| Background and objectiveThe hormesis effect and adaptive response of low dose radiation (LDR) received increasingly more attention since 1980s'. Hormesis effects include stimulation of growth of animal and plants, prolonging lifespan, reduce the chance of cancer, energizing lymph cells, enhancing immune system. Adaptive response means that the organism is subject to low dose radiation a certain period before high dose radiation. This will significantly reduce the damage from high dose radiation.In our experiment, we first let mouse subject to deep X-ray for low dose radiation. We discussed the effect of low dose radiation on hematopoiesis systems on several aspects: mRNA level, protein level, cell level. Results are:1. The amount of CFU-GM and BFU-E is significantly increased in hematopoiesis cells of the mouse exposed to radiation. 2. GM-CSF level increased in serum. 3. GM-CSF, G-CSF transcribe increased. The results showed that low dose radiation has hormesis effect on hematopoiesis systems. The strongest effect is recorded at 75mGy. This hormesis effect may come from the increase of the hematopoiesis-stimulating factor. We found that low dose radiation can not only stimulate the increase of hematopoiesis stem /progenitor cell in mesenchymal, also know as hormesis effect, but can also mobilize the additional stem /progenitor cells to peripheral blood system. The new discovery can generate a new method of mobilize peripheral stem/progenitor cells, which can be used in transplant of hematopoiesis stem cell and local injection on pathology part. The application in cure many malignant blood disease, other system malignant cancer, heredity/metabolize disease and autoimmune diseases is promising.Our work focused on the hormesis effect of hematopoiesis stem cells after LDR. Hematopoiesis system includes hematopoiesis stem cells and bone marrow stromal cells. With more research on the disease, we found that not all blood system disease can be cured by transplant of hematopoiesis stem cells, some of the disease require transplant of bone marrow stromal cells at the same time. Current research shows one kind of aplastic anema caused by over reaction from immunity function of T cell leading to bone marrow failure through apoptosis. The extremely strong T cell-mediated immunity not only damage hematopoiesis stem cell, but also damage the hematopoiesis microenvironment. The damage to bone marrow stromal cells will result in bone marrow stromal cells cease to secreate the growing factor needed in growth of hematopoiesis cells, which leads to the shortened lifespan of hematopoiesis stem cells. The collapse of microenvironment is one of the reasons the transplant of only hematopoiesis stem cells fails. For these kind of disease, the possibility of transplant of BMSCs simultaneously is worth further research.Mesenchymal stem cells can differentiate into bone marrow stromal cells. It is proved by many vitro experiments that mesenchymal stem cells in vitro can inhibit the proliferation of T lymphocyte responses. It is a good choice for those who simply hematopoietic stem cell transplantation failure patients to transplant hematopoietic stem cell at the same time infusion of mesenchymal stem cells. The advantages are MSC will not only rebuild damaged bone marrow stromal cells, but also too strong inhibition of T-lymphocyte proliferation.At present, low-dose radiation hormesis effect study focused on hematopoietic stem cells. There is not much LDR research on mesenchymal stem cells, another important part of bone marrow transplantation. We applied our experiences from LDR on hematopoietic stem cells to BMSCs research, observe the hormesis effect of BMSCs under LDR. If there is any, we study the mechanism on molecular level, and then we study the impact of LDR on the immune regulatory role of BMSCs. We are the first to study the hormesis effect of LDR on BMSCs and the impact of LDR on the immune regulatory role of BMSCs, thereby creating a new field of clinical application of low-dose radiation.MethodsI. Study mesenchymal stem cells in vitro culture and their biological characteristics. Mononuclear cells are obtained using density gradient centrifugation separation of fresh bone marrow cells. The mononuclear cells are cultured in low-sugar DMEM. The morphological appearances are observed under the microscope when the cells established a stable passage system. BMSCs growth curve is drawn using MTT. Flow cytometry is used to check expression. The quality and purity of cultivated BMSCs is evaluated. For functional testing, first the differentiation of mesenchymal stem cells is evaluated. Osteoblast induction: the osteoblast inducer contains fetal bovine serum (FBS) and basic fibroblast growth factor (BFGF); The inducer, together with dexamethasone, beta-glycerophosphate, 2-phosphate-L-ascorbic acid L-DMEM, is used to induce the cells for 3 weeks. Ethanol