The Study Of γ-ray Induced Cell And Tissue Injury And Its Intervention Measures

Exposure to radiation can cause varying degrees of damage to various tissues and cells.In clinical,radiation-induced heart disease（RIHD）and radiation-induced lung injury（RILI）are common complications in patients with chest tumor after radiation therapy.There are still no effective therapeutic stratege for the prevention and treatment of these two diseases,and most patients have poor prognosis and poor quality of life.Bone marrow hematopoietic system is also prone to radiation damage due to its radiosensitivity.The study foucsed on the y ray-induced injuries of cardiomyocytes,mouse lung tissue and bone marrow cells.Different radiation injury models in vivo and in vitro were established,and protective effects and potential mechanism of multiple interventions such as gene therapy,stem cell and extracellular vesicles injection on radiation injury were evaluated.This research including the following three parts.Part ⅠObjective To investigate the mechanism and possible intervention measures of radiation-induced cardiomyocyte necrotic apoptosis and mitochondrial damage.Methods Human and rat cardiomyocytes（iPSC-CMs and H9C2）were irradiated with 60Coγ rays at different doses.Western blotting and qPCR were used to detect the expression levels of markers related to necroptosis and mitochondrial injury of cardiomyocytes.The oxidative stress and mitochondrial damage of cardiomyocytes were detected by flow cytometry and laser confocal microscopy.Results The expression levels of RIP1 and RIP3,oxidative stress ROS,mitochondrial membrane potential and mPTP opening increased in iPSC-CMs cells after y ray irradiation.After treatment with RIP1 or ROS inhibitor,the ROS level decreased,and the mitochondrial membrane potential and mitochondrial permeability transition pore were maintained normal in irradiated iPSC-CMs cells.The expression of PTPMT1 in iPSC-CMs cells was decreased,and the expression of mitochondrial metabolism and division related proteins（AMPK,DRP1 and MFF）were abnormal in iPSC-CMs cells.Inhibition of PTPMT1 expression resulted in increased ROS levels,decreased mitochondrial membrane potential and open mitochondrial permeability transition pore in iPSC-CMs and H9C2 cells.Overexpression of PTPMT1 can reduce the ROS level,and inhibit the decrease of mitochondrial membrane potential and the opening of mitochondrial permeability transition pore of irradiated H9C2 cells.Conclusion γ rays can cause necroptosis,oxidative stress and mitochondrial damage in iPSC-CM.RIP1 or ROS inhibitor can protect cells from radiation induced damage.Rradiation can cause mitochondrial dysfunction and abnormal expression of related proteins in iPSC-CMs.PTPMT1 maybe involved in process of oxidative stress,mitochondrial damage and necroptosis in radiated iPSC-CMs and H9C2 cells.Part ⅡObjective To explore the effect and mechanism of Decorin modified-mesenchymal stem cells on radiation induced lung injury.Methods C57BL/6 mice were locally irradiated with 20Gy of 60Coγ ray to establish RILI model and MSCs were administrated via tail vein.HE and Masson staining were used to observe the lung structure and morphology of RILI mice.Immunohistochemistry and ELISA were used to detect markers of lung tissue injury.The changes of Treg cells of spleen were detected by flow cytometry.Multiple immunofluorescence was used to study the changes of local immune microenvironment in lung tissue.Results MSCs could inhibit the early inflammatory changes and late collagen fiber deposition in the lung tissues of mice.MSCs treatment could reduce the exudation of ALB and ALP in alveolar lavage fluid,decrease the expression of oxidative stress and hypoxia markers（8-OHDG and CA-9）in lung tissue,and inhibit the expression of apoptosis marker caspase3.MSCs could reduce the proportion of Treg cells in the spleen,and reduce the ratio of M1 to M2 macrophages and Th1 to Th2 lymphocytes in lung tissues.Conclusion MSCs can alleviate the radiation-induced lung injury and maintain the normal structure of lung tissue through inhibit irradiation-induced apoptosis,maintain normal permeability of alveolar vascular wall,and reduce oxidative stress and hypoxia.MSCs can regulate local and systemic immune response to inhibit the progression of inflammation and fibrosis.Decorin gene modification could enhance the preventive effect of MSCs.Part ⅢObjective To investigate the protective effect and mechanism of extracellular vesicles secreted from DPSCs on radiation-induced apoptosis of mouse bone marrow cells.Methods Mouse bone marrow cells（FDC-P1）were radiated with 60Coγ ray at different doses.The apoptosis level of FDC-P1 cells was detected by flow cytometry and the expression of apoptosis markers were detected by Western blotting.DPSCs-EVs were isolated by ultracentrifugation,and identified by Western blotting and NTA.The expression level of miRNA in bone marrow cells was detected by qPCR.Inhibitor or mimic was used to knocked down or overexpressed the expression of target miRNA.Results After radiation,the proportion of apoptotic cells of FDC-Plwas significantly increased,and the relative expression levels of cleaved caspase3 and cleaved PARP were significantly upregulated.DPSC-EVs treatment could reduced the percentage of apoptotic cells and the expression levels of cleaved caspase3 and cleaved PARP significantlywhen compared with the radiation group.The expression level of miR-1005p was significantly decreased in radiation group while increased in DPSC-EVs group.Cells with miR-100-5p knockdown were irradiated with 2Gy and then added with DPSC-EVs,the apoptosis ratio was significantly increased;After 2Gy γ ray radiation,the apoptosis level of miR-100-5p overexpressed cells was significantly reduced.Conclusion γray induced apoptosis of mouse bone marrow cells FDC-P1,and DPSCEVS intervention significantly reduced the level of apoptosis.The anti-apoptosis protective role of DPSC-EVS maybe mediated by up-regulating the expression level of miR-100-5p in FDC-P1.