The Effect Of Irradiated Haploidentical Lymphocyte Infusion Following Different Does Cyclophosphamide On Mice With Hepa1-6Tumor

Part I：Establishment of subcutaneous tumor mouse modelObjective: To establish a hepa1-6tumor-bearing mice model for irradiatedhaploidentical DLI.Methods:6-8weeks female CB6F1mice were divided to three groups (n=5foreach group). CB6F1mice were subcutaneously inoculated with4×10~6hepa1-6cells atthe right flank. Tumor size was measured with calipers every2-3days. Tumor volumewas calculated by the following formula: volume (mm~3)=length×width2/2(mm~3).The loss of total body weight was measured every2-3days. At the time of thetumor bearing mouse died, we take their ear skin, liver, small intestines and spleen tosubjected to histopathological examination.Results： CB6F1mice were subcutaneously inoculated with4×10~6hepa1-6cells atthe right flank. The subcutaneous tumor can be detected at day3or4. The threegroups had not any significant difference on the tumor growth and survival. We didn’tfind the spontaneous anesis in every group. In histopathological examination, thesubcutaneous tumor was confirmed a poorly differentiated hepatocellular carcinoma.The lung metastasis was found in tumor bearing mouse.Conclusions: The hepa1-6cell can grow in the CB6F1mouse subcutaneous.Part Ⅱ：The effect of irradiated haploidentical lymphocyte infusion followingdifferent does cyclophosphamide on mice with Hepa1-6tumorObjective: To analyze the effect of irradiated haploidentical lymphocyte infusion(HLI) following different dose cyclophosphamide （CTX） on hepatocellularcarcinoma Hepal-6.Methods: Female (BABL/c×C57BL)â†'F1(CB6F1H-2b/d) mice were subcutaneouslyinoculated with Hepa1-6cells to develop a solid tumor model that was taken asrecipients. The irradiated haploidentical lymphocytes were from female(BALB/c×C3H)â†'F1(CC3HF1H-2d/k). All the mice were divided to five groups:group PBS (mice received a intravenous of PBS), group CTX80mg/kg+SP/irr (micereceived the irradiated CC3HF1splenocytes following Intraperitoneal injection ofof cyclophosphamide at80mg/kg), group CTX200mg/kg+SP/irr (mice received the irradiated CC3HF1splenocytes following Intraperitoneal injection ofof cyclophosphamide at200mg/kg), group CTX300mg/kg+SP/irr (mice receivedthe irradiated CC3HF1splenocytes following Intraperitoneal injection ofof cyclophosphamide at80mg/kg) and group SP/irr (mice received the irradiatedCC3HF1splenocytes).1) The tumor size and loss of total body weight weremeasured every2-3days. The survival time was monitored.2) the graft-versus hostdisease (GvHD) was assessed by histology of the skin, small intestine, liver andspleen.3) The chimerism and the proliferation of tumor bearing mouse lymphocytessubset were measured after HLI.4) the IL-2and IFN-γ concentration of the peripheralblood were measured using specific ELISA kit for mouse IFN-γ and IL-2.5) Thekilling effect of host splenocytes was measured by LDH at the ten day after irradiatedhaploidentical lymphocytes infusion.Results: The tumor was significantly suppressed in group CTX80mg/kg+SP/irr andgroup CTX200mg/kg+SP/irr comparing with group PBS[(1.25±0.24)cm~3，（1.38±0.31) cm~3vs (2.03±0.24) cm~3，P<0.01]，so was the survival time [48d（39d,55d），45d（35d,50d） vs35d（18d,39d），P<0.05], of which, group CTX80mg/kg+SP/irr got significantly different antitumor effect from the groupSP/irr[(1.25±0.24)cm~3vs (1.76±0.40)cm~3，P<0.05]. GvHD was not found in anygroup. The chimerism of group80mg and200mg CTX combining with HLI waslower than group CTX300mg combining with HLI and the time of disappearing wasshorter. At the same time can increase secretion of the type I cytokines e.g IL-2, IFN-γ and enhances the proliferation of the lymphocyte subsets of CD8+T cells and NKcells.Conclusion: Transplantation of irradiated haploidentical lymphocyte following lowdoes CTX can induce antitumor effect on hepatocellular carcinoma of mice in F1â†'F1infusion model systems. Higher dose of CTX may not enhance antitumor effect. Atthe same time can increase secretion of the type I cytokines IL-2, IFN-γ and enhancesthe proliferation of the lymphocyte subsets of CD8+T cells and NK cells.