| BackgroundThe immune system has a function of controlling the occurrence and development of tumor, which is one of the oldest concepts of oncology research.The immune status of tumor microenvironment has a vital role in tumor genesis and treatment. With the development of science.technology and methodology, more and more tumor antigens have been identified. The researchers found that, the immunogenic ability of a lot of tumor antigens are not weak, antigen-presenting cells can rapidly render most tumor antigens, and the immune response to tumor antigen do exist in patients tumors. Unfortunately, the immune response products can not produce effective antitumor effect. Most immunotherapeutic approaches can enhance the immune function of patients systematically, but can not produce good antitumor effect as expected. The main reason may be that the immune function of cancer patients can not directly reflect the immunological effect of the body to anti-tumor. Even the systemic immune function has been improved in patients, the immune effect is still in a inhibited state within the tumor microenvironment. The tumor microenvironment is the internal environment of tumor cells needed in the course of development, including the tumor cells themselves, mesenchymal cells, angiogenesis, tissue fluid and a small amount of infiltrating cells, such as dendritic cells, macrophages and other common components. As the execution place in the tumor immune effector phase, there are many factors are involved in local immune inhibitory state within the tumor microenvironment, forming a peculiar immune mode. Tumor cells not only can avoid the immune system’s attack passively, but also actively inhibit the function of the normal immune cell in the environment. These factors provide a train of thought in order to open up new tumor immunotherapy regimens.With the development of molecular biomedicine, as tumor fourth treatment model, the important component of biological treatment, plays a more and more important role in the treatment of tumor increasingly. It is the dilemma and breakthrough point of the tumor immunotherapy to reverse immunosuppression state of tumor microenvironment.ObjectiveMake BALB/c mice produce antibody to blood group antigen B by intraperitoneal injection of health adult blood type B whole blood; building tumor models on BALB/c mouse with4T1breast cancer; intratumoral inject with blood group B antigen/Fas recombinant adenovirus, determine its expression in tumor tissue, and observe its function of mouse tumor growth inhibition and effect on survival period; determine indicators of tumor microenvironment immune function after intratumoral injection of recombinant virus in mice, in order to judge its effect on reversing the tumor microenvironment immunosuppressive state. Mathods1.Dilute healthy adult whole blood to different concentrations of red blood cell suspensions, and immune BALB/c mice by intraperitoneal injection of red blood cell suspensions,0.2ml each time,2times/week.3days,7days,14days,21days,28days after immunization, antibody to blood group antigen B can be measured in mice, then select the most suitable red cell concentration to immune, analyze antibody production trend to make a preparation for follow-up test;2. Culture4T1breast cancer cells in the sterile environment, and as its grow to the logarithmic growth phase, make mouse tumor model by subcutaneous injection of4T in breast cancer cells (106mmol/ml×0.2ml) in the mouse mammary fat pad area.7days after, the tumor can grow to about the size of1cm, then select tumor without necrosis and hemorrhage in mice for further experiments;3. Divide18BALB/c mice bearing tumor model into3groups randomly, each group of6, by intratumoral injection with the recombinant adenovirus Ad-P1/Fas (titer of7×1010pfu/mL), empty adenovirus (titer of2.21×1011pfu/mL),0.9%NaCL0.1ml, respectively, with intravenous injection for3days. On the tenth day after injection, sacrifice three groups of mice, remove the tumor tissue, weighing the weight, and determine the expression of B/Fas in tumor tissue with the method of Western blot. Examine related changes of immunity index with Flow cytometric. Determine immune function indicators in the tumor microenvironment;4. Randomly select36mice with age at4-6week age, at the same level of weight, then divide them into three groups, each group of12. Measure tumor diameter and vertical width by vernier, in order to compare tumor volume changes of mice in three groups before injection,3days,7days,14days after injection, and observe the survival time of tumor-bearing mice in the three groups during a period of60days. The calculation of tumor volume is:V (cm3)=1/2a×b2. Observe the effects of different treatment on tumor growth inhibition and survival period of murme.5. Statistic methods(1) Measurement results can be represented by mean±SD;(2) Apply repeated measurement data analysis of variance of SPSS13.0statistical software package line to analyze antibody titers of blood group antigen B, expression of B/Fas protein, tumor volume comparation in different subgroups of different time; apply one-way ANOVA analysis of SPSS13.0statistical software package to compare antibody titers of blood group antigen B, B/Fas protein expression level, tumor volume, tumor lymphocyte subsets, cytokines, tumor microvascular density at different time points in each group and apply LSD method to undergo comparison between groups; if variances is unequal, Welch method approximate the F test, and Dunnett’s T3method are recommended to undergo comparison between groups. P<0.05represent the significant difference.