| As nuclear technology was widely used in the field of military and civilian, the probability of nuclear radiation-induced acute radiation sickness(ARS) is increasing. ARS, in particular bone marrow type, the hematopoietic function of bone marrow is seriously injuried. The infection and bleeding, as a result of lacking of white blood cells and platelets in peripheral blood, are the major causes of death of ARS[1]. Interleukin 12 (IL-12) is a kind of known immunomodulatory effective cytokines [2], but its role in the promotion of bone marrow is less reported. Recent study reported that recombinant mouse interleukin 12 (rmIL-12) could promote bone marrow progenitor cells in each cell lineage proliferation and differentiation, and can promote the recovery of hematopoiesis in mice of ARS [3,4]. Using a mouse model of bone marrow type of ARS, our study was designed to observe the effect of rmIL-12 on hematopoietic function in mice of ARS, which may provide experimental basis on the development of treatment strategy for the ARS.Objective In this study, ARS mouse model was established after irradiation with sublethal doses of 6.0Gy 60Coγ-rays,and was used to study the effect of rmIL-12 in promotion of hematopoietic function in mice of ARS with different administration time schedules, different doses of rmIL-12,and combined with granulocyte colony stimulating factor (G -CSF) in the treatment of mice of ARS.Compared the role of rmIL-12 in promoting platelet recovery with recombinant mouse thrombopoietin (rmTPO),and the role of G-CSF in promoting white blood cell recovery.Methods1. Establishment of ARS model of mice: 6~8 weeks BALB/C male mice were given 6.0Gy60Coγ-rays total body irradiation(TBI) for one time, irradiation dose rate 187cGy/min, irradiation distance 80cm. Identification of ARS mouse model were relied on observing the clinical performance, peripheral blood cell counts and bone marrow pathology of mice after TBI.2. The effect of rmIL-12 on hematopoietic function in mice of ARS 2.1 rmIL-12 experiment with different administration time: 6-8 weeks BALB/C male mice irradiated with 6.0Gy60Coγ-rays TBI were given rmIL-12 using three dosing schedules,specifically:1) rmIL-12 pre-dosing at 24 hours before radiation,2) rmIL-12 post-dosing at 1 hours after radiation,and 3) a split dose of rmIL-12 pre-post dosing given both 24 hours before and 1 hours after radiation.RmIL12 5μg/kg was administrated intraperitoneally in rmIL-12 treatment groups,while mice of control group received solvent.The general conditions of mice,and changes in peripheral blood cell counts were observed.2.2 rmIL-12 experiments with different doses: 6-8 weeks BALB/C male mice were given rmIL-12 of different doses at 5,10,20,40,60μg/kg intraperitoneally,at 24 hours before and 1 hours after the 6.0Gy60Coγ-rays TBI,while mice of control group received solvent, The general conditions of mice,and changes in peripheral blood cell counts were observed.2.3 rmIL-12 experiments combined with G-CSF: 6-8 weeks BALB/C male mice were given rmIL12 20μg/kg intraperitoneally at 24 hours before and 1 hours after the 6.0Gy60Coγ-rays TBI,and rhG-CSF 100μg/kg were also given subcutaneously for consecutive 14 days in combination treatment group; G-CSF 100μg/kg were given subcutaneously 2 hours after irradiation for consecutive 14 days in G-CSF treatment group; rmIL-12 20μg/kg were given intraperitoneally at 24 hours before and 1 hours after radiation,while mice of control group received solvent.The general conditions of mice and changes in peripheral blood cell counts was observed.2.4 rmIL-12 compared with rmTPO experiments: 6-8 weeks BALB/C male mice were given rmIL12 20μg/kg intraperitoneally 24 hours before and 1 hours after radiation in rmIL-12 treatment group,while rmTPO 300μg was given 1 hours after radiation intravenously in rmTPO treatement group,mice of control group were given solvent, The general conditions of mice and changes in peripheral blood cell counts was observed.2.5 The bone marrow pathology and the ability of bone marrow cells to form colony forming unit(CFU) in culture comparison between rmIL-12 20μg/kg treatment group and control group: 6-8 weeks BALB/C male mice were irradiated with 6.0Gy60Coγ-rays TBI. Three mice were killed in each group after irradiation on 14th day and 28th day after irradiation, the bone marrow morphological changes were observed by the pathological specimens from the femur. 2×105 bone marrow cells were collected from femur in sterile conditions to observe its ability to form CFU.Results 1. Establishment of mouse model of acute radiation sickness: 6-8 weeks old BALB/C male mice were given 6.0Gy 60Coγ-rays TBI, the mice were poor-spirit, less active, the cell counts of peripheral blood decreased,WBC counts on 1th day after irradiation decreased to 26.3% (according to previous counts before irradiation%, the same below), dropped to 7% on 3th day, WBC counts decreased to a minimum counts of 3% on 11th day,and began to recover on 14th day, but the recovery speed of WBC is slow, which recovered to 40% on the 21th day after irradiation, restored to 68.9% on the 24th day. The hemoglobin (HGB) decrease slowly, which decreased to 87.8% on 7th day after irradiation,then to a minimum counts (49.9%) on 17th day,it,s recovery speed is fast,HGB began to recover to 72.3% on 21th day,and has been restored to 89.8% on 28th day after irradiation. The PLT counts on 1th day after irradiation decreased to 70.5%, 53.1% on 3th