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With the universal application of nuclear energy in the military field and in civilian areas, the huge benefit of mankind, but also to the health and safety of the public brought severe challenges. Explosion of the atomic bomb in Hiroshima and Nagasaki, Japan in 1954, former Soviet Chernobyl nuclear power plant accident, and, more recently, in Japan's Fukushima Daiichi nuclear power plant accident in 1986 caused a major disaster, all human and enormous social impact . China is a nuclear power, has a large number of nuclear bombs, strategic nuclear weapons, nuclear-powered ships in the military also has more than a dozen nuclear power plants in the civilian aspects, from 1991 of a nuclear power plant in Qinshan grid, there Qinshan, Zhejiang, Guangdong Daya Bay / Ridge Australia and Jiangsu Tianwan power station for a total of 11 nuclear power generating units put into commercial operation, with a total installed capacity of 9.12 million kilowatts, is expected to total amount of nuclear power in China will build 40 million kilowatts in 2020, nuclear power capacity will reach 150 million kilowatts in 2035, these the public the means radiation workers and nuclear facilities surrounding coverage will increase significantly, we are also faced with the test of the nuclear safety aspects. However, wounded in the treatment of high-dose irradiated nuclear accident, the most successful method is hematopoietic stem cell transplantation, but this method, the source of the difficulties and the long-term effects are not ideal hematopoietic stem cells with low. Develop a easy to produce, easy to use, the therapeutic effect of radiation injury treatment drugs or new ways of technology as a strategic reserve imminent. In recent years, foreign scholars in the study Toll-like receptor agonist, achieved incredible discovery. Such as Burdelya LG Science magazine in 2008 reported to the improved Toll like receptor 5 (TLR5) agonist (a Salmonella the hair proteins) activate TLR5 signaling molecules with strong mitigate the effects of radiation damage, and found that even in the experiment. 2 hours after irradiation administration of drugs is still better mitigate the effect of radiation damage that TLR5 agonist-mediated reduction of radiation damage through the activation of NF-κB (nuclear factor kappa B). This prompted the Toll-like receptor family members (Toll Like Receptors TLRs) agonists in the treatment of radiation damage have a wide range of research. Burdelya LG discovery undoubtedly opened up a new field for the study of radiation damage therapeutic agent. The TLRs family members belong to more conservative evolution of pattern recognition receptors (Pattern Recognition Receptor, PRR) be able to identify the pathogenic microorganisms associated molecular patterns (Pathogen Associated Molecular Patterns, PAMPs). Toll protein is by Hashimoto et al in 1988 for the first time found in Drosophila, followed by Jules A Hoffmann found that the Drosophila Toll protein mediated innate immunity, especially fungal infections can be suppressed Drosophila can feel the invading pathogens. Drosophila secretion of a variety of anti-microbial infection the polypeptide to clear pathogens. 1997 Medzhitov by homology analysis in the human body for the first time isolated homologs of the Drosophila Toll protein, called the human Toll protein. Opened the prelude to the research of mammalian TLRs and immune system. To date, the immunologist found in mammals, 11 TLR, and were named for TLR1 to 11, they are type I transmembrane protein, the common structural features of the membrane with a leucine-rich repeat sequences, film within a period of structurally conserved regions, starting site for intracellular signal transduction. TLRs recognition PAMPs are a class of highly conserved molecular structures, these PAMPs molecular structures present in the pathogen within. TLRs PAMPs interact initiate the immune response, activate the body's natural immune system, There are also differences in different TLRs recognition PAMPs, such as TLR1, 2,4 and 6, the identification of pathogens lipid fraction; TLR5 protein molecules of the pathogen recognition: TLR3, 7, 8 and 9, a nucleic acid molecule of the pathogen recognition. A common view of most of the structure of the TLRs family members of, signal transduction pathways and biological activity similar, theoretical analysis that TLRs other family members may also have a therapeutic effect of radiation damage, even more significant effect. TLR9 the TLRs family members, PAMP agonists CpG-ODN. CpG-ODN is endocytosed into the cell, the cytoplasm of TLR9 interaction complex is formed, and by myeloid differentiation factor (myeloid differentiation factor 88, MyD88) dependent signaling pathway to activate the immune response. MyD88 and tumor necrosis factor receptor-associated factor 6 (the of TNF receptor associated factor TRAF6) activate NF-κB-inducing kinase, which in turn activate NF-κB inhibitor kinase, resulting in IκB (Inhibitor of NF-κB) phosphorylation, NF-κB