(3) Apply Kaplan-Meier method to compare survival of each group of mice. P<0.05represent the significant difference.Results1. Successfully make the mice produce antibodies to blood group antigen B by red cell suspension of healthy adult type B blood.There is no statistical difference of the immune function with different concentrations of red blood cell suspension (F=1.871,P=0.196); antibody titers of blood group antigen B make to its maximum in14days and21days after immunization.2. The target protein B/Fas can stably express in the tumor tissue in3days,7days,10days,14days after intratumoral injection by recombinant adenovirus with B/Fas expression, and the expression levels at different time points were significantly different (F=36.322, P<0.001), in which the expression level is the highest in 7-10days after intratumoral injection.3. The comparation of the tumor volume changes of mice between different groups with intratumoral injection of B/Fas expression recombinant adenovirus, empty adenovirus, saline-treated respectively had significant difference (F=18.16, P <0.001). Tumor volume change has significant difference at different time points (F=129.291,P<0.001).4. The comparation of survival period of tumor-bearing mice in different group with Intratumoral injection of B/Fas expression recombinant adenovirus, adeno-associated virus, empty saline has no significant difference (P=0.154).5. Mouse tumor histopathology shows tumor cell necrosis and inflammatory cell infiltration can be significantly increased in mice immuned by red cell suspension and10days after intratumoral injection of peptide mimics of blood group B antigen/Fas recombinant adenovirus.while the control group has poorer effect.6. The ratio of CD3+lymphocytes, CD4+lymphocyte, CD8+lymphocyte, CD4+CD5+lymphocyte in mouse tumor tissue in different groups had remarkable difference (F=104.835, P<0.001; F=150.153, P<0.001; F=48.494, P<0.001; F=65.823, P<0.001) in10days after intratumoral injection of peptide mimics of blood group B antigen/Fas expression of recombinant adenovirus, intratumoral injection of empty adenovirus, intratumoral injection of saline treatment. The treatment of intratumoral injection with peptide mimics of blood group B antigen/Fas expression of recombinant adenovirus can significantly increase subset proportions of CD3+lymphocytes, CD4+lymphocytes, CD8+lymphocyte in the mice tumour tissue, and reduce the ratio of CD4+CD25+lymphocyte.7. The comparation of TNF-α,IFN-γ,IL-2in the tumor tissue in different groups has statistics significance (F=16.418,P<0.001; F=94.032, P<0.001; F=69.848 P<0.001) in10days after intratumoral injection of peptide mimics of blood group B antigen/Fas expression of recombinant adenovirus, intratumoral injection of empty adenovirus, intratumoral injection of saline treatment. Multiple comparisons between different groups indicated that, compared to the control group, the treatment with intratumoral injection of B/Fas expression recombinant adenovirus can significantly increase the content of TNF-α,IFN-γ,IL-2in the tumour tissue, enhancing local antitumor effect.8. The comparation of microvessel quantity between the groups with intratumoral injection of B/Fas expression of recombinant adenovirus and intratumoral injection of empty adenovirus or saline group in10days after treatment was significantly different (P=0.001, P=0.003), while the comparation between the group with intratumoral injection of empty adenovirus and intratumoral injection of saline group has no statistical significance (P=0.724). Compared to the control group, intratumor injection of recombinant adenovirus can significantly reduce tumor microvessel quantity.DiscussionAdenovirus can successfully transfect B/Fas expression of recombinant gene to be expressed in mouse tumor tissues; anti blood group antigen B antibody is successfully produced in the mice after immuned by intraperitoneal injection with type B red blood cells suspension of health adult; intratumoral injection of B/Fas expression of recombinant adenovirus after immunization with red cell suspension in BALB/c mice can inhibit4T1breast cancer growth, but did not make clear its beneficial effects on survival. Compared with the control group, intratumoral injection of peptide mimics of blood group B antigen/Fas dual expression of recombinant adenovirus can significantly increase the ratio of effector lymphocyte in tumor tissue, increase the proportion of CD3+, CD4+, CD8+lymphocyte subsets, and reduce the proportion of CD4+CD25+lymphocyte subset, in favor of reversal of tumor microenvironment inhibitory state; compared to the control group, intratumoral injection of B/Fas expression recombinant adenovirus can increase the content of TNF-α,IFN-γ,IL-2significantly in the tumor tissue, enhancing local antitumor effect; compared to the control group, intratumoral injection of recombinant adenovirus can significantly reduce tumor microvessel quantity, providing effective inhibition function on tumor growth. |
PDF Full Text Request