| Colorectal cancer,including colon and rectal cancer, is one of the most common malignant tumors in the digestive tract. In the worldwide scale, incidence rate of colorectal cancer ranks No.4 that has occupied 12%-15% of the whole body malignant tumor and there are 1,020,000 new cases and 530,000 death cases each year. Since the 70s of last century, China's morbidity and mortality of colorectal cancer have showed a rising trend (by colon cancer rise primarily), and the high incidence of colorectal cancer in adolescent (<30 years old) is a significant clinical feature in our country. The incipient symptoms of colon cancer are not obvious,so the regional or distant metastasis has occurred when most of patients come for treatment. Surgical treatment can not significantly improve the prognosis of patients alone, so currently the combined therapy is an important method. As an integral part of comprehensive treatment, biological treatment of colorectal cancer can be used independently, or combined with surgery, radiotherapy and chemotherapy,which has the merits of high efficacy, strong specificity, few side effects,etc. Now it has become one of the research hot spots.Dendritic cells (DC) is known as the most powerful professional antigen-presenting cells,and DC is the direct initiator and regulator of body's T cell-specific immune responses. The biggest feature of DC is the ability to stimulate the proliferation of naive T cells significantly which has a unique position in the immune response induction. DC uptake antigen in peripheral tissue, process and decompose it to antigenic peptides, which combined with MHC-I/II molecules, migrate to peripheral lymphoid organs, present antigen to T lymphocytes through the MHC molecules, then stimulate specific anti-tumor immune response. Compared to other antigen-presenting cells, DC expresses higher levels of MHC-I/II molecules, costimulatory molecules and adhesion molecules, therefore it plays an important role in stimulating T cell proliferations and anti-tumor immunity. As early as 1995, foreign scholars began to carry out the phase I clinical trials of cancer therapeutics with DC. DC vaccine has achieved remarkable effects in prostate cancer, malignant melanoma, colon cancer, breast cancer, ovarian cancer, multiple myeloma, brain glioma, renal cell carcinoma and other malignant tumors . Study of DC tumor vaccine for colorectal cancer therapy also shows that the safty and clinical effect. It can reduce carcinoembryonic antigen (CEA) levels of colorectal cancer patients, and even makes lung metastases disappeared. DC cancer vaccines have been one of the most advanced and promising cancer immunotherapy methods nowaday,.The colorectal cancer is one kind of weak immunogenicity tumor. The tumor cells lack costimulatory molecules or some adhesion molecules, and can arise tumor immune escape mediated by antigenic modulation,also can reduce expression of MHC molecule, thus affect the immune therapeutic effect. Another study shows that the reduction of DC and functional defects in cancer patients, so that it can not present tumor antigen effectively,which is also one of the main mechanism of tumor immune escape. Therefore, raising the number of DC in vivo of cancer patients, maintaining its strong antigen recognition and presenting function is one of the key measures to implement the anti-tumor immunotherapy. Because of this, application of sufficient quantity and functional DC for treatment or DC-based anti-tumor vaccine research is becoming increasingly important.Immunotherapy approaches for cancer patients with exogenous DC have developed through the following stages: earlier using the known tumor-associated antigen peptides or mRNA of tumor cells to modificate and allergize DC, but the effects are poor. With deeper study, switching over to use tumor entire antigen to sensitize DC. That is, use the integration of tumor cells with DC or DC loaded with tumor cell lysate to produce hybrid vaccine, which obtain a better anti-tumor effects in gastric cancer, hepatocellular carcinoma and colorectal cancer treatment. The recent study found that DC express a variety of heat shock protein (HSP) receptors (such as CD91, CD40, TLR2 / 4 or LOX1) in the surface. HSP can act on DC or monocytes to stimulate the production of cytokines (IL-12, TNF-α, IL-6, etc.) and enhance the non-specific immune response. In 2004,《Immunity》reported that DC loaded with HSP70-peptide complexes can stimulate CD8+ T much higher (1000 times) compared to DC loaded with peptide, which indicated that integrating HSP with DC to play their respective strengths will produce a stronger immune stimulating effect.In view of this, this study is to explore the methods both for preparation of auto-heat shocked apoptotic colorectal cancer cells as tumor antigens from fresh surgery tumor tissue and the induction of dendritic cell as well as the antigen loading system in vitro,and to compare the