Fluorescent Labeling Of Polysaccharide From Pine Pollen And Its Effect On TLR4 Of Cells

Our studies on polysaccharides from pollen mainly concentrated in the fat cells, cancer cells and immune cells in the last ten years. Among them, the studies on immune cells contain T lymphocytes, B lymphocytes, macrophages and researches on calcium ion related signaling pathways.Although our lab. has finished so much work, the specific function of polysaccharide on cell is in vivo or in vitro is still not very clear. In addition, due to having no glowing group and the complex role in cells of polysaccharide, so it is difficult in research. In view of the two reasons above, in this experiment,we are determined to label the polysaccharide by fluorescent marker and then research the effect on the cells.This paper mainly includes the following contents:1. Pollen polysaccharide was extracted using the method of hot water extraction. The crude polysaccharide had been removed the protein was precipitated by ethanol with different concentrations of 20%, 40%, 60%, 80%. Among them, 60% polysaccharide named PPM60 with better activity is needed for this experiment.2. PPM60 was labeled with fluorescein. Firstly, PPM60-Tyr, the reactants of PPM60 and tyramine were generated by Sephacryl S-400 HR, we could measure a strong absorption peak using ultraviolet spectrophotometer under 280 nm. Secondly, the production from the reaction of PPM60-Tyr with FITC named PPM60-Tyr–FITC were generated by Sephacryl S-400 HR, and we tested the result of the absorption peaks of the FITC using fluorescence spectrophotometer, then test the absorption peak of polysaccharide using the method of phenol-sulfuric acid. Finally, we contrasted the results, there were 30 tubes in the front of others had two peaks for our purpose named peak 1 and peak 4. Then, each of the two peaks was purification 2-3 times using Sephacryl S- 400 HR respectively. After the purification of peak 1 and peak 4, we calculated the substitution degree was 4.32% and 0.66% respectively, which had reached the requirement of detection machines such as flow cytometry instrument. Through the analysis of the results, we found that the experiment of labeling was successful.3. The effect of PPM60-Tyr-FITC on the proliferation of cells. According to the tests above, the fluorescence intensity of peak 1 was higher than peak 4, so we selected peak 1 in the experiment. From the results, PPM60-Tyr-FITC has a little influence on the activity of the cells, which could meet the requirement of the experiment on cells, 400 ?g/mL was the optimum concentration.4. The effect of PPM60-Tyr-FITC on TLR4 of macrophage RAW264.7. We tested the standard polysaccharide of FITC–dextran and PPM60-Tyr-FITC using flow cytometry and confocal laser scanning microscope,the results showed that FITC-dextran and PPM60-Tyr-FITC could enter cells from outside to inside with the change of time. We inhibited the key protein involved in endocytosis with inhibitors, the results showed that clathrin and TLR4 played an important role in the entering of FITC-dextran and PPM60-Tyr-FITC into cells.5. The effect of PPM60-Tyr-FITC on TLR4 of the rat small intestinal crypt epithelial cells, IEC-6. The results showed that clathrin and TLR4 played an important role in the entering of FITC-dextran and PPM60-Tyr-FITC into IEC-6 cells.