| PrefaceBladder cancer is the most common malignant disease of urology in China. Intravesical instillation of bacillus Calmette - Guerin (BCG) is considered the most effective treatment for tumor elimination and recurrence prevention of transitional cell carcinoma after transurethral resection of bladder tumor. However, some patients do not respond to BCG therapy, some patients experience serious adverse effects after BCG therapy. Therefore, more effective components of BCG for bladder cancer immunotherapy are needed to develop.Ag85A complex, a 30 ~ 32kDa family of three proteins (Ag85A, Ag85B and Ag85C) , constitutes a major portion of the secreted proteins in Mycobacteri-um tuberculosis and BCG culture filtrates. The DNA vaccine based on Ag85A protein significantly enhances cellular immune response in animal models.The main mechanism in antitumor immunity against bladder cancer is cell mediated immunity. BCG instillation induces local infiltration of the bladder wall with activated PBMCs, including T lymphocytes and natural killer (NK) cells. The CD4~+ T lymphocytes mainly secrete IL - 2, IL - 12 and IFN - γ to mediate delayed type hypersensitivity ( DTH ). After proliferation, the CD8~+ T lymphocytes , macrophages and NK cells also play an essential role in antitumor immunity. It seems that NK cells are required for successful BCG therapy.This study is undertaken to investigate whether intramuscular injection of Ag85A DNA vaccine could affect the T lymphocyte subsets and NK cell activity in tumor - bearing hosts, then evaluates the immunotherapy effect of plasmid DNA vaccines encoding the Ag85A component.Materials and methodsWith the method of alkaline lysis, Ag85A - VIJns. tPA plasmid and VI Jns. tPA plasmid were extracted and purified with Polyethylene Gly col, then i-solated on a 0. 8% agarose gel. Plasmid DNA was adjusted to a final concentration of lg/L in saline and stored at -20X1.MBT -2 cells(1 x 10 cells) were inoculated intradermally into the back of 6-8 week old C3H/HeN female mice, and tumors were allowed to develop to 5 to 8mm in diameter. Then mice were randomly divided into 3 groups;Ag85A -VI Jns. tPA group ( experiment group) , VI Jns. tPA group ( empty plasmid group) and blank control group ( normal saline group) . Each mouse was injected with lOOjxl Ag85A - VIJns. tpA or VIJns. tpA or saline on quadriceps femo-ris three times ( 2 - week intervals ) . 2 weeks after the third vaccination, mice were sacrificed and spleens were removed asepticly. The percentages of CD4+T cell and CD8 + T cell were measured by performing flow cytometry. In the same time, cytotoxicity of NK cells was detected. All data were expressed as mean ± SD, Student' s t - test was used for statistical analysis. Statistical significance was determined at P <0. 05.ResultsThe percentages of CD4+ T cell, CD8+ T cell and the relative value of CD4V CD8+ in the three groups were as below;8. 91 ± 6. 27% , 6. 57 ± 2. 83% and 1. 26 ± 0. 34 in the experiment group, 6. 53 ± 3. 50% , 5. 20 ± 1. 37% and 1.21 ±0. 37 in the empty plasmid group, 11. 94 ±3.74% , 8.97 ± 1. 92% and 1. 31 ±0. 23 in the normal saline group. The results showed no significant difference in the three groups (P > 0.05). The NK activity was 19.07 ± 5.8% , 11. 16 ± 10. 01% and 22. 06 ± 6. 02% , also showed no significant difference in the three groups (P > 0.05).DiscussionT lymphocytes play a key role in antitumor immunotherapy. The cellular immunity is predominantly carried out by CD4 + Thl cell ( T helper cell 1) and CD8 + cytotoxicity T cell ( CTL). NK cells are believed to be the most important effect cells during early tumor development, and it also play an essential role in the anti tumor effect mediated by BCG.When the Ag85A DNA vaccine enters into the body, it activates the Thl cells to secrete IL -2,IFN -7, et al, which activate CTL and NK cells. All the cells and cytokines will kill the tumor cells. But the immunotherapy effect of the Ag85A DNA vaccine on the MBT -2 bladder tumors is limited. Up to date, we did not see any reports about the treatment of Ag85A DNA vaccine on tumor models, especially on the bladder cancer models. In this study, we injected the single Ag85A DNA vaccine to tumor - bearing mice, but didn' t see the elevation of the immunity functions in this bladder cancer model.The injection methods and pathway of DNA vaccine will affect the expression of contained gene ( s) and the presentation of antigen ( s) , also the bias of immune response types. In this study, the intramuscular injection did not enhance the level of cellular immunity, which may be related to the different animal strains or the diversity injection methods and pathway.In short, more efforts should be made to choose the effective genes aimed directly at the diversity tumors, and select the appropriate injection methods and pathway in the anti tumor study by DNA vaccine.Conclusion1. Intramuscular injection of Ag85A DNA vaccine could not significantly affect the percentages of the T lymphocyte subsets of mice bearing MBT - 2 bladder tumor.2. Intramuscular injection of Ag85A DNA vaccine could not efficiently increase the activity of NK cells in the spleen cell of mice bearing MBT - 2 blad-der tumor.3. The single Ag85A DNA vaccine may not induce the effective anti -bladder tumor immune response. |
