| ObjectiveTo observe the humoral and cellular immune responses induced by MUC1 VNTR(variable number of tandem repeats) DNA vaccine in multiple myeloma mice model as well as to study whether the mice loading the 653-MUC1 cell line could be protected by the injection of pcDNA3.1-2VNTR plasmid which induced the tumor immune response.MethodsIn vitro,we used the Lipofectamine2000 to transfect the plasmid into the multiple myeloma cells.After transfection,the G418 was used to select the monoclonal multiple myeloma cell line 653-MUC1,then detected the expression of MUC1 by RT-PCR.Tweety normal 7-8 week female BALB/c mice were randomly divided into 2 groups,10 mice in Neo(pcDNA3.1) and other 10 in MUC1 (pcDNA3.1-2VNTR).A total of 100μg Plasmid DNA of pcDNA3.1-MUC1 in 100μl of PBS Plasmid DNA was injected into the spleen of the mice on week 0.On the fifth week a booster immunization was given by injecting the same plasmid DNA into the anterior tibialis muscle of BALB/c mice.Mice inoculated with the empty plasmid pcDNA3.1 were used as a control.After the spleen immunization,the blood from the inner canthus vein of the mice was collected for the ELISA assay of anti-VNTR antibodies in 1,2,4,6 and 8 weeks,respectively.Four weeks after the second immunization,the spleen cells of two groups were collected to detected CTL activity by the method of LDH.Spleen lymphocyte proliferation activity aslo detected at the same time by MTS/PMS method.Then the BALB/c female mice of 7-8 weeks age,were divided randomly into 2 groups,aslo 10 in MUC1 (pcDNA3.1-2VNTR) and other 10 in Neo(pcDNA3.1).The mice injected with plasmid DNA(100μl/100μg) were as the method mentioned above.Seven days after the second immunization,the mice were inoculated with 653-MUC1 at the interderm of the left anterior leg armpit(1×10~6/each mouse).The tumor development in individual mice was monitored every 2-3 days and the tumor size(in mm~3) was calculated by the following formula:0.5×length(mm)×width(mm~2).The survival time after the tumor challenge was recorded until all the mice death.ResultsThe constructed monoclonal multiple myeloma line 653-MUC1 could express MUC1 mRNA which demonstrated by RT-PCR.After immunization,specific antibody against MUC1 VNTR antigen was found significantly higher in pcDNA3.1-2VNTR immunized mice than the control group.Cytotoxicity assay showed that when the ratio of effector cell to target cell reached 80:1,specific CTL response could be detected significantly in immunized mice.Compared with those immunized with empty vector,the spleen lymphocythes,which immunized with pcDNA3.1-2VNTR,proliferated markedly.After the tumor challenging,the tumor growth rate in the pcDNA3.1-2VNTR immunized mice was much slower than the control group(P＜0.05) and the survival curve showed that the life time of the pcDNA3.1-2VNTR immunized mice was much longer than the control group (P＜0.01).ConclusionsMUC1 DNA immunization could elicited both humoral and cellular tumor specific immune response,and the MUC1 VNTR immunized mice could suppress the growth of the MUC1-positive cell line 653-MUC1,which could be a possible clinical trial for the treatment vaccine of multiple myeloma.
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