released into the activation of target genes in the nucleus, gene transcription start cytokines; CpG-ODN also through other signaling pathways induced and granulocyte colony-stimulating factor (Granulocyte Colony-Stimulating Factor G-CSF) tumor necrosis factor (Tumor necrosis factor-alpha, TNF-α), interleukin-6 (Interleukin-6, IL-6) the high level of transcription and expression, such as cytokines. Like other TLR agonists, CpG-ODN in immune modulation therapy resistant pathogens play an important role, in addition to CpG-ODN also have an important role in the treatment of tumors in combination with tumor antigen can induce strong CD4 and CD8 , T cell responses and rapid generation of antigen-specific antibody, killing tumor cells. CpG-ODN in the treatment of radiation damage is still an unknown area worthy of in-depth exploration. The existing literature suggests that TLR9 agonist CpG-ODN with a series of important biologically active, theoretically should be very useful to speculate that these biologically active against radiation damage, based on: ① TLR9 and TLR5 high homology (not shown), and the TLR9-having TLR5 signal transduction pathway similar to the activation of NF-κB [5]; ② TLR9-agonist CpG-ODN can stimulate the immune cells secretion of G-CSF, TNF-alpha, IL-6, etc., these cytokines promote hematopoietic system damage repair, reduce radiation damage. In addition, TLR9 agonist CpG-ODN with TLR5 agonist bacterial flagellin comparison of its lower toxicity, easier to obtain, CpG-ODN of the literature has been used as immunomodulatory agents used clinically. In the future if we are able to confirm that the TLR9 agonist CpG-ODN also have an extremely important role in reducing radiation damage, drug treatment for radiation damage undoubtedly bring a new direction. Explore the issues around the role of TLR9 agonist CpG-ODN radiation injury treatment, research topics include: the design and preparation of CpG-ODN, and preliminary evaluation of its toxicity to cells and mouse bone marrow hematopoietic system; CpG-ODN radiation damage the therapeutic effect; CpG-ODN treatment of radiation injury of the small intestine tissue; CpG-ODN treatment of radiation damage mechanism. The final results show that CpG-ODN has a better therapeutic effect of radiation damage, has good prospects for Applied Research. synthesis take CpG-ODN, entrusted Sangon (Shanghai) Co., Ltd.. (2) cytotoxicity test: use HIEC cells, respectively, one-time given different doses the CpG-ODN2395 (final concentration of 0.02,0.04,0.08,0.15,0.30,0.60,1.25,2.50,5.00 μM) after 48h incubation MTT assay survival; (3) mice acute toxicity testing: selection of 20 2 2gBALB / c mice were given once mice (n = 8 / group) of different doses of CpG-ODN2395 (450,900 , 1350μg / only), and continuous observation of 14d. 2. CpG-ODN2395 therapeutic effects of radiation damage in bone marrow hematopoietic system (1) survival, dose - survival Linear intends combined LD 50/30 and the DRF: mice treated with different doses of ionizing radiation (6.0Gy, 6.5Gy 8Gy 10Gy) dose rate 1.0Gy/min give different treatment to observe the 30-day survival rate of the mice in each group, and calculates the different LD 50/30 and DRF value (the exposure dose group LD 50/30 with radiation alone group LD 50/30 's ratio); (2) white blood cells and bone marrow nucleated cell count: small mouse 6Gy according to give different treatment after irradiation 1d, 9d, 28d (representing the early polar phase, recovery) to take the mouse peripheral blood and bone marrow nucleated cells, by CASY cell count and analysis system records the number of cells; ( 3) bone marrow HE staining: mouse 6Gy after irradiation to give different treatment, according to the day after, 9d, 28d mice femur fixed, decalcified, ultimately made into HE staining; (4) exogenous spleen colony formation The unit of measurement: reference to Till and McCulloch reported in the 1961 classic method; CpG-ODN2395 intestinal tissue radiation injury treatment effect (1) HE staining of the small intestine: mice 6Gy irradiation given different treatment, 5d take small intestine after irradiation samples with formalin-fixed, and ultimately made the HE sections; (2) apoptotic crypt cells in the small intestine in situ apoptosis: irradiation of mice 6Gy will be given different treatment of small intestine 5h after irradiation, the sample used in the sexual formalin-fixed, eventually made TUNEL slice; (3) BrdU crypt cell proliferation in the small intestine: mice 6Gy irradiation after giving different treatment in the days after irradiation of small intestine, small intestine before 2h intraperitoneal injection of BrdU (2.5mg / only), the samples with neutral formalin-fixed, and eventually made BrdU immunohistochemistry slice; (4) micronucleated cells: HIEC given different treatment according cytochalasin B (CB), and incubated for 36h After the cells were fixed, DAPI staining, counting micronucleated cells (the micronucleus Cell frequency MNCF); CpG-ODN2395 alleviate the mechanism of action of the radiation damage (1) cells, NF-κB activation: Western assay cells the NF-κB inhibiting factor IκB-α and NF-kappa B subunit p65 changes, and quantitative analysis of proteins by Gene Tools software. (2) blood oxidative stress indicators: mice 6Gy irradiation after giving different treatment after irradiation 2d, 4d, 7d the mice plasma to detect changes in plasma MDA, GSH and SOD; (3) the small intestine SOD given different immunohistochemical slice: mice 6Gy irradiation after 5h take small intestine after irradiation with neutral formalin-fixed, and eventually made SOD immunohistochemistry slice; (4) cytokine content: mice given CpG the-ODN2395 0.5,1,2,4,8,24,48 h after mice plasma, the by ELASA plasma G-CSF, TNF-α and IL-6 changes. 