| BackgroundMany experiments have proved that human-bodies' immune system can identify and clear tumor cells.Cell-mediated immunity is the most important anti-tumor immune effect.Especially,specific cytotoxic T lymphocyte(CTL)'s killing activity is critical among anti-tumor immune effects of human-body.At present,it is well known that dendritic cells(DC) have the most strong antigen-presenting function in vivo among antigen-presenting cells.Nevertheless,because of low immunogenicity of tumor and immune tolerance of human-body to tumor,tumor immunotherapy is limited.Studies have discovered that tumor microenvironment stay on an immune inhibitory state,and there have a complicated regulating network of cell immunity.In tumor patients' body,DC is devitalized,this can be proved by quantity's decreasing, phaenotype and function's defection of DC.Because DC's function defected,DC can't present tumor antigen effectively in immune reaction,that's why tumor patients' immune system can't identify and kill tumor cells.Therefore,it will be helpful to induce CTLs' killing activity by immunotherapy,breaking immune tolerance and improving immune inhibition of tumor microenvironment. PurposeUsing heteragenetic antigen to cure tumor through immuning generally and injecting intratumorally.We inlet anti-heteragenetic antigen immune response and secondary immune response in tumor region.Through computing index of spleen and index of thymus,measuring the killing rate of splenic lymphocytes against S180 cells and analysising the chang of quantity of spleen' CD4~+T and CD8~+T lymphocytes to fath the immune function state of mice of different treated groups.Furthermore,from the point of splenic immunologic cell,we can authenticate this method's usage, research for its principle.Method1.Using two heteragenetic antigen,inactivated streptococcus and blood group antigen to study.2.Grouping of experiment animals:4 groups are included in the inactivated streptococcus experiment:A.group of generally immuning and intratumorally injecting with inactivated streptococcus.B.group of generally immuning with inactivated streptococcus.C.group of intratumorally injecting with inactivated streptococcus.D.group of blank.4 groups are included in the experiment of blood group antigen:a.group of generally immuning and intratumorally injecting with blood group antigen,b.group of generally immuning with blood group antigen.c. group of intratumorally injecting with blood group antigen,d.group of blank.A total of 8 groups are required.As the group of blank can be shared by D and d,the two experiments can be divided into 7 groups,and the experiment required 42 Kunming mice in total.3.Modeling:The first two weeks the mice was generally immuned for 4 times,twice per week(each of the mice of group A and B was peritoneally injected with 0.1ml of inactivated streptococcus of 0.1g/ml;each of the mice of group a and b was peritoneally injected with 0.1ml of blood group antigen of 5mg/ml;each of the mice of groupC,D,c and d was peritoneally injected with 0.1ml of physiological saline). Each of the mice was subcutaneous inoculated with S180 cell of 1×10~7/ml on the back.About two weeks,the cells grew into a tumor with a size of about 1cm~3. Hetergeneic antigen was intratumorally injected into the tumor in the following 5 days(each of the mice of group A and C was intratumorally injected with 0.1ml of inactivated streptococcus of 0.1g/ml;each of the mice of group a and c was intratumorally injected with 0.1ml of blood group antigen of 5mg/ml;each of the mice of group B,D,b and d was intratumorally injected with 0.1ml of physiological saline).Five days later,the mice are put to death after the intratumorally injecting.4.Take out the spleen and thymus,then calculate the index of the spleen and thymus.5.Examination of the killing activity of spleenic lymphocytes against S180 cells: ratio of efficacy to target(E/T)= 20:1,using MTT to compute the killing activity in vitro of lymphocytes against S180 cells of groups treated by different methods.6.Analyze the change of the counts of CD4~+T and CD8~+T lymphocytes of spleen tissue by immunohistochemistry:Take the tissue immediately after the spleen was taken out.Conventional fixed,paraffin-embedded,sliced,using PAP immunohistochemistry method to dye.The Biopsy of each group samples was observed under the high power microscope and record the number of the cell of CD4 and CD8 positive.7.Statistical methods:All the results of data are expressed by((?)