| Objective:Dendritic cell is known as the most powerful antigen presenting cell(APC), whose capacity of antigen presenting is 10-100 times higher than the other types of APCs, e.g., the macrophage and the B-lymphocyte cells. It's the only type of the organism immune cells capable of acting as the external stimuli to the na?ve T-lymphocyte in initiating the immune recalls and responses. It thus plays a vital role in immune surveillance. For cancer patient, the dendritic cell defects and looses its normal functions in the microenvironment of tumors. They are incapable of delivering the antigens to the T cells efficiently, causing the immune tolerance and the immune anergy, which consequently enables the tumor cells to grow further by escaping from the immune surveillance. With the availability of the new technologies in vitro amplification and induction, the immunotherapy methods based on DC has aroused considerable interest. The DC-based tumor vaccine is expecte an effective treatment in tumor immunotherapy. The researches revolving DC-based anti-tumor immunotherapy have been widely tested both home and abroad. There have been DC-melanoma,prostate cancer,B-cell lymphoma ,multiple based vaccines applied in the treatments of malignant melanoma, prostate cancer, B-cell lymphoma, multiple myeloma, fibro sarcoma and kidney cancer etc. And have got very effective results in clinic. But in the treatment of oral squamous cell carcinoma(OSCC), the research of the DC-based vaccine is on the elementary step. The active immunotherapy that is based by DC tumor vaccine could overcome kinds of deficiencies upon surgery and other routine therapies and with the characteristic of highly efficiency and security. This therapy has become the new direction and new hotspot of the tumor immunotherapy home and abroad. The key of the DC-based treatment is the acquirement of the dendritic cells loaded by the tumor-lysate in vitro and to stimulate the T-lymphocyte activate and proliferate efficiently so as to induce the effect of anti-tumor in organism. It is much simpler to pulse the dendritic cells with tumor lysate comparing to other pulsing ways. The objective of this research is to observe the immune activity of the dendritic cell pulsed by Freeze-thaw antigen according to the comparison of the surface Molecules , the capacity of secreting IL-12p70 and activating autologous or homologous T-lymphocyte , the lethal effects to auto cancer cells before and after the loading of antigen to dendritic cells ,in order to provide the academic basis for the biologic therapy of the OSCC .Method: Study objects are 20 patients with OSCC that were testified by steady pathologist and accepted treatment from 2005.3 to 2006.10 in department of stomatology, the Fourth Hospital of Hebei Medical University without any anti- neoplaston, systemic diseases and acute or chronic in-flammation,including carcinoma of tongue,palate, gingival,et al. The clinical stage wasⅢandⅣ, age from 20 to 60, and sex ratio was 1:1.1. Peripheral blood monocyte-derived DCs isolation and culture Gathered the peripheral blood 20ml from each group (patient) respectively, then isolate the PBMC(peripheral blood mono- nuclear cells) by the method of Density Gradient Centrifugation as the progenitor cells. Then the PBMC were incubated 2 hours in RPMI1640 medium supplemented with 10% FBS(Fetal Bovine Serum)(complete medium)in the environment of 37℃,5% CO2 (tissue culture flask of 6 orifice with 3ml perorifice).2 hours later,non-adherent cells were removed gently and the adherent cells were left.The cells were cultured with RPMI1640 complete medium include 1000U/ml rhGM-CSF and 500U/ml rhIL-4. Exchang the half culture fluid with cell factor every other day and added TNF-α200U/ml into each unite at the fifth day to mature the DCs. They were divided into 2 groups then.2. The preparation of tumor-lysate and the pulse of DCs Cultured the Tca83 cells in the condition of 37℃,5% CO2 with the complete medium, exchanged the culture fluid every 2-3 days. Collected the cells during log phase growth and ensured the cell viability exceed 95%, then adjust the cell to 1×107/ml.Collected the tumor-lysate with freezing and thawing method for using. Add the tumor-lysate(The antigen to DCs is 10:1.The antigen amount is calculated by the quantity of tumor cells.) into one DCs group on the sixth day and collected the suspension cells and mild adherent cells on the tenth day which are DCs.3. The identification of each DC groupIn the process of DC culture,observed the cellular growth status under phase contrast microscope. Detected the expressions of CD1a,CD83,HLA-DR,CD86,CD80 to identify the mature de- gree of each DC group. Detect the level of IL-12p70 in the culture supernatant that is secreted by DCs before and after the pulse with the ELISA method.4. T cell isolation and cultureAt the beginning of the experiment, cultured the PBMC with the complete medium in the condition of 37℃5% CO2 for 2 hours. Collected the culture supernatant and removed non-adherent cells, adjusted the cells to 2×106/ml and freeze them with gradient method in freezing media. Cultured the thawed tumor cells in RPMI1640 medium supplemented with 10% FBS,200U/ml rhIL-12 and 2mmol/l glutamine for 7 days when using.5.Mixed lymphocyte reaction(MLR)Coculture T cells that is thawed as effecter cells and DCs which is pulsed before and after the loading tumor-lysate as activator cells at the rate of 1:5,1:10,1:20. Induced the T cell to perliferate and differentiate and detected the proliferous activity by MTT.6. CTL lethal activity induced by dendritic cells We cultured the Tca83 and 3AO cells as the leptocytes and the the CTLs activiated by DCs before and after antigen-loading of each group as the effecter cells. The target cells were incubated with effector cells in 5:1,10:1 and 20:1.The vitro cytotoxic activity to Tca83 and 3AO cells of CTL that stimulated by tumor-lysate DCs were measured by LDH.Results: The dendritic cells which be obtained by culturing the peripheral blood of OSCC patients displayed typical morphology and demonstrated the high expression of the surface molecules CD1a(83.3±6.8)%,CD83(80.6±7.1)%,HLA-DR(88.17±10.79)%,CD86(89.2±10.2)%,CD80(81.2±6.3)%. The DCs excreting capability of IL-12p70 were increased with the extension of culturing days. The secretion volume coming from the DC stimulated by tumor lysate was stronger than that coming from pure DC groups(P<0.001). Tumor lysate-loaded DC of patients with OSCC could induce the generation of T lymphocytes potently(P < 0.001), and the specific cytotoxic CTL against OSCC cells was highly enhanced (P<0.001)as compared with the unloaded DC. The DCs loaded with the Tca83 antigen is cytotoxially more active to the Tca83 cells than to the 3AO cancer cells(P<0.001).Conclusion: Monocytes were isolated from the patients of OSCC peripheral blood mononuclear cells(PBMC),cultured with cytokines including IL-4 ,GM-CSF and TNF-a can induce and expand mature dendritic cells. The tumor lysate had no influence on the maturity of the dendritic cells. The dendritic cells that be stimulated by tumor lysate could secrete more IL-12p70 whom play an important effects in the process of inducing CTLs. The dendritic cells that be stimulated by tumor lysate could stimulate strong proliferation of allogeneic T lymphocytes and activate the CTL efficiently against OSCC Tca83 cells. All the results above could provide us the experimental foundation for the clinical therapy of OSCC. |
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