lymphocytic antitumor activity stimulated in vitro by dendritic cell (DC) loaded with colorectal cancer cell strain in two different ways and to investigate the effects of dendritic cell (DC) loaded with autologous heat shock-induced apoptotic colorectal cancer cell on the immune function of the postoperative patients with colorectal carcinoma.This study provides a technical basis for the DC vaccines preparation of colorectal cancer.Study is divided into the following three parts:Part I Study on the method of Dendritic Cells Loaded with Auto Colorectal Cancer CellsObjective: To establish the methods both for preparation of auto-heat shocked apoptotic colorectal cancer (colon carcinoma, rectal carcinoma) cells as tumor antigens from fresh surgery tumor tissue and in vitro dendritic cell induction as well as the antigen loading system through which dendritic cell based tumor vaccine could be developed.Methods: Single colorectal cancer cell suspension was made from 14 fresh colorectal tissue by using enzyme digestion assay. The cell suspension was heated and induced into apoptosis with adding BA (Betulinic Acid). Monocyte derived dendritic cells was prepared from peripheral blood mononuclear cells (PBMC) of the patients with GM-CSF and IL-4 cytokine combination culture. Co-culture of imDC and apoptotic colorectal cancer cells was harvested and its phenotype was analyzed by FACS. Results: (1)The yield rate of single colorectal cancer cell from fresh colorectal cancer tissue was (11.81±0.65)×106/g and average apoptotic rate was (93.16±2.31)%;(2)im DC average yield rate from PBMC was(9.75±0.82)×106/(121.64)×106,and the percentage of live cells was more than 95%. The cell percentages of CD11c+CD14- , CD11c+HLA+DR+ , CD11c+CD80+ , CD11c+CD83+ , CD11c+CD86+ among total population were ( 87.58±2.56 ) % , (87.97±0.98)%,(2.21±0.69)%,(4.85±1.22)%, (5.02±0.95)%, respectively.(3)The average yield rate of DC from co-culture of imDC and tumor cells was(6.76±0.98)×106/(9.73±0.84)×106 with 95% alive cells. The cell percentage of CD11c+CD14-, CD11c+HLA+DR+, CD11c+CD80+, CD11c+CD83+, CD11c+CD86+ were (93.45±1.25)%, (89.79±1.35)%, (87.85±1.62)%, (70.74±6.45)%, (95.54±2.18)%, respectively.Conclusions: This method is steady, safe and credible by which potential DC could be developed.Part IIThe comparison of lymphocytic antitumor activity stimulated in vitro by dendritic cells (DC) loaded with colorectal cancer cell strain in two different waysObjective: To compare the lymphocytic antitumor activity stimulated in vitro by dendritic cells (DC) loaded with colorectal cancer cell strain in two different ways. Methods: DC isolated from peripheral blood mononuclear cell of 30 colorectal cancer peoples were loaded with heat shock-induced apoptotic colorectal cancer cell strain and tumor cell lysates separately. Using lymphocytes stimulated in vitro by DC as effector cells and colorectal cancer cell strain as target cells. The cell-killing effect of effector cells on target cells was determined by MTT method.Results: DC loaded with heat shock-induced apoptotic colorectal cancer cell strain and tumor cell lysates both showed the killing activity on target cells , but the killing rate of the former was significantly higher than the latter (P <0.05).Conclusions: The method of loading with heat shock-induced apoptotic colorectal cancer cell strain was more effective. Part III Effects of Dendritic Cells on the Immune Function of Postoperative Patients with Colorectal CancerObjective: To investigate the effects of dendritic cells (DC) induceded by autologous heat shock-induced apoptotic colorectal cancer cell on the immune function of the postoperative patients with colorectal carcinoma.Methods: DC isolated from peripheral blood mononuclear cell of the patients with colorectal cancer were cultured and proliferated in vitro by using rhGM-CSF and rhIL-4,and then were pulsed with autologous heat shock-induced apoptotic colorectal cancer cell itself. 28 postoperative patients with colorectal carcinoma were randomly divided into 2 groups. 14 cases were treated with dendritic cell vaccine. The other 14 cases received only chemotherapy and were established as control group. Immune function, clinical effects in two groups of patients were compared and observed.Results: The levels of CD3+, CD4+/CD8+ and NK cells in the DC vaccine group significantly increased after vaccination (all P values < 0.05), while those of the control group had no significant changes. The levels of IL-2, IL-12 and IFN-γin the DC vaccine group significantly increased and life time was delayed significantly after the vaccination. (all P values < 0.05).Conclusions: The autologous heat shock-induced apoptotic colorectal cancer cell pulsed DC vaccine can improve the immune function of the postoperative patients with colorectal carcinoma. This method results in fewer side effects and should be recommended.
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