Study The Effect Of CIK Combined With DC Pulsed Breast Cancer Stem Cell Antigen On MCF-7/ADR In Vivo And In Vitro

ObjectiveTo investigate the tumor-inhibitory effect of cytokine-induced killer cells (CIK) co-cultured with dendritic cells (DC) pulsde breast cancer stem cell antigen on the cell line of MCF-7/ADR in vivo and in vitro.Methods1. Culturing MCF-7/ADR cells, then sorting CD44+CD24-breast cancer stem cells by MACS.2. Established tumor-bearing mice which was injected by CD44+CD24-breast cancer stem cells.3. Extracted the peripheral blood mononuclear cells and induced to DC and CIK cells.Then identified DC by transmission electron microscopy ultrafine morphology, CIK by flow cytometry to identify the proportion of CD3+CD56+cells.4. Extracted frozen-thawed antigen from cell lysates and stem cell lysate, and the pulsed DC.5. Make up the group:BCSC-AP-DC+CIK group,AP-DC+CIK group, DC+CIK group, CIK group and NS group.6. Detected the cytotoxic activity by MTT in vitro.7. In vivo, the DC and CIK will injected to tumor-bearing mice, and evaluated through the tumor size, TUNEL and the expression of bcl-2and bax.Results1. CD44+CD24-breast cancer stem cells can be stored by MACS.2. Tumor-bearing mice can be established by CD44+CD24-breast cancer stem cells.3. Peripheral blood mononuclear cells can be induced to DC and CIK cells. The induced DCs were with typical DC morphology and cell organelles. The proportion of CD3+CD56+CIK cells BCSC-AP-DC+CIK group and AP-DC+CIK group was higher than DC+CIK group and CIK group. The difference was statistically significant (P <0.05). 4. In vitro, the CIK co-cultured with DC that pulsed antigen was with higher cytotoxic activity than DC+CIK group, CIK group and NS group, but there was no signs shows the anti-tumor effection of BCSC-AP-DC+CIK group was better than AP-DC+CIK group. In vivo, Except for CIK group, the tumor size in each group before and after therapy and the tumor size after therapy between each group was of difference in significance(P<0.05).The minimum size is BCSC-AP-DC+CIK group, which is(1.234±0.131)cm3.5. In TUNEL, the apoptotic index in five groups is:BCSC-AP-DC+CIK group> AP-DC+CIK group> DC+CIK group> CIK group>NS group.6. The result of immunohistochemistry is:the BCSC-AP-DC+CIK group is with highest IOD of bax expression, the lowest IOD bcl-2expression.Conclusion1. Peripheral blood mononuclear cells were able to induce DC and CIK cells, and when CIK cells co-cultured with the DC that pulsed by antigen have the higher proportion of CD3+CD56+cells.2. In vitro, the CIK co-cultured with DC pulsed breast cancer cell antigen was more effective to kill breast cancer cells than those of CIK cells co-cultured with DC unpulsed breast cancer cell antigen, and just CIK cells. But the CIK co-cultured with DC pulsed breast cancer stem cell antigen was not more effective than those of CIK cells co-cultured with DC pulsed breast cancer cell antigen in MTT. In vivo,the CIK co-cultured with DC pulsed breast cancer stem cell antigen was more effective to induce apoptosis of breast cancer cells than those of CIK cells co-cultured with DC pulsed breast cancer cell antigen, CIK cells co-cultured with DC and those of pure CIK cells, and the ability of induce apoptosis was related to bcl-2-associated X protein.