day, fell to 20.4% on the 7th day, and decreased to a minimum counts(8%) on 14th day after irradiation, it began to recover on the 17th day,which has been restored to 60.4% on 28th day after irradiation.The pathological morphology on 14th day after irradiation showed pathological femur bone marrow cavity was empty state, only to see the few remaining blood cells, filled almost entirely by fat tissue,the hematopoietic function of bone marrow pathology gradually returned to normal on 28th day after irradiation. No mice died during the experiment, no diarrhea, convulsions was obeserved,which indicating that the establishment of bone marrow-type ARS mouse model was successful.2. The effect of rmIL-12 on hemopoiesis in mice of acute radiation sickness.2.1 rmIL-12 experiments with different administration times: the generally conditions in rmIL-12 treated ARS mice were improved.rmIL-12 administrated before irradiation, after irradiation or comibination between pre-irradiation and post-irradiation all promoted the recovery of PLT counts in mice of ARS, rmIL-12 administrated before irradiation and combination between pre-irradiation and post-irradiation promoted the recovery of WBC, there are no significant difference between the post-irradiation group and irradiation control group (P>0.05), the HGB nadir of rmIL-12 treated groups was higher than that of the control irradiation group (P<0.05), and the recovery speed of HGB is faster than that of the control group.2.2 rmIL-12 experiments with different administration doses: the promotion role of rmIL-12 in PLT recovery did not increased as the dose increased. when the dose of rmIL-12 was 20μg/kg and 40μg/kg,the PLT recovery speed is faster than other doses groups,and all doses groups were statistically significantly effective than the control group (P <0.05).The WBC in rmIL-12 5,20,60μg/kg treated groups on 21th day after exposure were significantly different than that of the control group(P <0.05),and the WBC in all rmIL-12 different doses groups on 24 day after exposure were significantly different than that of the control group.The HGB nadir of all dose groups was significantly higer than that of the control groups(P<0.05). Instead, the initial decline speed of HGB in terms of 20 and 40μg/kg groups was faster than the control group(P<0.05).2.3 rmIL-12 experiments combined with G-CSF: the PLT recovery in combined treatment group were faster than that of merely G-CSF or rmIL-12 20μg/kg treatment group,which is most obvious on 17th day after exposure. The WBC recovery time of combined treatment group was started as the same time as that of G-CSF group,and faster than the single rmIL-12 20μg/kg treatment group.There were significantly difference between all three treatment groups with the control group(P<0.05).The HGB nadirs of combined treatment group and single G-CSF group were higer than that of the control group(P<0.05),and the recovery speed was faster than that of the control group.Instead,the HGB decline speed of single rmIL-12 20μg/kg treatment group was faster than that of the control group,but the nadir was no significantly difference,the HGB recovery speed was slowly than the control group.2.4 rmIL-12 compared with rmTPO experiments: the PLT nadirs in single rmIL-12 treatment group and rmTPO treatment group were significantly higer than that of the control group,and no significant difference between the two groups (P>0.05).In the PLT recovery process,the PLT counts on 21th,24th,28th were significantly higer than those of rmTPO treatment group(P<0.05),and the two treatment groups were significantly different than the control group(P<0.05).2.5 The morphological changes in ARS mice after rmIL-12 treated suggested that hematopoiesis in mice treated with rmIL-12 was better than that of control irradiation group. And the results of bone marrow cell colony culture also showed that rmIL-12 could promote bone marrow cells to form more CFU-Mix.Conclusion1. ARS mouse model was successfully established after healthy BALB/C male mice were given 6.0Gy60Coγ-rays whole body irradiation, which provided an ideal platform for the drug efficacy trials, MSC and HSCT treatment for the ARS.2. Different administration time schedules,different doses of rmIL-12 could promote the recovery of WBC and PLT in mice of ARS, especially in the aspects of PLT, and it's role in promoting PLT recovery was equal or better than the efficacy of TPO,which have recognized role of promoting platelet recovery, but it's role in promoting recovery of WBC was weaker than the current widely used G-CSF. Different doses of rmIL-12 had different effects in the recovery of HGB, 5,10,60μg/kg doses group were better than the irradiated control group,while 20,40μg/kg doses group were worse than the control group. Collaborative application with rmIL-12 and G-CSF had not better promote the recovery of WBC, HGB, and PLT.3. Histomorphological changes in bone marrow showed that hematopoietic recovery in the mice of ARS treated with rmIL-12 were better than those in the mice of irradiated control group, Semi-solid bone marrow cell culture also demonstrated that rmIL-12 could stimulate bone marrow cells to form more CFU-Mix than those of the irradiation group in vitro.4. This experiment suggested that rmIL-12 could promote the recovery of hematopoietic system in mice of ARS, and provide experimental basis for the IL-12 used in the treatment of ARS. |
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