The Changes Of Immune-related Factors Within The Mice Muscle Cells After Immunized With The Plasmid

Objective: Since the discovery of smallpox vaccine in1798, thousandsof vaccines have been developed to prevent and control of various diseases.Classic vaccines include attenuated live vaccine, inactivated vaccine, toxoidand subunit vaccines. Although these vaccines make many infectious diseasesget controlled and prevented. But traditional vaccine was found to have manydefects, such as incomplete inactivation of dead vaccine and virulencerecovery of live vaccine. So people faced with a new challenge to developvaccines, which may be safer, more effective, inexpensive, and have betterimmunity. With the advance and mature of tuerecombinant DNA technology,genetic engineering seems to be the best way to solve these problemscurrently.Genetically engineered vaccine is developed by molecular biology techniques,which transform the genomes of pathogenic microorganisms to reduce itspathogenicity and improve its immunogenicit, or clone one or more genes,which may be useful for prevention and treatment of diseases, into prokaryoticor eukaryotic expression vector to make vaccine. DNA vaccine is one kind ofgenetic vaccines.Muscle injection is the routine way for vaccination. Skeletal muscle is thelargest group of cells, and also as the common vaccination site for DNAvaccine. After vaccination of skeletal muscle, the protein encoded by it canexpression on a variety of cells including skeletal muscle cells. Theprofessional antigen presenting cells can be transfected directly and thentransferred to the draining lymph nodes and initiating an immune response.Dendritic cells could induce an immune response by cross-presenting.Whilein the existing research results, the role of muscle cells in the DNAimmunization is conflicts. Some studies have found that the expression of MHCâ…  molecules and B7-1is increase in the muscle cells transfected, whichwas consigned may be effective on the initiating and maintain of immuneresponse induced by muscle cells as a non-professional antigen presentingcells. But other studies suggest that the antigen expressed on the muscle cellsleads to the immume tolerance of CD8+T cell. In general, the role of musclecells in DNA immunization remains to be studed and exploredPD-1is a negative regulator in immune respond. There are two kinds ofligands of PD-1, PD-L1and PD-L2. PD-L1can express on professionalantigen-presenting cells, non-professional antigen-presenting cells and musclecells.while PD-L2can be induced to express on dendritic cells andmacrophages. PD-1/PD-L1is a negative signal regulatory pathway, highexpression of PD-1on immunocompetence cells can induce aenergy of thecells in the procedure of Chronic virus infection, suggests that, on the onehand, the DNA vaccined muscle cells play a role in inducing an immuneresponse by cross-presenting, on the other hand, it is likely to provide negativecontrol signals and suppress immune response in the process of buildingself-preservation. This might be one of the reasons why the protective effect ofthe DNA vaccine is not strong enough.Based on the above considerations, in this experiment, mice muscle cellswas used as a model. Mice were immunized with plasmid, and then theexpression changes of PD-L1and CD80within the mice muscle cells wereobserved in order to investigate its role in gene vaccine and to pave a new wayto improve the protection effective of DNA vaccine.Methods:â‘´C57BL/6mice were immunized with plasmidpcDNA3/VP-1by injection in muscle of quadriceps,100ug each.(2)Extraction of total cellular RNA: total cellular RNA was extracted in differenttimepoints after the mice immunized with plasmid,(in1day,3days,5days,7days,) the expression of PD-L1and CD80were detected by real time PCR.Results:(1) the results of real time PCR showed that the expression ofPD-L1mRNA in the experimental group increased to peak at3days afterimmunized with plasmid, then began to slowly decline at5days and7days; The expression of CD80in the experimental group increased to peak at3daysafter immunized with plasmid, and the rest of the time remained at a low level;Compared with the experimental group, the expression of PD-L1andCD80in control group were generally maintained at a low level; In the controlgroup the levels of CD80is higher than PD-L1.Conclusions:(1) immunized with plasmid DNA made the PD-L1andCD80mRNA expression in mouse muscle cells increased.(2) Compared withthe control group, mRNA expression of PD-L1and CD80in the experimentalgroup were generally increase.(3) Normal muscle tissue also expressedPD-L1and CD80, and the CD80expression levels was higher than the PD-L1.