Intervention Study On Immune Cells Loaded By Antigen Of HT29 Colon Cancer Stem Cell And Its Combined Traditional Chinese Medicine

Colon cancer is one of the most common digestive tract cancers.There are about 600 thousand people dying from colon cancer each year in the world.Cancer stem cells are considered as the source of tumorigenesis,metastasis and recurrence.However,there is still a lack of systemic study on the effects and mechanisms of inhibiting colon cancer HT-29 stem cells in vitro and in vivo.Traditional Chinese medicine is based on the theoretical treatment of traditional Chinese medicine and has been used in clinical practice for a long time.Based on this,the research focuses on killing effect and mechanism of DC-CIK cells loaded by colon cancer stem cells HT-29 cells as a specific antigen and DC-CIK cells line combined with traditional Chinese medicine Huaier granule and the cinnamomvine extract system in vivo and in vitro experiment.First,CD133~+cells in the HT29 cell line were selected by magnetic beads sorting method,and the cloned formation and nude mice tumor induced tumor experiments were carried out.The results showed that the clone formation rate of CD133~+cells was significantly higher than that of CD133~-cells at the same inoculation density,and CD133~+cells could induce tumor formation in nude mice.These studies have demonstrated the feasibility of CD133 as a specific marker for cancer stem cells derived from HT29 cells.On the basis of these CD133~+cells as cancer stem cells of colon cancer,3specific antigens were prepared:(1)to construct a prokaryotic expression plasmid containing the AC133 encoding sequence of CD133 antigen epitope and obtain the purified recombinant protein of AC133,It was confirmed that the immune response could be produced in mice,and then the eukaryotic expression vector of AC133lentivirus was constructed.(2)the total RNA extract of colon cancer stem cells;(3)all cell lysates of colon cancer stem cells.DC-CIK cells were loaded with 3 kinds of colon cancer stem cell antigens,and HT29 cells and their stem cells were killed in vitro.The results showed that the cytotoxicity of the 3 systems was stronger than that of the unloaded DC-CIK cells and the single CIK cells.In addition,the killing effect of traditional Chinese medicine Huaier granule cinnamomvine and Yam extract on HT29 cells and stem cells.The results showed that Huaier granule and yam extract can inhibit the proliferation rate of colon cancer HT-29 cell,stem cells(CD133~+cells)decreased significantly.Traditional Chinese medicine Yam and the cinnamomvine extract Huaier granule combined with DC-CIK cells on HT29 cells and stem cells is stronger than the use of Huaier granule and yam extract.After that,a nude mouse model of colon cancer stem cells was established,and the pharmacodynamics and mechanism of the mice were studied in vivo.The results show that the 3 kinds of antigen loaded DC-CIK cell system,traditional Chinese medicine or the cinnamomvine extract Huaier granule combined with DC-CIK cells and 5-Fu cells combined with DC-CIK system,system of inhibitory effect on tumor bearing mice was stronger than 5-Fu alone,Chinese medicine or the cinnamomvine extract of Huaier granule and without load of DC-CIK cells;Its mechanisms include CD4~+T,CD4~+T/CD8~+T and Th1of cytokine up regulation,and PI3K/Akt,Wnt/?-catenin and key genes in Notch pathway by down regulated mRNA expression of PI3KR1,Wnt1 and Notch1,and down regulation of Bcl-2 protein and up regulation of Bax protein expression,then induce apoptosis.In conclusion,this paper has demonstrated that CD133 can be used as a specific marker for cancer stem cell stem cells derived from HT29 cells in vitro and in vivo.The corresponding antigen of DC-CIK load cell system,and traditional Chinese medicine Yam and the cinnamomvine extract Huaier granule combined with DC-CIK cells has good killing effect on colon cancer stem cells,which lays a foundation for its clinical application.