| Objective: Chest radioactive treatments often lead to radiation-induced lung diseases, such as acute radiation-induced pneumonitis and fibrosis and other pulmonary radiation injury, severe cases, respiratory failure. There is no particularly effective treatment approach, therefore, concentrated its research and protection mechanisms and measures, this study in the establishment of radiation injury in the rat lung model, based on CT imaging combined with pathological means testing , immunohistochemistry, Western blotting techniques, from various angles, multi-level analysis and observation of radiation injury in rat lung and the relationship between radiation dose and the occurrence of radiation injury of the lung and development of mechanisms, in particular concerned about the whole process of radiation-induced lung injury of vascular endothelial the case of damage and repair; and further the use of human umbilical vein endothelial cells to establish in vitro model of radiation injury, observation of ionizing radiation on endothelial cells in vitro effect and mechanism of injury, in particular radioactive induced vascular endothelial cell apoptosis; and in vitro the basis of experiments in vivo, design of the tea polyphenols in vivo, vascular endothelial cells outside the protection of the experimental study of radiation injury, comprehensive observation and analysis of tea polyphenols on radiation-induced lung injury and mechanisms of intervention effects. Methods: The purpose of this study, this study is divided into three parts, the first is radioactive rat lung injury model and a multi-level research, the second part of the radiation damage of vascular endothelial cell apoptosis in vitro studies, the third part of the tea polyphenols on radiation injury in the experimental intervention; Among them, the first part of the radioactive rat lung injury model and a multi-level observational study of experimental methods include: the use of machine generated by the use of 60Coγ-ray line unilateral right lung irradiation, dose of 7.0 Gy, 14.4Gy, observed for the irradiation point 1d, 7 d (1 weeks), 30 d (1 mon), 90d (3 months), a total of four time-point, taking the control group and the irradiated rat lung organizations related to testing, including rat lung radiation injury①pathological observations, including the whole lesion, HE staining under the light microscope and electron microscope changes in the micro-changes,②pulmonary fibrosis changes include: spectrophotometric method with acid determination in lung tissue hydroxyproline proline concentration was detected by immunohistochemistry the expression of TGF-β1,③observation of lung cell apoptosis using TUNEL assay,④pulmonary vascular endothelial cell markers CD34, CD105 and VEGF expression levels detected using Western blotting,⑤lung inflammation TNF-αfactor detected by immunohistochemistry. The second part of radiation damage of vascular endothelial cell apoptosis in vitro, using human umbilical vein endothelial cells in vitro 60Coγ-ray irradiation, a dose of 7.0 Gy, 14.4Gy and 30.0Gy, detection of apoptosis indicators include:①vascular endothelial cell DNA injuries "comet" experiment,②the rate of vascular endothelial cell apoptosis using Annexin V / PI staining by flow cytometry after,③vascular endothelial cells of Caspase-3 activity and cellular changes in mitochondrial membrane potential using flow cytometry. The third part is the effects of tea polyphenols on radiation injury in the experimental intervention, the use of radiation dose 14.4Gy lung injury model in rats and in vitro human umbilical vein endothelial cell apoptosis model of 500mg/kg dose, respectively, the use of tea polyphenols in vivo intervention and 10μg / L, 100μg / L, 1000μg / L three orders of magnitude of the intervention of tea polyphenols in vitro, were used to detect apoptosis in vitro test①indicators: the use of Annexin V / PI staining, the rate of detection of vascular endothelial cell apoptosis and streaming cells, endothelial cells were used to detect Caspase-3 activity and mitochondrial membrane potential changes in vascular endothelial cells to reflect the changes of apoptosis;②vivo indicators: effects of tea polyphenols on radiation- induced pulmonary injury in rat serum SOD and MDA detection of the impact of the use of Colorimetry, radiation-induced pulmonary vascular effects of tea polyphenols on radiation injury in CD34, CD105 and VEGF expression changes detected by Western blotting method, effects of tea polyphenols on radiation- induced pulmonary injury in the detection of TNF expression by immunohistochemical method.Results: The first part of the radioactive rat lung injury model and a multi-level observation of the results of the study are as follows:①lung specimens changes the whole rat lung 7.0Gy group sheet no obvious significant bleeding and edema, and pulmonary 14.4Gy rats sheet can be seen bleeding and obvious edema and swelling.