitation can increase protein solubility and enhance the number of protein. At the same time, the horizontal and vertical tailing was ouviously decreased after optimizing the condition of the isoelectric focusing electrophoresis (IEF), a high definition 2-DE gel with more complete protein separation and distinct protein spots was obtained. 3. The analysis results of the 2-DE maps of two groups by the image analysis software named PDQuest 8.0 showed that:the total protein points of HPS group was 415±16 and the control group was 397±14, there were 16 spots changed significantly in HPS group, including eleven up regulated and one down regulated and four disappeared.Conclusions:1. HPS have the effect of improving the ability of mouse spleen T lymphocyte and the level of IL-2, so HPS can improve the immunity of mice.2. Standard pyrolysis combined with acetone precipitation is a sutable method for protein extraction of mouse spleen lymphocytes, the 2-DE maps of mouse spleen lymphocytes treated with HPS have been obtained after optimizing experimental conditions.3. The expression of mouse spleen lymphocytes protein can be impacted significantly.