| Lipoteichoic acid (LTA) as a common and unique component of cellula and cytoderm of gram positive bateria, play an important role in the pathogenic process of Staphylococcus aureus. This paper aims to study the effect of LTA extracted by TrionX-114on the process of Staphylococcus aureus interact with intestinal epithelial cells.Trial1:TrionX-114method is used to extract the LTA by DEAE-Sephacel, Sephedex G-10gel column chromatography. The contents of LTA is detected phosphorus by phosphomolybdic acid and the residues of protein and nucleic acid is measured by UV scanner. In addition, we analyze and test the structure of LTA by Infrared Spectroscopy and Nuclear Magnetic Resonance Spectroscopy. Results show that the yield of LTA extracted by TrionX-114is low, only0.83%o of the weight of bacteria (m/m), but there are almost no residues of protein and nucleic acid. What more, LTA has a complete structure especially the D-alaine construction which play a role on the adhesion and immune stimulation. Therefore, using TrionX-114can extract the complete structure and high purity of LTA.Trial2:First pretreatment HT-29cells using different concentrations of LTA, and then adhere to cells with different concentrations of Staphylococcus aureus marked by FITC. Results show that Staphylococcus aureus ahhesional rate of the experimental group decline along with the increase of LTA’s concentration compared with the blank group.This result indicates that LTA plays an important role on the process of Staphylococcus aureus adhere to HT-29cells.Tria13:Pretreatment HT-29cells using different concentrations of LTA or Staphylococcus aureus for a certain period of time, then measure the expression level and extracellular concertration of immune factors.Results show that both experimental groups significantly promote the level of inflammatory factors NF-κB, IL-6, IL-8, IL-12, TNF-a and inhibitory effect of proinflammatory factor IL-10. However another inhibitory effect of proinflammatory factor Hsp70has no obvious change. We measure those cytokines IL-6, IL-10, TNF-a, IL-12of cell culture medium by ELISA technology. The results show that the concentrations of IL-6, IL-10, TNF-a are at highest when the concentration of LTA is50μg mL-1and Staphylococcus aureus is1×107CFU mL-1; The level of IL-12in cell culture medium is at highest when the concentration of LTA is25μg mL-1and Staphylococcus aureus is1×109CFU mL-1. Results indicate that LTA plays an important role on the immune stimulation in the process of Staphylococcus aureus interact with HT-29cells with different trends for different immune factors. |