5 by SPSS13.0 statistical analysis of experimental data. The 1. CpG-ODN2395 design and preparation of the experimental results, and preliminary evaluation of its toxic to cells and mice (1) The subject chosen type CpG-ODN CpG-ODN2395 composed of a total of 22 nucleotide sequence. Its sequence is: 5-TCGTCGTTTTCGGCGCGCGCCG-3. And part of the base phosphorothioate (blackbody underlined base phosphorothioate, other bases for phosphorylation). (2) Cytotoxicity test: HIEC cell culture solution was added with different concentrations of CpG-ODN2395 (0μM 5 .00 μM), found that the CpG-ODN2395 of low concentration on the cell survival by MTT assay did not affect the cell survival only appears when the concentration of up to 2.5μM reduce the the the CpG-ODN2395 concentration used in the test was 0.1 μm, far below the cytotoxic doses, it can be considered the the CpG-ODN2395 cells are safe or few toxic side effects; (3) animal acute toxicity test [17]: mice were given high doses of CpG-ODN2395 (450,900,1350 μg / only) to observe the 14d found no mouse weight loss and death, and the trial CpG-ODN dose mice were given only 50μg / PCS, far lower than the dose in the acute toxicity test, it can be considered the CpG-ODN2395 is little or no acute toxicity. 2. CpG-ODN2395 treatment of mouse bone marrow hematopoietic system radiation injury (1) after different doses of irradiation, the survival rate of mice: mice were given 6.0Gy, 6.5Gy, 8.0Gy and 10Gy irradiation, the survival rate of the radiation alone group 95%, 58%, 35% and 0%, respectively; irradiation dose group in the survival rate of 100%, 89%, 81% and 25%. Dose - survival fitting a straight line calculated the pure radiation group LD 50/30 7.6Gy, irradiation dose group LD 50/30 9.0Gy, DRF value 1.2; (2) peripheral blood leukocytes and bone marrow nucleated cell count: 6.0Gy irradiation 1d (early), 9d (very period), 28d (recovery) detection in peripheral blood leukocytes and bone marrow nucleated cells and found that irradiation to the drugs set of peripheral leukocytes and bone marrow nucleated cells after irradiation 1d, 9d and 28d than radiation alone group, especially after irradiation 9d and 28d statistics, is particularly significant; (3) bone marrow HE staining: 6.0Gy irradiation after irradiation dose group with the radiation alone group compared with the lighter bone marrow damage, faster recovery of hematopoietic function; slice bone marrow nucleated cell count results coincide; (4) exogenous spleen colony-forming unit assay: by spleen nodular quantity reflects the content of pluripotent hematopoietic stem cells in the bone marrow, the experimental results show that the number of the spleen nodules formed by irradiation administered bone marrow cells in the recipient mice is 2.1 times the radiation alone group; 3. CpG-ODN2395 mice role of intestinal tissue radiation injury treatment (1) HE staining of the small intestine: 6.0Gy irradiation 5d, HE staining of the small intestine, according to the pathological changes, found that the integrity of the intestinal tissue morphology of irradiation dose group than in the radiation alone group; (2) small intestine TUNEL implicit nest apoptotic cells in situ: 6.0Gy irradiation after 5h, TUNEL in situ apoptosis staining and quantitative irradiation dose group of the small intestine crypt cells and epithelial cells positive rate is lower than the radiation alone group; (3) small intestine BrdU cell proliferation assay: 6.0Gy irradiation 3d, slice observation and quantitative immunohistochemistry of BrdU cell proliferation irradiation dose group of intestinal crypt cells positive rate is higher than the radiation alone group. (4) micronucleated cells: count HIEC cells in the formation rate of micronucleus cells after different doses of radiation, results showed that irradiation dose group can significantly reduce the incidence of micronucleated cells; 4 the CpG-ODN2395 radiation injury possible mechanism of the protective effect (1 ) cells, NF-κB activation: the content of IκB-α, NF-κB activation inhibitory molecules the CpG-ODN2395 added after irradiation appeared to significantly lower; the content of the a subunit of NF-κB molecule of p65 protein molecules significantly increased. After irradiation 30min; (2) changes in the level of oxidative stress in the blood: after irradiation, 2d, 4d and 7d plasma