Studies On Anti-radiation Activities And Its Mechanisms Of IL-12

To study the effects of rmIL-12on survival and bone marrow hematopoieticfunction of gamma-ray irradiated mice. And the effect of rmIL-12on irradiated mousebone marrow mesenchymal stem cells survival in vitro. We randomly divided the miceinto five groups according to weight, irradiation control group, positive control groupand rmIL-12treatment group （200ng/mouse,100ng/mouse,50ng/mouse）. One hundredmice, twenty mice in each group （male:female=1:1）, were used to study the survival rateafter total body irradiation （TBI） in8.0Gy and fifty female mice, ten mice in each group,were used to study the effect of hematopoietic function after TBI in4.0Gy. Alladministrated24h and1h before the delivery of irradiation time, after irradiation ofmedication once a day, total7days, administration routes for subcutaneous injection,and observed the protection effect of survival in mice bone marrow mesenchymal stemcells （MSCs） cultured in vitro in different irradiation doses. In⁶⁰Co gamma-rayradiation dose for8.0Gy, after TBI in mice, rmIL-12can be obviously increasedsurvival rate, and lifted and right-shifted its survival curve （Kaplan-meier curve）. In⁶⁰Co gamma-ray radiation dose for4.0Gy. The numbers of white blood cells, plateletsand reticulocytes in peripheral blood were increased and recovered in advance byrmIL-12, and the contents of DNA in bone marrow were obviously higher than which incontrol group. And meanwhile, the final concentration of rmIL-12was between0.125ng/mL and32ng/mL, we observed protection effect of the MSCs survival indifferent doses in vitro.Twenty rhesus monkeys are divided into four groups. In each group, there wereequal diffenent gender animals. Two animals were in normal control group, anothereighteen animals were distributed averagely into irradiation control group, low dosegroup and high dose group. The last two groups were administrated with rhIL-1224h and1h before irradiation, and then treatment once a day after irradiation, total9days,administration routes for subcutaneous injection.⁶⁰Co irradiation dose was3.0Gy. Afterirradiation of animals, index changes of the blood system in accordance with the generalperformance characteristics of bone marrow form of radiation sickness, mainlyexpressed as white blood cells count, red blood cells count and platelet counts down.We did not observe that rhIL-12inhibited three-line cell count dropped or deterred, highdose group even increased. But in the hemogram recovery phase, white cell countcurves, high dose group was higher than the control group. From reticulocyte countcurves and platelet count curves, high and low dose group were all higher than thecontrol group. In the latter part of the experimental analysis of lymphocyte distortion,lymphocytes distortion rate in low dose group was significantly lower than radiationcontrol group of animals’.rmIL-12can increase survival rate and accelerate the recovery of hematopieticfunction after total body irradiation （TBI） in mice, and also has the protective effect inthe survival of vitro cultured MSCs. In⁶⁰Co preliminary study on radiation-inducedacute irradiation sickness of rhesus monkeys, administrating rhIL-12in the recovery ofbone marrow hematopoietic function played an important role in promotive effect, andon the lymphocytes repair of the genome it also takes a positive role in recovering. To investigate the anti-tumor pharmacodynamics evaluation of ACT-001on C6glioma model in situ and relative functional mechanism initially. The anti-tumor activityof ACT-001to C6and U87MG in vitro was inspected by MTT method, compared withthe two strains in different concentrations, the effect of cell cycle and apoptosis in FCM.Using the establishment of C6glioma model in situ, we assayed the research ofanti-tumor effect in different concentrations, Observed tumor-bearing rats dailybehavior, body weight and survival and HE staining to pathologic change. Finally usingRT-PCR detected C6cells and tumor-related genes in Nestin and apoptosis in rats’brain gene expression of Bax and Bcl-2.The replicated MTT results of each cell showed ACT-001a optimal dose relatedeffect, which was on the subjected cell growth inhibition effect of C6and U87MG. Thedesigned concentration of essential drugs covered the potency windows. Half of theinhibitory concentration （IC50） ranged27.2²8.6μM and20.6²7.9μM, respectively.Flow Cytometry analysed, as ACT-001effects of the concentration increased, the C6and U87MG cell apoptosis rate has gradually increased, while induced cell cycleparameter G0/G1and G2/M blocked of C6compared with control cells. But there wereno difference in different concentrations and no obvious influence is to U87MG cell. Invivo experimental results showed that ACT-001inhibited the growth of the C6cells inrats’ brain. In the first experiment （80mg/kg and160mg/kg） the inhibition ratios ofACT-001were69.4%（P<0.01） and63.9%（P<0.01） respectively.5animals died inmodel control group. Meanwhile,80mg/kg group also died only1animal, and160mg/kg group in the late stage of experiment, animals’ weight also has reduced. It didnot display good dose-effect relationship. Therefore in the second test, the ACT-001dose was adjusted for25,50and100mg/kg, inhibition ratio was respectively for59.9% （P<0.01）,78.7%（P<0.001） and87.5%（P<0.001）.5animals died in model controlgroup, and in low dose group and middle dose group only1animal died, till toexperiment was over, the weight still has increased, both rendering better dose-effectrelationship, and increased animals survival rate （P<0.05）. HE staining results displayedtumor cell in rats’ cranial proliferated actively in control group. Edge invasionphenomenon was distinct. Nevertheless, the animals in the group of administratingACT-001relieved the symptoms. RT-PCR assayed that Quantity One software analysedthe gray scale, high expression of Nestin gene in tumor-bearing rats brain tissue, andlow expression of normal ones’. Different concentrations of ACT-001at the same timepromoted apoptosis gene Bax expression in C6cells and inhibited the expression ofBcl-2.ACT-001in vitro can inhibit C6cell and U87MG cell growth, promote cellapoptosis and induce the cell cycle changed. In vivo, we can obviously suppress C6cellin rats brain growing, the effect of100mg/kg group is the best. Simultaneously,animals’ survival time can extend and survival rate increase after administratingACT-001, did not produce drug toxicity. ACT-001can induce apoptosis gene Bax andBcl-2expression.