Expression Of Mouse REG3α And REG3α Dimer In Vitro And The Inhibition Of Human Hepatoma Cells

Objectives: The experiment was designed to express the mouse recombinant REG3α and REG3α dimer using Pichia pastoris expression system, and hope to explore the anti-tumor function of REG3α and REG3α dimer on mice.Methods: We expressed and purified the mouse recombinant REG3α and REG3α dimer using Pichia pastoris expression system. MTT test were used to determine maximum no-cytotoxic concentration (MNTC) of REG3α and REG3α dimer on HL-7702 cells and RAW264.7 cells,50% effective concentration (EC50) of REG3α and REG3α dimer on SMMC-7721 cells were also tested using MTT. Acute toxicity test: the mice were divided randomly into three groups and injected intraperitoneally with REG3α (10mg/kg), REG3α dimer (10mg/kg) and PBS, monitored for 14 days. Chronic toxicity test, the mice were divided randomly into three groups injected intraperitoneally with REG3α (7.25mg/kg), REG3α dimer (3.625 mg/kg) and PBS for 28 days. Tumor inhibition test: the mice subcutaneously implanted SMMC-7721 cells were injected intraperitoneally with REG3α (7.25mg/kg), REG3α dimer (3.625mg/kg) and PBS for 22 days, once two days.Results:The recombinant protein REG3α and REG3α dimer are expressed as expected. SMCC-7721 cells were treated with the indicated concentration of REG3α and REG3α dimer for 24 h, EC50 of REG3α and REG3α dimer were 0.145mg /ml and 0.0725mg/ml. The maximum no-cytotoxic concentration of REG3α and REG3α dimer in HL7702 cells were 0.015313 mg/ml and 0.008938 mg/ml, the maximum no-cytotoxic concentration of REG3α and REG3α dimer in RAW264.7 cells were 0.001133 mg/ml and 0.004531 mg/ ml. Chronic toxicity test showed that when compared with PBS control group, the CRE, AST, BUN, TBIL and DBIL level of serum of REG3α group were significant difference, the AST, TBIL, BUN and DBIL were significant difference. Tumor inhibition test: The tumor weight and volume of treatment group mice showed no significant difference compared with the PBS control group.Conclusion: We expressed the mouse recombinant protein REG3α and REG3α dimer successfully; REG3α and REG3α dimer had no obvious effect on the tumor of the mice implanted with SMMC-7721.