fixation, detection of calcium deposition using Von Kossa staining. For induction into fat: FBS solution, isobutyl-methylxanthine, indomethacin Sim, dexamethasone, insulin and high glucose DMEM. After 2 weeks, formalin is used for fixation and Oil red-"0"staining is applied. Second, the immune function of BMSCs is evaluated: observation of T-lymphocyte inhibition under the conditions of BMSCs as trophoblast cells. Select BMSCs after 3 stable passages, digest, spread on 96-well culture plate, and adjust cell concentration. Groups are set according to the ratio of BMSCs: T, three groups with 1: 20, 1: 10, and 1: 5 respectively. Subjects are placed in a 37℃5% CO2 incubator for 72 hours, wait until fully adherent, inject mitomycin 20ug/cell, keep 37℃for 30 minutes, remove and dispose supernatants. The BMSCs is tempered with mitomycin and then inoculated with T-lymphocytes by the above ratio. ConA is added to stimulate T-lymphocyte transformation. Evaluation is performed using MTT.II. Study the effects of low-dose radiation on biological characteristics of human bone marrow mesenchymal stem cell. Compare BMSCs growth curves from irradiation group and control group: obtain 3rd-8th generation of stable passaged cells, digest, spread on a 96-wcell culture plate, wait until cells adhere to the surface, then set control group and irradiation group, expose the irradiation group to 75mGy radiation, use MTT to evaluate at different time point. Use Double-antibody sandwich ABC-ELISA to evaluate the secreted SCF, GM-CSF, IL-6 and IL-11. Flow cytometry is used to evaluate the changes in cell cycle for the two groups. To study the effects of BMSCs irradiated group and control group on T-lymphocyte inhibition, MTT is used to evaluate groups with BMSCs: T ratio at 1: 20, 1: 10, and 1: 5 respectively.III. Study cell signal transduction of BMSCs after low-dose radiation. Use proteomic array to select phosphorylated protein and activated cyclins.Results1. In the original cell culture, log growth appeared in about 10 days, cells reached 90% confluent at 14 days. 2. the purity BMSCs with 3 passages and above reached over 98%. Phenotypic analysis showed that the percentage of CD34+ cells is at (0.7±0.1)%, CD45 cells at (0.4±0.2)%, CD166+ cells at (96.7±0.5)%, CD105+ cells at (97.0±0.2)%, CD29+ cells at (98.5±0.3)%, and CD44+ cells at (97.3±0.2)%. 3. the BMSCs had stable cell passages and could differentiate into adipocytes and osteoblasts when inducers is added to the culture. 4. BMSCs inhibition of T-lymphocyte proliferation analysis showed that there was apparent inhibition response when BMSCs: T ratio was set at 1: 10 and 1: 5. There was no difference between the two groups (P>0.05). 5. When mesenchymal stem cells were exposed to the low-dose radiation, a rapid growth peak appeared in the first 3-7 days of exposure. The cells achieved convergence 3 days earlier. There was a significant difference (P<0.05) between the two groups at this phase. 6. After 24 hours irradiation, cells began proliferating in advance. Percentage of S-phase cells in the irradiation was 7% higher than that of the control. 7. in the supernatants that was used to detect the irradiated group and the control group cultured 4-7 days, increased level of SCF, GM-CSF, and IL-11 were discovered in the irradiation group, while there were no difference in IL-6 level between the two groups. 8. comparison of the irradiation group and control group in the inhibition of T-lymphocyte transformation experiment showed that, when BMSCs: T=1: 20, the inhibition rate of T-lymphocytes reached over 50%, at the ratio of 1: 10 or 1: 5, inhibition rate was above 80% with no significant differences between the two groups. But between irradiated group and the control group, the inhibition of T lymphocytes proliferation is significantly stronger in irradiation group (P<0.05). 9. for the first it was discovered that after 24 hours of low-dose radiation, five proteins were phosphorylated and 8 cyclins increased.Conclusions1. This study for the first time discovers that LDR also has hormesis effect on BMSCs. After BMSCs was exposed to 24h of low dose radiation, cells expand more quickly, and cells reached confluent sooner. Percentage of S-phase and G2-phase cells in the irradiation was higher than that of the control.Using Proteomic Array method and exposing BMSCs to 24h of LDR, 5 proteins are shown to have phosphorylation and the level of 8 cyclins increases. This provides empirical evidence that LDR has hormesis effect.2. The mixed T-lymphocyte reaction suppressive effect of BMSCs obviously increased after BMSCs exposed to MLR.3. MLR can activize Rb/E2F-cyclinE signal transduction pathway, thus induce cells entering G1/S phase.4. MLR can activize CDK1-cyclinB1 compound, increase the expression of CDC25B, and accelerate cells entering G2/M phase.
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