The Research Of Colon Cancer Stem Cells Killed By Their Own Gp96 Pulsed DC-CIK Cells And Analysis Of Gp96 By Mass Speetrometry

[Objectives]The research ofcolon cancer stem cells killed by their own gp96-peptide complexes pulsed DC-CIK cells,and analysis of gp96-peptide complexes from HT29 and HT29CSCs by mass speetrometry. This experiment establish basis for the deep research.[Methods]1.The colon cancer stem cells were enriched from HT29 cells by serum free medium added a variety of cytokines .After microspheres wereformed ,we passage them by trypsin.2. The detection of enrichment: flow cytometry were used to detect of CD133 + cells from HT29 microsphere?3. Firstly,Thetotal protein were preliminary purified by ammonium sulfateprecipitat-ion.Then ConA-Sepharose affinity Chromatography were used to further purification.Lastly,the gp96-peptide complexes were obtainedby DEAE anion exchange Chromatography.To identify the purpose proteinthrough SDS-PAGE and Western-blot.The BCA method was used to measured the protein obtained from every step.4.DC and CIK were obtained from human peripheral blood mononuclear cells. The gp96-peptide complexes from colon cancer stem cells were used to promote maturation of DC cells, then co-cultured with CIK cells in the ration of 1:5.5.Experiments included the experimental groupand control group; With the gp96-peptide complexes were used as experimental group.Without as control group.6. The gp96-peptide complexes from HT29 and HT29CSCs were analyzed by mass speetrometry.[Results]1.We inoculate HT29 cell in serum free medium.After cultured for about 10 days in serum free medium, the HT29 could produce cell spheres suspended which state was optical.2. The flow cytometry showed that around the fifth generation of HT29 sphere cells expressed higher levels of stem cell markers CD 133+ (73.92±2.76) % than HT29 cells CD133+ (30.45±4.50) %.3. The proteins were identified as gp96-peptide complexes through SDS-PAGE and Western blotting.4. The study showed that no matter the effector-target ratio was 10:1 or 20:1 or 40:1,the killing rate of the group with the gp96-peptide is higher than the group without[P<0.05].5. Finally, 14 common contain proteins were identified in sample HT29 and sample HT29CSCs.[Conclusion]1. Suspension culture method can enrich HT29 colon cancer stem cells.We isolated from human peripheral blood mononuclear cells, and then we cultured them through adding proper growth factor to form DC and CIK repectively,then we get the mature DC and CIK cells..2. The gp96-peptide complexes were obtained from total proteins through ammonium sulfate precipitation. two classic chromatography.3. The group with the gp96-peptide had higher killing rate than the group without the gp96-peptide. This conclusion may prompt that gp96-peptide complexes play an important role in it. And this lays a good experimental basis for new study in the treatment of tumor.4. It's different in gp96-peptide complexes of HT29 and HT29CSCs.Compared the two proteins from those 14 common contains proteins, they had differences except the basic properties of protein.It's worth of further exploration.