The Study Of Antitumor Effect Of Human Peripheral Blood Mononuclear Cells (PBMC)

Background and ObjectiveMalignant lymphoma is a malignant proliferative disease generates from the human immune system cells and their precursor cells. Although malignant lymphoma in China is relatively rare, yet new cases have been increasing in recent years, at least more than 25,000 cases a year, which often occurs in young and seriously endangers the health and lives of human beings. Currently, combined chemotherapy is the common modality of the treatment of malignant lymphoma. It plays an important role in improving the curative rate and remarkably prolonging the disease-free interval of sufferers, but it has inevitably adverse reactions, such as bone marrow suppression and the decreasing of immune system, which make it difficult for the patients to complete the full course of treatment. Moreover, Primary or secondary drug resistance during treatment has become one of the tough problems of clinical therapy of the malignant tumor. Therefore, it is time to seek new ways and means of treatment for malignant lymphoma. More current researches are focus on immunotherapy of cancer and CpG ODN. A number of articles have been published that treatment with CpG ODN on solid tumors was significantly. However, the study of malignant tumors of lymphoid system is less.DNAs in bacteria and virus, which contain numethylated CPG motifs activate host defense mechanisms leading to innate and acquired immune responses. Synthetic oligodeoxynucleotides(ODNs) containing unmethylated CPG motifs Minic the immunostimulatory qualities of bacterial DNA and elicit a coordinated set of T helper 1 (Thl)-like immune responses, including direct activation of B cells, plasmacytoid dendritic cells (PDCs), macrophages, antigen present cell (APC) and indirect activation of T cells, natural Killer (NK) cells. CpG ODN-induced activation of innate immunity protects against lethal challenge with a wide variety of pathogens. However, we can only find some reports on antitumor activity of CPG ODN in solid tumors.So, in this study, we will investigate the antitumor effects of PBMC activated by Synthetic oligodeoxynucleotides (ODN) containing unmethylated CPG dinucleotides (CPG ODN) in vitro and explore the mechanisms of anti-tumor action of CPG ODN, which offers a new way for the treatment of the lymphoma.MethodsSeparating the circumference anti-hemoglutination of healthy persons with Lymphocyte separation medium, human peripheral blood mononuclear cells (PBMC) were cultured with technology of cell culture in vitro. We established four groups in the experiment:control group:PBMC+PBS; Positive control group:PBMC+LPS; ODN2006 group:PBMC+ODN2006; Combination group:PBMC+ODN2006+LPS. The secretions of INF-γand IL-12 from PBMC determined using ELISA method. After treated with these groups for 48 hours, Co-stimulatory molecules of CD80, CD86 on the surface of PBMC were analyzed by flow cytometry. PBMC activated by CPG ODN was co-cultured with hut-78 cells for 48 hours, and then MTT method was used to detect PBMC-mediated antitumor activity. We examined cell morphology with the inverted microscope.Results1. The effect of CpG ODN on INF-γand IL-12 of PBMCThe secretions of INF-γand IL-12 from PBMC treated with CpG ODN were increased significantly. The levers of IL-12 from PBMC of control group, Positive control group, CpG ODN group and Combination group was 15.50±10.12, 60.54±1.82 pg-mL-1,115.50±19.34 pg·mL-1,120.75±13.71 pg·mL-1,respectively; The levers of INF-γfrom PBMC of these four groups was 85.50±10.01, 158.74±18.46 pg·ml-1,262.75±30.21 pg·mL-1,265.25±29.26 pg·mL-1 separately.2. The effect of PBMC treated with CpG ODN on Co-stimulatory molecules of CD80, CD86 of PBMCPBMC treated with CPG ODN and Combination group exhibited more expression of Co-stimulatory molecules of CD80 and CD 86 than control group and Positive control group.3. The effects of PBMC Stimulated by CPG ODN on hut-78 cellsThe growth of hut-78 cells could be inhibited partially after being treated by PBMC activated by CPG ODN for 48h. The Cytotoxicity of control group, Positive control group, CPG ODN group, and Combination group was 24.93±7.81%, 41.86±2.93%,63.65±7.98%,67.05±3.56%.4. The influence of PBMC stimulated by CPG ODN on cell morphology of hut-78 cells co-cultured PBMCAfter co-cultured with PBMC stimulated by CpG ODN, morphological changes of hut-78 cells were more obvious:some cell boundaries unclear, and some cells shows nuclear enrichment.Conclusions1. CpG ODN could increase the secretions of INF-γand IL-12 of PBMC2. CpG ODN could significantly exhibited more Co-stimulatory molecules of CD80 and CD 86.3. CpG ODN could increase the cytotoxicity of PBMC on HUT-78 cells in vitro.