±s).SPSS 13.0 statistical analysis software is used to deal with the variance analysis of factorial design data of the four groups of every experiment.If there is reciprocation,fix one factor and use t test to compare the two levels of another factor.At last,use one-way ANOVA to find the best group.Between the two experiments,t-test is used to comparise averages of two seperate samples.Significant level:a=0.05.Result1.inactivated streptococcus experiment(1) Thymus indexs(mg/g) of the groups of A.B.C and D are 2.50±1.07 mg/g,1.67±0.37 mg/g,2.33±1.04 mg/g and 2.25±0.91 mg/g.Generally immuning and intratumorally injecting with inactivated streptococcus all have no effect on thymus index(F=0.328,P=0.573 and F=1.585,P=0.223 ).Generally immuning and intratumorally injecting with inactivated streptococcus have no reciprocation (F=1.061,P=0.315).There is no significant difference between the four treated groups(F=0.991,P=0.417).(2) Spleen indexs(mg/g) of the groups of A.B.C and D are 14.42±2.63 mg/g,15.83±2.28 mg/g,15.28±4.51mg/g and 5.67±1.31 mg/g.Generally immuning and intratumorally injecting with inactivated streptococcus all have significant effect on spleen index(F=15.163,P=0.001 and F=11.788,P=0.003).Generally immuning and intratumorally injecting with inactivated streptococcus have reciprocation(F=21.342, P=0.000).When there is general immunization,intratumoral injection have no effect on spleen index(t=0.995,P=0.343).When there is no general immunization, intratumoral injection can boost spleen index(t=5.014,P=0.001).When there is intratumoral injection,general immunization also have no effect on spleen index (t=0.406,P=0.693).When there is no intratumoral injection,general immunization can boost spleen index(t=9.449,P=0.000).There is significant difference between the four treated groups(F=16.097,P=0.000).The spleen index of groupA.B and C are higher than that of group D(P values are all less than 0.05 ).(3) When the E/T=20:1,the killing rates of spleen lymphocytes against S180 cells of groups of A.B.C and D are 63.17%±3.94%,10.26%±1.95%,33.30%±3.82%and 9.85%±2.76%.Generally immuning and intratumorally injecting with inactivated streptococcus all have significant effect on killing rate(F=132.499,P=0.000 and F=842.219,P=0.00 ).Generally immuning and intratumorally injecting with inactivated streptococcus have reciprocation(F=125.308,P=0.000).Whether there is general immunization,intratumoral injection all can boost killing rate(t=29.455, P=0.000 and t=12.198,P=0.000).When there is intratumoral injection,general immunization can boost killing rate(t=13.330,P=0.000).When there is no intratumoral injection,general immunization have no effect on killing rate(t=0.302, P=0.769).There is significant difference between the four treated groups (F=366.675,P=0.000).The killing rate of group A is the highest. 2.blood group antigen experiment(1) Thymus indexs(mg/g) of groups of a.b.c and d are 1.73±0.51 mg/g,3.25±2.23 mg/g,2.26±1.38 mg/g and 2.25±0.91mg/g.Generally immuning and intratumorally injecting with blood group antigen all have no effect on thymus index(F=0.164, P=0.690 and F=1.692,P=0.208).Generally immuning and intratumorally injecting with blood group antigen have no reciprocation(F=1.768,P=0.199).There is no significant difference between the four treated groups(F=1.208,P=0.333 )(2) Spleen indexs(mg/g) of groups of a.b.c and d are 11.60±2.61 mg/g,11.78±1.58 mg/g,11.86±3.62 mg/g and 5.67±1.31mg/g.Generally immuning and intratumorally injecting with blood group antigen all have significant effect on spleen index (F=8.504,P=0.009 and F=8.995,P=0.007).Generally immuning and intratumorally injecting with blood group antigen have reciprocation(F=10.125,P=0.005).When there is general immunization,intratumoral injection have no effect on spleen index (t=0.147,P=0.886).When there is no general immunization,intratumoral injection can boost spleen index(t=3.943,P=0.003).When there is intratumoral injection, general immunization have no effect on spleen index(t=0.146,P=0.887).When there is no intratumoral injection,general immunization can boost spleen index(t=7.311, P=0.000).There is significant difference between the four treated groups(F=9.208, P=0.000).The spleen indexs of group a.b and c are higher than that of group d(P values are all less than 0.05 ).