Effects Of Ag85A DNA Vaccine On The Proliferation Ability Of Lymphocyte And Expression Level Of FasL MRNA On Mice With Bladder Cancer

PrefaceBladder cancer is one of the most common tumors that ranked fourth in male, tenth in female. 90% of bladder cancers are transitional cell carcinoma and some of them are superficial bladder cancer. Intravesical BCG therapy is considered the most successful immunotherapy to date. Nevertheless, a significant proportion of patients do not respond to BCG therapy. Adverse effects are quite common. Therefore it is necessary to find a more effective and safer strategy to replace this therapy.Ag85 complex, a 30 -32kDa family of three proteins (Ag85A, Ag85B and Ag85C) , constitutes a major portion of the secreted proteins in M. tuberculosis and BCG culture filtrates. Ag85A may be the most essential component for bacterial survival and virulence. Genetic vaccination with plasmid DNA encoding Ag85A is found to generate robust Th1 response and CD8 CTL response. This study is undertaken to investigate whether Ag85A DNA vaccine could influence the proliferation ability of lymphocyte and expression of FasL in mice with bladder cancer by muscular injection.Materials and MethodsWith the method of Alkaline Lysis, Ag85A - V1Jns. tPA plasmids and V1Jns. tPA plasmids were extracted from E. coli JM109. The plasmids were digested by BglII restriction endonuclease. The digested plasmids were analyzed by agarose gel electrophoresis. Large quantity of plasmids were extracted andpurified. Then the concentration of plasmids was modified to 1 g/L, stored at -C3H/HeN female mice, 6-8 weeks, were randomly divided into 3 groups: Ag85A DNA plasmid group, empty plasmid control group and saline control group. MBT -2 cells (1 x 106 cells) were inoculated subcutaneously into the back of mice. For muscular immunization, 100jxg Ag85A DNA vaccine in 100|xL saline was administered on quadriceps three times at 14 - day interval when the diameters of the tumors grow up to 5 -7 mm. As for negative control groups, mice were injected with empty plasmids and saline respectively. Two weeks after the third vaccination, mice were killed. Tumors were removed immediately and net weights of individual tumors were measured. Spleens were removed aseptically. Spleen cells were cultured and proliferation abilities were detected using MTT assay. Total RNA was isolated from lmL spleen cells (1 x 107 cells/mL) using Trizol and analyzed by UV300 ultraviolet spectrophotometer. RNA was reverse transcribed to cDNA according to the manufacturer' s protocol. PCR was performed to amplify FasL gene with housekeeping gene f$ - actin as internal control. PCR products were visualized in 1.5% agarose gel following staining with EB. FasL - encoding mRNA expression levels were determined by the ratio of band density between FasL and p - actin after gel scanned by Chemi-Imager 5500 gel analyzer. All data were expressed as mean ± SD. Significance of differences was determined by t - test. A p value less than 0. 05 is considered statistically significant.ResultsAg85A - VI Jns. tPA plasmids were extracted from E. coli JM109 and digested by Bglll restriction endonuclease. Ag85 A gene band was visualized in 0. 8% agarose gel at the site of 1014bp.On the 14th day after last immunization, the proliferation abilities of lymphocytes , net weights of tumors and the expression levels of FasL mRNA were compared with the saline control group and empty plasmid control group. There is no significant difference between them ( p > 0. 05 ).DiscussionThe DNA vaccines are simple rings of DNA containing a gene encoding an antigen and a promoter or terminator to make the gene express in mammalian cells. When introduced into the body, this material ultimately leads to synthesis of proteins inside the cells and then activates host immune responses. As a result , the host immune system responds in a protective manner to produce antigen molecule frequently almost the same way as would occur if the host were actually infected by the true organism itself. Most importantly, DNA vaccine can stimulate not only humoral immunity but also cellular immunity in host. DNA vaccination has been a promising new approach for auto - immune diseases, allergic diseases and tumor diseases.In our research, there is no change on the proliferation ability of lymphocytes and expression level of FasL mRNA in lymphocytes of mice with bladder tumor, following muscular injection of Ag85A DNA vaccine, which coincides with the result of tumor net weights. All of the above shows that the lymphocytes haven t received enough stimulation that can improve the protective immune response in mice with bladder cancer. The reasons may be as following: First, Ag85A DNA vaccination following muscular injection cannot induce enough expression of Ag85A;Second, Ag85A alone cannot improve the anti -tumor effect of cellular immune response.Therefore, it is necessary to construct recombinant DNA vaccine which combines Ag85A gene and other adjuvant gene followed by further research. Appropriate attention also should be focused on inoculation method to improve the tumor therapy.