| Objective: The one cause of difficult to cure Malignant tumors relates to tumor immune escape.The human anti-tumor immune almost is cellular immunity by T cell mediated and the activation of T cell needs bisignal stimulation: Which the first signal is provided by the compound of TCR combined MHC,the second signal is provided by costimulatories on the APC and the corresponding ligands on T cell. Only when stimulated by two signals specific T cell can be activated to take cell immunity and kill tumor cell. A lot of studies show that tumor cell can escape body immune by absence of costimulatory.The dendritic cells (DC) play an essential role in induction, regulation and maintenance of body's anti-tumor immunity, and it has been regarded as the most efficient and potent antigen-presenting cell (APC). DCs are uniquely potent in their ability to capture, process and present antigens to T cells. The ability to culture sufficient numbers of DCs from human bone marrow or blood progenitors has attracted a great deal of interest in their potential utilization in human tumor vaccination. Many studies in dendritic cell-based tumor immunity have been reported in recent years. For these reports, DC vaccine showed satisfactory efficacy in the treatment of some malignant tumorsBesides CD28/B7, 4-1BB/4-1BBL is another pair of important costimulatory signal, which plays an important role in immune response, tumor immunity and autoimmune diseases. It has various roles in cell-to-cell interaction, cell adhesiveness, antigen present, T cell costimulation and signal transmission. The costimulatory signal mediated by 4-1BB and 4-1BB ligand is necessary for effective anti-tumor immune response, particularly in the long-term anti-tumor response, which may provide a new biological therapy of cancer.This study was that the co-stimulatory molecule 4-1BBL gene was transfected into gastric cancer cells, then the total RNA of gastric cancer cells transfected DC, production of tumor vaccines, to observe the in vivo anti-tumor effect. As for DC-based immunotherapy of gastric cancer to provide a new strategy, while laying the theoretical foundation for further clinical application.Methods:1 Insert the liposome-mediated pMKITneo/4-1BBL gene into the gastric cancer cells of the 615 mice, the positive cloning then turn into MFC/4-1BBL cell through G418 select.2 Assay the existence of 4-1BBL gene mRNA in the transfectional cell with reverse transcription-PCR (RT-PCR).3 Seperate the dendritic cells from 615 mice's myeloid tissue, then culture in vitro to increase the quantity and promote maturation.4 Wrap up the total RNA of mfc/4-1bbl cell with liposome and transfect mouse dendritic cells to make the DC/4-1BBL/RNA vaccine.5 Inoculate MFC cell into 615 mice to set up tumor-bearing mouse gastric cancer model. The mice were randomly divided into control group, DC group, DC/MC group, DC/MFC/4-1BBL group. Immunized by subcutaneous injection of vaccine. To observe the growth of the tumor and the immune status.All statistical analyses were carried out with SPSS 13.0 and P<0.05 were considered significant.Results:1 Identification of pMKITneo/4-1BBL plasmidAfter the extracted plasmid was cut by XhoI and NotI enzyme,we found 927bp segment exfoliation from pMKITneo/4-1BBL plasmid and this plasmid became 5.8kb linearity vector,which match to theory value and confirm pMKITneo/4-1BBL plasmid existed,amplificated successfully and was a recombination colone.2 The expression of 4-1BBL mRNA by RT-PCR and cell shape observation of cell selecting after 4-1BBL gene transfered into MFC cellsWe could get positive colone called MFC/4-1BBL after 4-1BBL gene transfered into 615 mouse cells MFC by the method of liposomes and selected by G418 .Total RNA was extracted from MFC/4-1BBL and MFC cells. The cDNA was syntheted by reverse transcription. 616bp specific strap was gotten by PCR amplification with mouse 4-1BBL specific primers in MFC/4-1BBL cells which indicated 4-1BBL mRNA was expressed stronger in this cell line; meanwhile we could get the 4-1BBl mRNA was expressed weak in MFC cells.3 The preparation of bone marrow-derived DC, and identification of cell phenotypeThe mouse bone marrow mononuclear cells will differentiate into normal DC after seven days cultured in 10% FBS, rmGM-CSF and rmIL-4 system. The expression of CD11c is up to 80% analyzed by flow cytometry.4 The influence of the tumor vaccine acting on the growth of tumor-bearing mice and the immune status4.1 General states of tumor-bearing mouseNormal 615 mouse responses quick, forages normal, and has strong motoricity. Two weeks after tumor cell inoculation, mice in each group decreased activity, lagged the response and loss of appetite, especially in the control group and the DC groups. One week after the last immunization, mice were sacrificed and then anatomized. The tumor tissue grows in the subcutaneous in each group, has a slight adhesion with surrounding tissue and an integrity capsule, is soft, easy to peel, and densely covered with blood vessel.4.2 comparison of tumor weight and tumor inhibitory rate in each groupTumor weight of each group in decreasing order is control group (3.82g±0.57), DC group (3.63g±0.51), DC/MFC group (2.67g±0.32), DC/MFC/4-1BBL group (2.06g±0.39). The mean tumor weight of DC/MFC/4-1BBL group was lowest (P<0.05), and the inhibition rate is highest, has the best anti-tumor effects; DC/MFC group average tumor weight was significantly smaller than the control group (P<0.05), its inhibitory rate was significantly higher than DC group; DC group, the average tumor weight less than the control group, but no statistically significant difference between them (P>0.05).4.3 comparison of apoptosis rate in tumor tissue in each groupThe apoptosis of the tumor tissue was detected by flow cytometry. The rate of apoptosis from high to low was the DC/MFC/4-1BBL group (28.58%±2.84), DC / MFC group (25.03%±1.88), DC group (20.66%±2.71), control group (19.09%±2.73). There is significant difference (P<0.05) between DC/MFC/4-1BBL group and other groups. DC / MFC group has statistical difference (P<0.05) compared with DC group and control group. No statistically significant difference (P> 0.05) showed between DC group and control group.4.4 The observation Systemic immune status of mice in each groupCD4~+T cell level: there is statistical significance (P<0.05) between DC/MFC/4-1BBL group and other groups. But there is no statistical significance (P<0.05) between DC group and control group. CD8+ T cell level: there is no statistical significance between each treatment group and control group (P>0.05).NK cell level: the DC/MFC/4-1BBL group has statistics difference (P<0.05) compared with other treatment group and the control group. DC group and control group were no significant difference. (P>0.05).Conclusion:1 The 4-1BBL gene can be transfered into MFC cells by liposomes and this gene can express stably.2 Compared with other groups, DC/MFC/4-1BBL vaccine can significantly delay the growth of gastric cancer cell in tumor-bearing mice, and reduce the tumor weight. The inhibition rate is higher than other treatment group. The DC/MFC/4-1BBL group showed a stronger inhibition of tumor growth in vivo.3 The tumor apoptosis rates of DC/MFC/4-1BBL treated group is higher than other groups, suggesting that DC/MFC/4-1BBL vaccine can promote apoptosis of tumor cells.4 Compared with other groups, the function of CD4~+T cells and NK cells in peripheral blood of DC/MFC/4-1BBL treated mice is at a higher level, suggesting that this vaccine can improve the immunity of tumor-bearing mice. |
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