Primary Study Of The Effection Of CIK Combined With DC Pulsed Gp96-peptide Complexes From Colon Cancer Stem Cell On Homologous Cancer Stem Cells

Objectives: To investigate the tumor-inhibitory effect of cytokine-induced killer cells(CIK) co-cultured with dendritic cells(DC) pulsed gp96-peptide complexes on HT29 colon cancer stem cells(CSC). This experiment establish basis for the deep research.Methods: 1. Hoechst 33342 staining test were used to assess the proportion of side population(SP) cells in cultured HT29 cells;Flow cytometry、Western blotting analysis were used to observe the expression levels of CD133 and ALDH1 in adherent cells. 2. HT29 cells were cultured in serum-free condition medium containing growth factors.It was better to use trypsin to digest for preparation of single cell suspension to passage after microspheres were formed. 3. Flow cytometry、Western blotting analysis and Colony formation assay were used to observe the expression levels of stem cell markers and the colony formation ability of HT29 sphere cells. 4. gp96-peptide complexes were purified from colon cancer HT29 sphere cells by ammonium sulfate precipitation,Con A-Sepharose affinity Chromatography and DEAE anion exchange Chromatography.Then it was identified by SDS-PAGE and Western blotting.BCA kit was used to measure the concentration of gp96-peptide complexes. 5. DC and CIK were respectively cultured which were separated from umbilical cord blood.It was better to add gp96-peptide complexes to DC on the 5th day and continue to culture for 72 h which purpose was to promote maturation of DC.DC and CIK were co-cultured in the ratio of 1:5 on the 10 th day,and were carried on to test killing activity after 2 or 3 days by the means of LDH.Results: 1. The percentage of SP cells in HT29 cells was(3.45 ±0.05)%;The HT29 cells contained CD133+ which percentage was(45.38%±10.65%) and ALDH1+subpopulation of colon cancer stem cell. 2. After cultured for 7-10 days in SFM,it could produce cell spheres suspended which morphology were regular and state was optimal.Serum could promote the differentiation of HT29 sphere cells.In addition,it could transform in SSM and SFM. 3. The results showed that there was significantly higher expression of ALDH1 in HT29 sphere cells(P<0.05); HT29 sphere cells expressed higher levels of stem cell markers CD133+ than HT29 cells(P<0.05); The Clone Formation Efficiency of the HT29 sphere cells was higher than HT29 cells(P<0.01). 4. The protein purified from HT29 sphere cells was identified as gp96-peptide complexes by SDS-PAGE and Western blotting.40μg gp96-peptide complexes which purity was 95% could be obtained from 1g wet weight of HT29 sphere cells. 5. CIK co-cultured with DC which pulsed gp96-peptide complexes was owning hig- her killing activity[(33.07±2.79)%] than DC+CIK group[(15.20±1.67)% ](P<0.05).when the effector-target ratio was 40:1.Conclusion: 1. Suspension culture method can enrich HT29 colon cancer CSC and it is easy to operate and suitable for experimental study. 2. Gp96-peptide complexes can be gained from HT29 sphere cells by ammonium s- ulfate precipitation,Con A-Sepharose affinity Chromatography and DEAE anion exchange Chromatography. 3. CIK co-cultured with DC which pulsed gp96-peptide complexes kills colon canc- er CSC effectively which establish basis for new strategy study of CSC...