| Objective： Deinococcus radiodurans (DR）is one of the microorganisms whichexhibits an extraordinary ability to withstand the lethal and mutagenic effects ofionizing radiation, ultraviolet radiation, strong oxidizer and chemical mutagen. It is oneof the most radioresistance life-forms which have been found so far. Research has so farproved inconclusive about the specific mechanism for this super resistant. A variety ofrepair proteins play an important role about their specific radiation resistance in therepair mechanisms of Deinococcus radiodurans.PprI is one of the unique gene in D.radiodurans, also known as irrE, is considered to be a master switch of Deinococcusradiodurans DNA repair and protective pathway, PprI protein,the expression productionof the PprI gene, is one kind of promoting DNA repair efficiency protein inducer.Inrecent years, researchers of our department confirmed that PprI gene transfection has asignificant role in the prevention and treatment of acute neutron and gamma-rayradiation injury of the mammals,and used pCMV-HA-PprI as a template, constructedthe recombinant vector pET-28a-His-PprI and transfered it into E.coliBL21(DE3)RP,after induced by IPTG and purified,got the PprI fusion protein ofDeinococcus radiodurans.And the study confirmed that this protein has a certainresistance to theγ-ray radiation damage.This subject made a preliminary study of itscytotoxicity and immune toxicity on the basis of the mechanism of this protein in orderto provide the basis for the future preclinical safety evaluation. It has not been reportedin the literature yet about the toxicity of this protein.Materials and Methods: PprI protein was provided by Zhang Yongqin of ourdepartment.Human umbilical vein endothelial cells were used as the research object,theinfluence on the cell proliferation and cell survival about the protein was detectedrespectively by the MTT assay and cell colony formation method, meanwhile drew thecell survival curve. For the other hand,the ICR mice were used as the immune object,the experimental group was divided into physiological salineimmune group and ppr protein immune group. Further study of this protein on the influence to mice immunefunctions was observed through the immune organs viscera coefficient, lymphocytetransformation ability (MTT assay), cytokines released (ELISA kit),T lymphocytesubsets analysis (Flow cytometric).Results: The study indicated that when the PprI protein concentration reached to2.5,5,10ug/mL,it has inhibition to cell proliferation,and the inhibition and proteinconcentrations were positively correlated.When the cell density was0.5×104and theeffect time was24h,cells inhibition rate IC50is6.40ug/mL.When PprI proteinconcentration were100,200,400,600ng/mL, it has promotion to cellproliferation.When protein concentration was400ng/mL and the effect time was36h, ithas the most obvious promotion to cell proliferation.From the cell survival curves fittedby multi-target click model,we got in the PprI protein group D0value is1.33, the Dqvalue of1.17, N value of2.41, SF2value is0.51, each value is higher than theirradiation group.Within the observation period of the immune,immune Organcoefficient calculation result showed that,14days after the primary immunization, forhigh-dose group kidney weight coefficient is lower than that of the saline controlgroup;thymus weight coefficient of the the medium and high dose group is highercompared with saline control group;Three days after the last immunization, kidneyweight coefficient of the low-dose group is lower than that of the saline controlgroup;thymus weight coefficient of the high dose group is higher compared with salinecontrol group;three weeks after the last immunization, liver weight coefficient of thehigh dose group is higher than that of the saline control group;thymus weight coefficientof low-dose group is higher compared with saline control group;all results hadsignificant difference between control saline group anddifferent test groups(P<0.05). On the other hand, lymphocyte transformation isenhanced while immune dose reaching medium and high dose;CD3+T lymphocyte ofspleen increases at the medium dose; of CD3+T-lymphocytes of thymus decreases atthe low, medium and high dose; IL-4antibody secretion of the PprI protein immunegroup increases in the end of immune period at the high dose compared with salinecontrol group(P<0.05); there is no significant difference at the level of IFN-γantibody-secreting.Conclusions:1.The study indicated that when the PprI protein concentration reached to 2.5,5,10ug/mL,it has inhibition to cell proliferation,and the inhibition and proteinconcentrations were positively correlated.When the cell density was0.5×104and theeffect time was24h,cells inhibition rate IC50is6.40ug/mL.When PprI proteinconcentration were100,200,400,600ng/mL, it has promotion to cellproliferation.When protein concentration was400ng/mL and the effect time was36h, ithas the most obvious promotion to cell proliferation.2. The cell survival curve showed that, PprI protein increases the average lethaldose of the cells, enhances the radiation resistance and the cell repair ability.3. PprI protein mainly influence central immune organs thymus and the maininternal organs kidney, and the stimulus is reversible.And it almost has no toxic effectsto liver and spleen.4. While the immune dose reached to100μg/kg、200μg/kg，PprI protein canstrengthen the lymphocyte transformation function of the spleen and the thymus inmice.5. About the immune way stimulated by the PprI protein，late the mice mainlyshow cellular immunity，recovery period mainly show humoral immune.6. PprI protein can raise the proportion of spleen CD3+T cells in T cellssubsets,CD4/CD8ratio has been in the normal range; meantime reduce the proportionof thymus CD3+T cells in T cells subsets, it showed that cellular immune is dominantin the recovery period.7. In conclusion,it showed that immune function of the mice is enhanced,however,there is no adverse effects to the immune organs and immune function,almost noimmune toxic effects.It has a good safety profile and the results provided a referencebasis for future clinical application of this protein. |
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