| Objective To explore the possible mechanism of CTX induced enhanced immunity of adoptive cells immunotherapy(ACI) by detection the influence under single or cyclical CTX injection plan on regulatory T cells( Tregs) and it,s anti-tumor curative effect combined with cytokine induced killer cells(CIKs) on hepatocellular carcinoma(HCC) in mice, and we assumed that the study could provide evidence and suggestions for us to expore new ways to improve anti-tumor therapy.Methods In vivo C57BL/6 mice were subcutaneous injected with Hepa1-6 cells to construct hepatocellular carcinoma animal model.After the model were successful generated,the mice were randomly divided into 4 groups, 40 mice for each group, there were normal control group, single tumor group, single CTX group and cyclical CTX group.After corresponding treatment with intervention, the single cells suspensions of spleen was carried out to observe the CD4+CD25+Foxp3+/CD4+ cell proportion by flow cytometry. We measured the length and width of tumor every other day to calculated the volume and the growth curve was drawn.In vitro we get the mononuclear cells of spleen in mice by density gradient centrifugation to culture CIKs with IFN-γ, IL-2 and anti-CD3 e Mab and the cell growth curve was drew.In the end of the incubation the proportion of main effector cell phenotype CD8+ NK1.1+ in CIKs was detected and killing activity was also detected by cell counting kit-8. In the last part of the study we combined CTX with CIKs to cure mice with hepatocellular carcinoma. After the model were successful generated, the mice were randomly divided into 6 groups, 5 mice for each group, there were single tumor group(untreatment control group), single CTX group, cyclical CTX group, single CIKs group, single CTX + CIKs group and cyclical CTX + CIKs group. After corresponding treatment with intervention,we measured the length and width of tumor every other day to calculated the volume and the growth curve was drawn. At the end of the experiment, the tumor was dissected and weighed to calculated tumor inhibition rate..Results Firstly,in vivo part of the study, the Tregs increased gradually with time last in tumor control group and transitory downregulated of Tregs was appeared then which returned to the initial level; The Tregs in cyclical CTX group maintained at low level; In the last day, the volume of single tumor group, single CTX group and cyclical CTX group was respectively 4056.5 ± 865.4mm3, 3517.9 ± 756.5mm3, 2256.4 ± 620.3mm3, and there was no statistical difference in tumor volume between single CTX group and single tumor group(P>0.050), the tumor volume in cyclical CTX group was less then the single tumor group(P<0.01). Secondly, in vitro the CIKs increased by 14.4 times after 2 weeks of culture,in which the proportion of CD8 + NK1.1 + double positive cells increased from the initial 0.7 ± 0.3% to 17.7 ± 2.3%. The different effector-target ratio in 10:1, 20:1, 40:1 corresponding the killing activity was respectively 25.99 ± 3.53%, 38.26 ± 3.65%, 54.77 ± 5.35%. In the end of the experiment, tumor volume in single CTX group close to the single tumor group(3764.8 ± 537.7 vs 3800.4 ± 605.5 mm3, P=0.784); the tumor volume in single CIKs group less then the single tumor control group(3352.2 ± 485.4 vs 3800.4 ± 605.5 mm3, P=0.02); the tumor volume in cyclical CTX group less then the single CTX group(2076.6 ± 620.2 vs 3764.8 ± 537.7 mm3, P<0.001); the tumor volume in single CTX + CIKs group less then the single CTX group(1867.1 ± 533.6 vs 3764.8 ± 537.7 mm3, P<0.001) and single CIKs group(1867.1 ± 533.6 vs 3352.2 ± 485.4 mm3, P<0.001); the tumor volume in cyclical CTX + CIKs group less then the cyclical CTX group(970.7 ± 135.3 vs 2076.6 ± 620.2 mm3, P<0.001), single CIKs group(970.7 ± 135.3 vs 3352.2 ± 485.4 mm3, P<0.001) and single CTX + CIKs group(970.7 ± 135.3 vs 1867.1 ± 533.6 mm3, P<0.001). Tumor volume in single CTX or single CIKs group grew rapidly in the later stage, while tumor volume in CTX combined CIKs treatment group remains moderate upward trend. Inhibition rate in single CTX group, single CIK group,cyclical CTX group, single CTX + CIKs group and cyclical CTX + CIKs group was respectively 3.7%, 5.2%, 48.1%, 54.4 %, 78.6%.Conclusion With the development of tumors, the proportion of Tregs gradually increased in the spleen-bearing mice; Cyclical administration of low-dose CTX may be of more clinical value, which could prolong the period in which Tregs was kept at low level and tumor volume was inhibited; The CIKs can be successfully cultured from spleen of C57BL/6 mice with mouse cytokine IFN-γ, IL-2 and murine Anti-CD3 Mab, which provide a basis for the design of relevant animal experiments; Low-dose CTX could enhance the anti-tumor effect of CIKs by regulating Tregs, which provided a theoretical basis for the clinical application of CIKs based on CTX regulating Tregs. |
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