②Pathological changes in lung tissue: I.radiation early after 1d, 7d histopathological changes under light microscope: 7.0Gy with rats 14.4Gy between alveolar space and no significant exudation, and edema in congestive. II. medium-term 30 d after irradiation histopathological changes under light microscope: widened alveolar septal edema, and infiltration of inflammatory cells. III. late 90 d after irradiation, histopathological changes under light microscope: we can see real changes in lung tissue, alveolar septal further widened, and some alveolar space collapse, atelectasis, and bulla formation, it can be seen that the formation of spindle fibers from the cells fibroblastic lesions;③changes in pulmonary fibrosis related to indicators of changes in TGF-β1 for the 7.0 Gy irradiation group in 30d and 90d shows that the weak expression, 14.4 Gy group rat lung tissue TGF-β1 in the whole observation period showed a positive expression, 14.4 Gy group and the lungs in rats 7 d time point 1d and hydroxyl proline content changed little, with 14.4 Gy group 90d rat lung hydroxyl proline in the lungs were significantly higher than the normal control group;④cytokines main group for the 7.0 Gy each time pointin lung tissue expression of TNF-αbasic at a low level of expression or negative, lung tissue expression of TNF-αonly 7.0 Gy radiation group in 1d after the expression of positive, 14.4Gy group the TNF-αat all time points were positive.⑤pulmonary vascular endothelial cell markers CD34, CD105 and VEGF expression levels: 14.4 Gy group rats lungs do quantitative Western blot determination of CD34, CD105, was found after radiation injury in 1 day with the first 7 days, CD34, reduce the level of CD105 protein expression, compared with Day 1, radiation injury to the first 30 days after the first 90 days with no significant change in CD34, and CD105 may have a higher trend, but lower than the normal level group. In addition, the group also found that 14.4 Gy radiation lung injury in rats 1 day after the first 7 days, there are increased levels of VEGF, but the first 30 days and 90 days, reduce the level of VEGF and significantly lower than the level of the first 7 days . The second part of radiation damage of vascular endothelial cells in vitro apoptosis in the main results are as follows:①comet assay: With the increase in radiation dose, "comet" tail length, the cells showed a higher percentage increase in radiation dose group of the "comet "cell rate as well as the" comet "tail length were significantly higher than that;②after radiation injury of vascular endothelial cell apoptosis, in 7.0 Gy group, endothelial cell apoptosis positive rate of early apoptosis and late-positive rates were 16.5%±6.6% and 10.8%±5.1%, and 14.4 Gy group and 30.0 Gy group the positive rate of the rate of early apoptosis and late apoptosis-positive rate is significantly increased, namely 36.8%±8.2% and 16.8%±6.1% and 43.1%±6.2% and 18.2%±5.8%;③Caspase-3-positive rate and the mitochondrial injury rates were 1.6%±0.9% and 2.2%±0.8%, and 7.0 Gy in the group, Caspase-3-positive rate and mitochondrial damage rate: 9.9%±2.2% and 8.4%±1.8%,and 14.4 Gy group and 30.0 Gy group Caspase-3-positive rate and the mitochondrial injury rates were: 26.8%±4.6%,36.2%±4.1%, and 35.2%±3.6% and 38.3%±3.1%. The third part is the effects of tea polyphenols on radiation injury of experimental results of intervention,①vitro found that 10μg / L intervention group no significant change, 100μg / L intervention group and 1000μg / L in the early intervention group were apoptotic rate 23.1%±5.8 % and 20.2%±4.6%, significantly lower than 14.4 Gy radiation injury group, 1000μg / L the late intervention group positive rate of apoptosis 8.9%±2.6%, also significantly lower than 14.4 Gy radiation injury group, and 100μg / L intervention group and 1000μg / L intervention group of Caspase-3-positive rate and the mitochondrial injury rates were 23.2%±6.5% and 24.3%±4.8%, significantly lower than 14.4 Gy radiation injury group, 1000μg / L intervention group Caspase-3 positive rate and the mitochondrial injury rates were 16.2%±3.9% and 18.3%±5.8%, two indices were significantly lower than 14.4 Gy radiation injury group. To do further tests found the body,①tea polyphenols intervention, MDA levels decreased significantly 1d and 7d, and 90d to 30 d and there is a trend to lower; compared with the control group, 14.4 Gy level of SOD in rats significantly or 1d high, 7d are changed little, 30d and 90d were significantly reduced after the intervention in the tea polyphenol, SOD rats were significantly higher than the normal control group, which 7d, 30d and 90d are also significantly higher than 14.4 Gy rats SOD levels;②14.4 Gy group and the intervention of tea polyphenols in rats lungs do quantitative Western blot determination of CD34, CD105, VEGF expression levels and found that tea polyphenols intervention group and 14.4 Gy radiation injury of the expression levels between no significant change,③intervention by tea polyphenols, the lungs have a large number of TNF-positive phagocytic cells and inflammatory cells did not significantly reduce the trend remains positive.Conclusion:⑴Radiation-induced pulmonary injury shows time- and dose-dependent markedly,and is a dynamic process which involves in several cytokines. The radiation-induced injury and disorder of regeneration of vascular endothelial cells are the crucial factor of acute radiation-induced pneumonitis and fibrosis and other pulmonary radiation injury.⑵Radiation injury through mitochondrial pathway, as well as family Caspase-mediated apoptosis signal transduction induced by vascular endothelial cell apoptosis.⑶Tea polyphenols can increase the body's antioxidant capacity to reduce radiation-induced pulmonary injury, and have a certain resistance to radiation-induced pulmonary injury.
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