The Research Of Adenovirus Mediated IL-28B Combined With 3-bromopyruvic Acid On Regulatory T Cells And Antitumor Effect Of U14 Cervical Subcutaneous Transplantation Tumor

Objective:1.To evaluate the therapeutic effect of adenovirus-mediated interleukin-28 B combined with 3-bromopyruvate on transplanted cervical cancer in mice.2.The gene sequence of IL-28 B mature peptide was constructed on eukaryotic expression vector by using the cloning technique.Methods:1.(1)U14 cervical cancer cells and HEK293 cells were cultrured in vitro.(2)the adenovirus of Ad-IL-28 B and Ad-EGFP were amplified by HEK293 cells,and the titer of Ad-IL-28 B and Ad-EGFP were determined by CPE method.(3)Establishment of Babl/C mouse U14 model of cervical cancer ascites,and extraction of ascites for subcutaneous xenografts.(4)The experimental mice divided into five groups with the transplanted tumor diameter about 3mm were completely random distribution based on random number table,including 3-bromine pyruvic acid treatment group,3-bromine pyruvic acid + Ad-mIL-28 B treatment group,IL-2 treatment group,Ad-mIL-28 B,and PBS treatment group.The size of the transplanted tumor was observed and measured,and the tumor inhibition rate was calculated.(5)Mice were euthanized after 20 days and their spleen,lung and liver were isolated were removed from the peritoneal cavity and minced,and the changes of cytokines CD4+CD25+Fox P3+Tregs in spleen were detected by flow cytometry.2.The eukaryotic expression protein Ppic9k-IL-28 B was constructed by molecular cloning technique,and then expressed in BMMY medium and optimized for protein expression.Result:1.The therapeutic effect of adenovirus-mediated interleukin-28 B combined with3-bromopyruvate on transplanted cervical cancer in mice.(1)The titers of Ad-mIL-28 B and Ad-EGFP were 108PFU/100 ul by CPE method.(2)The U14 cervical cancer ascites mode was established successfully.(3)The proportion of CD4~+CD25~+FoxP3~+Tregs Tregs in the spleen of Babl/C mice treated with Ad-mIL-28 B,IL-2,3-bromine pyruvic acid,and 3-bromine pyruvic acid + Ad-mIL-28 B were 7.66%,5.69%,6.63%,2.64%,respectively,the 3-bromine pyruvicacid + Ad-m IL-28 B was significantly reduced compared with PBS group.(4)there was a significant reduction in the volume of transplanted tumor in the 3-bromine pyruvic acid + Ad-mIL-28 B treated groups compared with the PBS control group.2.(1)The recombinant plasmid Ppic9k-IL-28 B was successfully constructed.(2)The expression vector of recombinant plasmid Ppic9k-IL-28 B was established.Conclusion:1.The CD4~+CD25~+FoxP3~+Tregs in the spleen of mice treated with Ad-mIL-28,IL-2,three bromo pyruvic acid,and the group of three bromo pyruvic acid +Ad-mIL-28 B were decreased.Ad-mIL-28,IL-2,tribromopyruvic acid,tribromopyruvate + Ad-m IL-28 B could inhibit the growth of cervical cancer transplanted tumor.2.The Ppic9k-IL-28 B was successfully constructed,and it can be expressed under certain conditions.