| Cancer is a worldwide health issue. Nowadays even undergoing surgery supplemented by radiotherapy and chemotherapy, the treatment of malignant tumors still cannot completely prevent tumor recurrence or metastases. Stem cells can differentiate into more than one kind of mature tumor cells, and have the ability of pluripotent embryonic development and organogenesis. In recent years, the cancer stem cells theory holds that tumors derived from pluripotent stem cells whose gene mutated, and these cells have a permanent self-renewal and differentiation capacity, so that the tumor can continue to grow. The treatment (radiotherapy and chemotherapy) is just against differentiated tumor cell subsets, rather than the cancer stem cells. But it is these cancer stem cells that really determine the biological characteristics of malignant tumors, though its proportion is very low. So far, cancer stem cells can be isolated in many tumor tissues. These cells are able to successfully replicate tumor tissues with the same biological characteristics as the primary tumor in immunosuppressed animals. The antigen applicated in the ordinary tumor immunotherapy is just against differentiated tumor cells, but the cancer stem cells do not express such antigens, so the immune reactions do not direct to the cancer stem cells. It is expected to make up the lack of tumor treatment that applicating tumor stem cell-specific antigen. The current separation of cancer stem cells mainly relies on its surface specific markers, aldehyde dehydrogenase (ALDH) as a hallmark of cancer stem cells has been in-depth study.In our study, we will use aldehyde dehydrogenase (ALDH) as flow cytometry hallmarks to identify the ALDH+cells from Hepal-6hepatoma cell line and B16melanoma cell line. Then verify that these cells are just the cancer stem cells and prepare the cancer stem cell-specific antigens. We will study the role of cancer stem cells antigen in mouse tumor immunity and the potential mechanisms by using flow cytometry and bioluminescence imaging in vivo technology, then pave way for the further basic research and clinical applications. Part I. Preparation of cancer stem cells antigenObjective:To identify the ALDH+cells from Hepal-6hepatoma cell line and B16melanoma cell line. To verify that such cells are the cancer stem cells, and prepare the cancer stem cells antigen.Method:We will identify the ALDH+cells from Hepal-6hepatoma cell line and B16melanoma cell line by using ALDEFLUOR stem cell identification kit and flow cytometry. Tumor clone ball culture experiments and subcutaneous tumor formation experiments can test whether the ALDH+cells have the characteristics of cancer stem cells or not. After then the cancer stem cells antigen will be prepared by repeating freezing and thawing cells, also the antigens of ALDH-cells and the unfiltered ordinary tumor cells.Results:The flow cytometry figures show:the proportion of Hepal-6ALDH+is3.27%, ALDH" is4.09%; the proportion of B16ALDH+is3.40%, ALDH" is15.11%.10days after cultured in serum-free medium, we can find that, under high-power field (400X), ALDK+cells are able to form many cell clone balls while the unfiltered ordinary tumor cells don’t have the ability nor the ALDH+cells do. We got the same results in both Hepal-6cell line and B16cell line experiments. Mouse subcutaneous tumor inoculation experiments in different quantity of tumor cells:Subcutaneous tumor can be found after4weeks inoculated with50000、2000、500Hepal-6ALDH+cells while the unfiltered ordinary tumor cells or the ALDH-cells don’t have the same tumorigenic ability. The result of B16cell line experiments is consistent with that of Hepal-6cell line experiments.Conclusion:Obtain Hepal-6ALDH+cells and B16ALDH+cells and prove that they have the characteristics of cancer stem cells. Afterwards obtain the cancer stem cells antigen after repeating freezing and thawing such cells.Part Ⅱ. The influence of dendritic cells loaded with the cancer stem cells antigen on the mouse immune cells in vitroObjective:To study the effects of cancer stem cells antigen on the functions of mouse immune cells in vitro.Method:Four groups are divided with different antigens in vitro:A. PBS control group; B. loaded with unfiltered ordinary tumor cells lysate as antigen group; C. loaded with ALDH" tumor cells lysate as antigen group; D. loaded with ALDH4+tumor cells lysate as antigen group. Co-cultured with mouse DC cells7days, and then test the content of IL-12in the supernatant. Afterwards DC cells loaded with different antigens co-culture with mouse spleen cells (mainly T cells) in vitro. After that undergo the cytotoxity assay, target cells are the unfiltered ordinary cells and ALDH+tumor cells (cancer stem cells).Results:In Hepal-6cell line experiments A. PBS control group; B. loaded with unfiltered ordinary tumor cells lysate as antigen group; C. loaded with ALDH" tumor cells lysate as antigen group; D. loaded with ALDH+tumor cells lysate as antigen group; we test the contents of IL-12in the supernatant. Results are:54.45±3.976pg/ml,84.82±5.320pg/ml,83.38±5.889pg/ml,83.68±4.507pg/ml. There are statistically significant differences between B1、C、D group and control group (P<0.05), but no statistically significant difference among B、C、D group. In B16cell line experiments, the contents of IL-12in the supernatant are54.53±3.705pg/ml,84.12±5.156pg/ml,80.47±4.328pg/ml,85.43±5.260pg/ml. We got the same conclusion as in Hepal-6cell line experiments:There are statistically significant differences between B、C、D group and control group (P<0.05), but no statistically significant difference among B、C、D group. In the cytotoxity assay, there are also four groups:A. PBS control group; B. loaded with unfiltered ordinary tumor cells lysate as antigen group; C. loaded with ALDH" tumor cells lysate as antigen group; D. loaded with ALDH+tumor cells lysate as antigen group. When the target cells are the unfiltered ordinary Hepal-6cells and B16cells and E:T value reach20:1,40:1, the killing rate of group B is better than other groups (P<0.05), no statistically significant difference between group C and group D, but better than the control group(P<0.05)(When the target cells are unfiltered ordinary Hepal-6cells and B16cells, cytotoxicity assay have the same conclusion). When the target cells are Hepal-6ALDH+cells and B16ALDH+cells and E:T value reach10:1,20:1,40:1, the killing rate of group D is obviously better than other groups (P<0.05), and the killing rate of group B is better than that of group A and group C (P<0.05), but no statistically significant difference between group A and group C (When the target cells are Hepal-6ALDH+cells and B16ALDH+cells, cytotoxicity assay have the same conclusion).Conclusion:No matter which kind of tumor cells lysate as antigen can make DC cells secrete more IL-12than the control group. All kinds of tumor lysate as antigen can enhance the cytotoxicity against the unfiltered ordinary tumor cells. Cancer stem cells antigen has a specific effect on cancer stem cell.Part Ⅲ. The establishment of Hepal-6orthotopic liver tumor model and B16lung metastases tumor model for bioluminescence imaging in vivoObjective:To establish the animal models for the experiments in vivo.Method:Create Hepal-6cell line and B16cell line for the experiments in vivo by using plasmid transfection method. Establish the Hepal-6orthotopic liver tumor model by using the mouse subcutaneous tissue. Establish the B16lung metastases tumor model by injecting mouse tail vein. We’ll observe tumor fluorescence and dissect to further observe in vitro by using a small animal imaging in vivo system in both tumor models, and then validate the animal models.Results:The fluoscence can be observed as the cells reach104. Hepal-6orthotopic liver tumor model and B16lung metastases tumor model are validated very well, and further dissected to prove that the animal models are accurate and reliable.Conclusion:Have established Hepal-6orthotopic liver tumor model and B16lung metastases tumor model for imaging mouse in vivo, providing good support for the further experiments in vivo. |
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