| The delayed Haematopoiesis and immunologic reconstitution are two major causes of death of acute radiation sydrom(ARS). A Concerted Action called"Medical Treatment Protocols for Radiation Accident Victims as a Basis for a Computerised Guidance System", in short METREPOL, was accepted within the framework of the Nuclear Fission Safety Program (DG XII Science) of the European Atomic Energy Community on October 25–27, 2005 METREPOL identifies 1–4 response categories (RC) based on the grading of the effects observed in the four most important organ systems N (neurovascular system), H (haematopoietic system), C (cutaneous system) and G (gastrointestinal system). 1 is defined as mild damage means Autologous recovery certain and 4 as very severe damage means Autologous recovery most unlikely. According to METREPOL grading system, H1 patients can be followed on an outpatient basis. H2 to H4 patients needing maximum medical effort to be rescued. Because of the it's severe complications such as graft verse host disease (GVHD) and delayed immunologic reconstitution there was consensus that allogeneic haemopoietic stem cell transplantation(HSCT )using BM,mobilized peripheral blood (PB) and cord blood (CB) cells should not be done on any radiation accident victim with the potential of endogeneous haematopoietic recovery,it only be recommended in radiation casualties exhibiting no signs of autologous hematopoietic recovery (i.e. exposed to 7 to 10 Gy),and who are not affected by severe trauma, burns and GI syndrome, and also have an appropriate donor. For the patients whose autologous haematopoiesis recovery is likely(H2) and those whose autologous recovery is possible(H3) Supportive care, including blood transfusion,fluids and electrolytes administration,antibiotic and antiviral therapy remains the basis of medical management. Cytokine treatment including growth factor(GF) is recommended for moderate to severe myelosuppression .However, the higher the radiation dose, the weaker the efficacy of pure hematopoietic GFs, because there are too few residual HSCs left to respond tocytokine administration. So the seccessful management of this part of patients is the focus of our research.Apart of the general treatment , many researches attempt to use haemopoietic stem cells(HSCs) for supportive therapy. Harvesting of residual autologous haematopoietic stem cells for ex vivo expansion and transplantation was not considered a practical option. Allogeneic HSCs can serve as an alternative choice.But as we all know, radiation can heavily damage the patients'immune system, so the active allogeneic HSCs can easily become engraftmented and lead to severe complication such as GVHD. The occurrence of GVHD is closely related to engraftment proportion ,for the higher the engraftment proportion, the likeness happening of GVHD.So in our research we tried to find a way to reduces the HSCs multiplication activity, and make them have a transient and low proportion of engraftment to avoid GVHD but retain the ability to support the hemopoiesis.Compared with other ways, in vitro radiation served as the simplest and most effective way to reduce the HSCs'activity. In the first part of our research, we used a haplo-identical mouse model with male CB6F1 as the donor and femal BALB/c as the recipient. After mobilized with rh-GCSF, the donor's spleen HSCs were collected and exposed to different dose ofγ-radiation with a range from2.5Gy,5Gy,7.5Gy to 10Gy. 3x107 irradiated HSCs were transplanted into the sublethally irradiated(6Gy) recipient mice through intravenous infusion immediately after the radiation. We found that irradiated HSCs can speed up the recipients'hemopoiesis.. Significant hematopoietic supportive effect can be observed in the 2.5Gy irradiated HSCs, with 5Gy,7.5Gy irradiated HSCs to be a little inferior and 10Gy irradiated HSCs hardly had any obvious promotion. one month after HSCs infusion, we use flow cytometry and quantity real time quantitation PCR(QRT-PCR) to detect the donor cell engraftment in the recipients'peripheral blood. we found that 2.5Gy irradiated HSCs had a high proportion and stable engraftment in some of the recipients and led to chronic GVHD. But 5Gy irradiated HSCs can only forms a microchimerism in the recipients'peripheral blood with a level of 10-3-10-4 continuing for 2 to 4 months and finally disappeared. no incidence of GVHD could be observed. 7.5Gy,10Gy irradiated donor HSCs had no engraftment in the recipients'peripheral blood one month after the infusion and also no GVHD was observed in these two groups. The results hinted that 5Gy and 7.5Gy in vitro irradiated HSCs could enhance the autologous hematopoietic recover with transient donor cell engraftment without causing GVHD. Then we combined 5Gy and 7.5Gy irradiated HSCs with G-CSF to investigate whether they have synergistic effect together. Our observations suggested that G-CSF acts synergistically with 5Gy and 7.5Gy irradiated HSCs to shorten the period of marrow surpression.The combination of G-CSF with 5Gy irradiated HSCs protected 90% and 70% of recipient mice from sublathel(6Gy) and lathel(8Gy) radiation respectively, which is much better than any of them used separately. Using QRT-PCR we could detect microchimerism engraftment of 7.5Gy irradiated donor HSCs in the peripheral blood of the 8Gy irradiated recipients one month after the stem cell infusion and the microchimerism lasting for about 2-4months and finally donor cells been cleared from the recipients. The engraftment of 5Gy irradiated donor HSCs in the peripheral blood of the 8Gy irradiated recipients varied greatly from completely donor cell chimerism to microchimerism one month after the stem cell infusion. But the engraftment was unstable and declined with time.Donor engraftment can be eliminated from the recipients'peripheral blood 2-4months later when the autologous haematopoietic recovered.what is more,there is not any signs of caute or chronic GVHD could be detected in both cell therapy groups.so we further to investgate whether G-CSF combined with irradiated HSCs can protected recipient mice from superlathel dose(10Gy) of radiation.we found that only the 5Gy irradiated HSCs combined with G-CSF can protect 60% of the recipients from that high dose radiation at the cost of autologous haematopoiesis recovery. 5Gy irradiated HSCs formed completely stable donor cell chimerism in the recipients and some of the mice dead of cGVHD. In first part of our research we also assayed the colony forming ability of the irradiated HSCs in vitro and their homing and proliferating ability in vivo,and their potential mechanism.we also made a detailed discussion on the relationship between recipient immunosuppression,donor cell engraftment and the incidence of GVHD.In the second part of our research we focus on the effect of 5Gy irradiated HSCs combined with G-CSF on the immunological reconstitution of the sublathelly irradiated recipients.Our results sugested that combined cell therapy can modulate the subsets of thymocyte by promoting the double negative thymocytes multiplication and differentiation, reducing apoptosis of the double positive thymocytes and accelerating their differentiation to CD4/ CD8 single positve cells.Combined cell therapy can also have influence on the peripheral lymphocyte subsets and improve their function.we also found that the proportion of na?ve cell in the peripheral blood and the TRECs copies of the thymus was significant higher than control group which suggested that combined cell therapy can enhance the thymus immunological reconstitution by promoting thymus recent emigrat.Through the above experimental study, we found that certain dose of in vitro irradiation can partly reduces the allogeneic HSCs'activition and make them have a transient engraftment in lathelly or sublathelly irradiated recipients to support the recipient's own hematopoietic recover and restores the recipient's immunologic function without causing GVHD.Our resaerch provided a new method for ARS therapy.
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