| Objective：Tumors, especially malignant tumors, which threat to humanhealth, has been paid more and more attention. Because of its biologicalcharacteristics of easy invasion, metastasis and recurrence, makes people feelvery difficult in the treatment of tumor. But the traditional method oftreatment can not effectively meet the patients needs, therefore, it isimminently to find and develop a new efficiency treatment. How could theoccurrence and development of tumors in the body and escape the immunesurveillance, so that the body’s immune response to the tumor in a low energystate? Elucidating the mechanism involved in this process, no matter for theresearch of pathogenesis and development of tumors or exploring efficienttreatment method might play a significance role.PD-L1(programmed death1ligand1), also known as B7-H1or CD274,is a member of B7/CD28family. PD-L1expression is found in a variety ofimmune cells, and tumor cells. PD-L1is expected to play a role inautoimmune disease, transplantation immunity, chronic infection andimmunological response. It is found that, the expression of PD-L1issignificantly associated with histopathologic grade in various malignanttumors. The over expressed PD-L1molecules on the surface of tumor cells,can bind to PD-1(programmed death1) receptors on the activated T cell, andtransmit negative regulatory signals, resulting in tumor antigen-specificCTL(Cytotoxic T lymphocytes cell) apoptosis and immune anergy. Make thetumor cells escape from immune surveillance and destruction successfully.Although it has confirmed the expression of PD-L1take place in bothmembrane and cytoplasm of multiple tumor cell, the regulatory mechanism ofexpression of PD-L1on tumor cell surface is not clear yet. B7.1, known as CD80, which also belong to B7/CD28family, hasattracted more attention in the anti-tumor research in recent years. Whencombined with CD28/CTLA-4(cytotoxic T-lymphocyte antigen-4) ligands onCD4+T cells，it plays an important role as costimulatory molecules. It canpromote the proliferation and differentiation of T cells and prevent the cellsfrom apoptosis. Most tumor cells do not express or low express of B7.1, oronly express in the intracellular but not on the cell membrane, which leads tothe body cannot produce specific and effective anti-tumor immune response.Besides, a large number of studies show that, tumor vaccine made by usinggene transfection method to prepare tumour cells over expressing of B7.1, caninduce a obviously immune response, such as tumors shrinked or evendisappeared. Meanwhile, without B7.1expression, tumor cells usually induceimmune tolerance. Therefore, the lack of B7.1costimulatory signal isconsidered to be one of the important reasons for tumor cells to escape fromimmune surveillance.In this study, The plasmds of recombinant mouse pcDNA3/PD-L1andpcDNA3/B7.1were constructed. The mouse lung carcinoma cell line, Lewiscell, were transfected with the plasmids and selected by cultured with G418.The cells selected, which constantly express PD-L1and B7.1, were injectedinto mice to construct mouse tumor model. By compare the differences ofthe rate of tumor forming and tumor size in different groups. a preliminarystudy on the effect of PD-L1and B7.1molecules on immune response wascarried out, to provide ideas and direction for the development of tumortherapy and gene vaccine.Medthods:(1) Amplification of PD-L1and B7.1DNA fragments andconstruction of the plasmid.(2) Transfection C57BL/6mice Lewis cells with pcDNA3/PD-L1,pcDNA3/B7.1and pcDNA3plasmid by LipofectamineTM2000. Screeningstable cell lines by200μg/ml G418and degrade it to100μg/ml25~30dayslater. Detect the expressions of objective DNA fragments in transfected cellsby using RT-PCR and FCM technology. (3) Male C57BL/6mice aged6~8weeks were divided into A, B, C, Dfour groups randomly, with10mice in each group. Including normal Lewiscells group, pcDNA3-Lewis cells group, PDL1-Lewis cells group andB7.1-Lewis cells group. Washing the cells with serum-free RPMI1640medium three times, subcutaneous injection of5×105/0.2ml/mouse in leftaxilary. The growth of tumors, and tumor size between groups were compared30days later after inoculation.Results:(1) Extracted the total RNA of NOR-10cells. The objectiveDNA fragments were amplified by RT-PCR and PCR. Construction ofeukaryotic expression plasmid pcDNA3/PD-L1and pcDNA3/B7.1successfully, sharing100%homology with the secquences published inGenbank (PD-L1：NM-021893.3，B7.1：NM-009855.2).(2) Lewis cells were transfected with plasmid of pcDNA3,pcDNA3/PD-L1, and pcDNA3/B7.1. Stable transfected cells were select withG418. PD-L1and B7.1gene can be detected by RT-PCR and FCM in thetransfected Lewis cells.(3) The statistics analysis show that A and B group have no significantdifference in the mean time of tumor-forming(P>0.05). So as C group amounA and B group(P>0.05), and D group amoun A and B group(P>0.05).(4) The differences of tumor-forming rate among A, B and C group(100%)were not significant(P>0.05). D group was80%, the differences werenot significant(P>0.05).(5) The statistics analysis show that the differences of tumor size amongA, B,C and D group were not significant(P>0.05).Conclusions：(1) Expression vector pcDNA3/PD-L1and pcDNA3/B7.1has been constructed successfully.(2) PD-L1was expressed stably in Lewis cells, B7.1was also expressedstably in Lewis cell.(3) PDL1-Lewis cells and Lewis cells have no significant difference intumor-forming.(4) B7.1-Lewis cells and Lewis cells have no significant difference in tumor-forming. |
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