oxidative stress indicators (MDA, GSH, SOD), results showed that irradiation dose group blood system oxidative stress level is lower than the radiation alone group; (3) small intestine SOD immunohistochemistry slice: after irradiation, 5h, SOD immunohistochemical staining and quantitative found higher than irradiation, irradiation dose group of small intestinal tissue SOD content group. (4) changes in cytokine content: injection the CpG-ODN2395 within hours after can induce blood cell factor, G-CSF, TNF-α and IL-6 levels increased. Experimental discuss radiation injury treatment has been the problems facing biology and medicine, in many countries of the world have invested heavily in research in radiation protection drugs, but so far, only a radioprotector WR2721 American FBA approved to enter clinical as preventive protection measures in radiotherapy in the application, but its side effects. Therefore, good way so far there is no special treatment in the treatment of large doses of radiation damage, rely mainly on hematopoietic stem cell transplantation and injection of a large number of hematopoietic growth factors. In recent years, immunologists found that there is an important part of the innate immune system: Toll-like receptor family. Studies have shown that TLRs signaling pathways can be dependent on MyD88 pathway activation of NF-? B or stimulate immune cells release certain cytokines, which inhibit apoptosis and promote cell proliferation, scavenging free radicals. TLR5 agonists such as Burdelya LG Science magazine in 2008 first reported improvement the strong radiation protective effects, and even two hours after irradiation, the administration still has a strong mitigating the effects of radiation damage. Prompted the the TLRs family agonists members have a wide range of research in the treatment of radiation damage prospects [2]. CpG-ODN TLR9 agonist artificial analog bacterial DNA synthetic oligonucleotide molecules having the activity of bacterial DNA, can stimulate the immune response induced by the activation of the immune system in mammals oligonucleotide fragments. As early as in the 1890s, people discovered the cancer patients were injected with bacterial extract can significantly alleviate the condition, but later found that bacterial DNA composition is mainly mediated by the substance of this anti-tumor effect, further findings affirmed the bacterial DNA has a direct immune stimulation and anti-tumor effects [18] [19]. Depending on the structure of CpG-ODN and immune activation mainly divided into three types: type A CpG-ODN such as ODN2216, its structural characteristics of ODN 3 and 5, the end of the sequence of the poly-G, in addition to both ends from a small amount of phosphorothioate, and the remaining major phosphorylation modification, can induce IFN-α released from the DC (dendritic cell), thereby activating the NK cells and T cells, but a weak ability to activate B cells. Type B CpG-ODN, such as ODN2006, its nucleotide soley by thio modified, is the activation of Th1 cells and adjuvant and a strong role in the stimulation of B cell activation, but weaker stimulus pDCs produce IFN-α. C-type CpG-ODN ODN2395, part of the modified base by thio modification, both of the first two immune activity, and in vivo stability [6] In the experimental study, we preferred type C and select with the typical representatives the CpG-ODN2395 as the main object of study of this topic. We found that after irradiation in mice given CpG-ODN2395, higher survival rate, alleviate bone marrow hematopoietic system and the degree of acute radiation injury of the small intestine, which show that of CpG-ODN2395 on radiation damage with good therapeutic effect. Although the mechanism of the therapeutic effect of radiation damage is still not entirely clear, however, we find that given CpG-ODN activation of NF-κB in cells after irradiation can inhibit the expression of molecules IκB-α content more significantly reduced NF-κB molecule subunit of the p65 protein molecule content corresponding to more elevated; micronucleus cell number is reduced accordingly; observation of MDA, GSH, SOD and oxidative stress indicators found in the blood system and intestinal tissue irradiation dose group animals oxidative stress level is lower than the radiation alone group; blood of G-CSF, TNF-α and IL-6 cytokine content after injection of CpG-ODN2395 few hours was significantly elevated. These experimental results show that the mechanism the CpG-ODN2395 mitigate radiation damage may activate NF-κB, improve the body's antioxidant ability and the promotion of G-CSF, TNF-α and IL-6 secretion of cytokines with hematopoietic and immune function. In addition, reported in the literature CpG-ODN as immunomodulatory agents for clinical use, the research of the CpG-ODN2395 preliminary toxicity analysis display no obvious acute toxicity, suggesting that the TLR9 agonist CpG-ODN in clinical applications of radiation damage in the future may There are good prospects for development.
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