Effect Of Ag85A DNA Vaccine On The Subsets Of Intestinal Intraepithelial Lymphocytes And Cytotoxic Activity Of NK Cells In Mice

PrefaceIn my laboratory, we have completed National Natural Science Foundation《Changes of biological activity of Intestinal Epithelial Lymphocytes in The DigestiveUlceration》and Science and Technology Foundation of Liaoning province《Theresearch of Ag85A gene vaccine curing the bladder cancer》, we have established aseries of experimental technique on ilEL, such as the ilEL subsets separation technique,examine the CK, cytotoxic activity and NK cells activity etc. This experiment is a partof National Natural Science Foundation《Expression in the mucosa and Effecctivemechanisms of inducing IEL of self-prepared DNA vaccine administrated orally》,intend to adopt the Ag85A gene plasmid which can express in the eukaryoticcell,prepare the DNA vaccine for oral,taking orally it to the mice, regarding observatingthe influence of biological activity of IEL as point, proceeding the research of theimmunological effect after taking orally the DNA vaccine, elucidating the mechanismof the bowel mucosa immunological reaction after taking orally the DNA vaccine.Thisexperiment will help us understand the IEL function and partial mucosa immunologicalreaction after taking orally the DNA vaccine, for providing the new way of regulatingthe partial bowel immune function, and will provide the further theories and theexperiment basis for the clinical application of DNA vaccine.Materials and methodsAccording to protocol of Pure Yield Plasmid Maxiprep System Kit,Ag85A-pcDNA3.1~+ Plasmid were extracted and digested by BglⅡrestriction endonuclease. The digested plasmid was analyzed by electrophoresis.30 C57BL/6 mice,male, 6 to 8 weeks, were randomly divided into 3 groups. (Ⅰ) 10 mice for normalcontrol group. (Ⅱ) 10 mice for empty plasmid control group. (Ⅲ) 10 mice for Ag85Aplasmid group. For oral immunization, Salmonella typhimurium (harboring the emptyexpression plasmid or the Ag85A-encoding plasmid) were once administeredintragastrically in a 100μ1 volume into each mouse from empty plasmid control groupand normal control group. Immunization for 3 times at 14 days intervals, iIEL andspleen lymphocytes in each group were collected on day 10th after last immunization.The percentage of CD4+CD8 iIEL and CD4~-CD8~+ iIEL were measured by performingflow cytometry.In the same time, cytotoxic activity of NK cells was detected. All datawere expressed as mean ((?)±s) , T-test was used for statistical analysis.Statisticalsignificance was determined at P＜0.05.Results10 days after the third vaccination,the percentage of CD4~+CD8~- iIEL in the threegroup were similar, CD4~-CD8~+ iIEL in the three group were different,and the cytotoxicactivity of NK cells showed significance changes. (P＜0.05)。The results indicated thatoral of Ag85A DNA vaccine could affect the CD4~-CD8~+ iIEL,and the cytotoxicactivity of NK cells in mice.DiscussionIn this experiment after taking orally the Ag85A DNA vaccine, the immunityfunction of iIEL decreased and the cytotoxic activity of NK cells increased in mice,we calculate the possible mechanism as follow, the inoculating method and the path ofDNA vaccine will affect the expression of purpose gene,angtigen presentation and thetype of immune response..The inoculating of DNA vaccine includes the direct muscleinject, the lump inside inject and the gene gun method,et al. Tanghe once injected themuscle and the gene gun bombarded the skin with Ag85A DNA vaccine in the mice ofBALB/c and C57BL. Consequently discovering, that gene gun bombarding can induce antibody level of BALB/c go up and the CTL response strengthen, but the levelwith Th1 type CK IL-2 and IFN-γsecret lower;But the mice of C57BL responds togene gun weakly, which was injected by muscle respond strong, IL-2 and IFN-γsecrete more than the mice of BALB/c obviously.Moreover,the oral tolerance is closely related with the mucosal-associatedlymphoid tissue,a large quantity research show the possible mechanism ofimmunological tolerance is active suppression,clonal deletion and clonal anergy, etc.By taking high dose orally antigen will make active suppression,low dose will makeclonal deletion and clonal anergy.Oral antigen affects on the regulating CD4~+T cell ofGALT, induce it secreting the depressing CK, such as Th2 or Th3 CK inducing theimmunity depress.Tsuji etc give the BALB/C mice theβ- LG orally, the intestinal PPlymph node appears specific antigen CD25~+ regulating cells clone, these clones isanergy, and can active suppress the producing ofβ- LG specific antibody. Theactivited CD8~+γd T cell oneself can secrete TGF-β, IL-10 depressing CK, can makethe CD4~+ aβT cell change to Th2/Th3 type, and make the secreting of TGF-β, IL-4,IL-10 increase, develop the immune suppression function.CD8~+T cell also expressesdepressing types, after cultivating with the enterocytes, CD8~+ Tr cell can proceedoligoclonal amplification, and these cells are dismissed in the bowel of the patients whohave inflammatory bowel disease,we can know that the CD8~+ T cell attendsimmunological tolerance.Conclusion1.Oral of Ag85A DNA vaccine could reduce the CD4~-CD8~+ iIEL in mice,but notsignificant affect the CD4~+CD8~- iIEL in mice.2.Oral of Ag85A DNA vaccine could increase cytotoxic activity of NK cells.