(3) When the E/T=20:1,the killing rates of spleen lymphocytes against S180 cells of groups of a.b.c and d are 76.27%±4.07%,10.55%±2.29%,40.92%±4.98%and 9.85%±2.76%.Generally immuning and intratumorally injecting with blood group antigen all have significant effect on killing rate(F=143.829,P=0.000 and F=1036.665, P=0.000).Generally immuning and intratumorally injecting with blood group antigen have reciprocation(F=132.875,P=0.000).Whether there is general immunization, intratumoral injection all can boost killing rate(t=34.453,P=0.000 and t=13.372,P=0.000).When there is intratumoral injection,general immunization can boost killing rate(t=13.463,P=0.000).When there is no intratumoral injection,general immunization have no effect on killing rate(t=0.478,P=0.643).There is significant difference between the four treated groups(F=437.790,P=0.000).The killing rate of group a is the highest.(4) Under the high power microscope(10×40 times),The counts of CD4~+T lymphocytes of groups of a.b.c and d are 59.00±3.00,55.00±7.21,58.67±7.51 and 39.00±3.00.The counts of CD8~+T lymphocytes of groups of a.b.c and d are 41.33±4.51,23.67±2.52,32.33±1.53 and 23.00±3.00.Generally immuning and intratumorally injecting with blood group antigen have significant effect on the count of CD4~+T lymphocytes(F=6.335,P=0.036 and F=13.301,P=0.007).Generally immuning and intratumorally injecting with blood group antigen also have significant effect on the count of CD8~+T lymphocytes(F=7.377,P=0.026 and F=57.553, P=0.000).For CD4~+T and CD8~+T lymphocytes,generally immuning and intratumorally injecting with blood group antigen all have reciprocation(F=5.828, P=0.042 and F=5.482,P=0.047).When there is general immunization,intratumoral injection have no effect on the count of CD4~+T lymphocytes(t=0.887,P=0.425). When there is no general immunization,intratumoral injection can boost it(t=4.214, P=0.014).When there is intratumoral injection,general immunization also have no effect on it(t=0.071,P=0.946).When there is no intratumoral injection,general immunization can boost it(t=3.548,P=0.024).Whether there is general immunization, intratumoral injection can boost the count of CD8~+T lymphocytes(t=5.926,P=0.004 and t=4.802,P=0.009).When there is intratumoral injection,general immunization can boost it(t=3.274,P=0.031).When there is no intratumoral injection,general immunization have no effect on it(t=0.295,P=0.783).There is significant difference of CD4~+T and CD8~+T lymphocytes between the four treated groups(F=8.488, P=0.007 and F=23.471,P=0.000).The counts of CD4~+T lymphocytes of groups a.b and c are higher than that of group d(P values are all less than 0.05).The count of CD8~+T lymphocytes of group a is the highest. 3.Compare the results of inactivated Streptococcus experiment and blood group antigen experiment:the killing rate of splenic lymphocytes against S180 cells of group a is higher than that of group A(t=5.662,P=0.000),and the killing rate of splenic lymphocytes against S180 cells of group c is higher than that of group C (t=2.973,P=0.014).Conclusion1.Generally immuning and intratumorally injecting or only generally immuning or only intratumorally injecting with heteragenetic antigen,the thymus index of mice all can't be raised.2.Generally immuning and intratumorally injecting or only generally immuning or only intratumorally injecting with heteragenetic antigen,the spleen indexs of mice all was raised.As we can say,they improve the whole immune function of mice.3.Generally immuning and intratumorally injecting or only intratumorally injecting with heteragenetic antigen can boost the killing activity of splenic lymphocytes of mice against S180 cells,but only generally immuning with heteragenetic antigen can't boost the killing activity of splenic lymphocytes of mice against S180 cells.4.Generally immuning and intratumorally injecting or only intratumorally injecting or only generally immuning with blood group antigen can boost the count of CD4~+T lymphocytes of spleen.Generally immuning and intratumorally injecting or only intratumorally injecting with blood group antigen can boost the count of CD8~+T lymphocytes of spleen intensively.But,generally immuning with blood group antigen have weak ability to boost the count of CD8~+T lymphocytes.5.Generally immuning and intratumorally injecting or only intratumorally injecting with blood group antigen have better ability to boost the killing activity of splenic lymphocytes of mice against S180 cells than generally immuning and intratumorally injecting or only intratumorally injecting with inactivated streptococcus.
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