淋巴结组织和T细胞辐射损伤效应及机理的初步研究

发布时间：2018-11-04 17:53

【摘要】：目的和意义：本实验以小鼠淋巴结淋巴细胞和正常人淋巴母细胞作为研究对象，以6 Gy γ射线照射作为凋亡的诱发因素，探讨凋亡的发生及调控机理，以期能为淋巴细胞凋亡信号转导机制提出一些新的观点和见解，，为临床上肿瘤放疗患者和急性核事故病人放射线损伤的防治提供一条新的思路。材料和方法：本实验从整体和离体两部分进行。在整体实验中，用C57BL／6小鼠作为研究对象，于γ线照后不同时间点检测淋巴结组织和淋巴细胞损伤效应、淋巴细胞凋亡率和凋亡相关基因及蛋白的变化规律，为Bcl-X_L调控机理的进一步研究提供依据。在体外实验中，人AHH-1 T淋巴细胞经6 Cy γ线照射后在不同时间点分别进行细胞凋亡率、存活率、细胞内活性氧和线粒体跨膜电位的检测，然后用转基因细胞模型对Bcl-X_L调控凋亡的分子机制进行探讨。结果：(1)不同剂量γ线照射后，淋巴结组织严重受损，淋巴细胞显著减少，在6-12 Gy范围内，淋巴细胞死亡方式以凋亡为主，15-20 Gy范围内以凋亡和坏死为主；(2)6 Gy γ线照射后淋巴结淋巴细胞出现大量凋亡，抗凋亡基因及其蛋白Bcl-2表达下降，促凋亡基因及其蛋白Bax表达上升；(3)6 Gy γ线照射后AHH-1细胞出现大量凋亡，细胞内活性氧水平升高；(4)值得注意的是，用于检测线粒体跨膜电位的Rh123荧光强度在凋亡早期没有发生改变；(5)Bcl-X_L能有效抑制辐射诱发的T淋巴细胞活性氧产生及Caspase 3的切割活化。结论：(1)凋亡是中等和中等以上剂量辐射诱导淋巴结淋巴细胞减少的主要原因；(2)辐射诱导淋巴细胞发生凋亡与Bcl-2的表达增加、Bax表达减少和活性氧水平的升高有关；(3)用于检测线粒体跨膜电位的Rh123荧光强度在凋亡早期没有发生改变，有关机制尚需进一步研究；(4)Bcl-X_L过表达能通过抑制活性氧的产生以及凋亡执行蛋白Caspase 3的切割活化等方式有效抑制细胞凋亡的发生。
[Abstract]:Objective and significance: in this study, the lymphocytic cells of mouse lymph nodes and normal human lymphoblastic cells were used as the object of study, and 6 Gy 纬 -ray irradiation was used as the inducing factor of apoptosis, and the mechanism of the occurrence and regulation of apoptosis was discussed. In order to provide some new ideas for the mechanism of lymphocyte apoptosis signal transduction and provide a new idea for the prevention and treatment of radiation injury in patients with tumor radiotherapy and acute nuclear accident. Materials and methods: the experiment was carried out both in whole and in vitro. In the whole experiment, C57BL/6 mice were used as the research objects. At different time points after 纬 -ray irradiation, the damage effect of lymph nodes and lymphocytes, the apoptosis rate of lymphocytes and the changes of apoptosis-related genes and proteins were detected. It provides the basis for the further study of the regulation mechanism of Bcl-X_L. In vitro, the apoptosis rate, survival rate, intracellular reactive oxygen species and mitochondrial transmembrane potential of human AHH-1 T lymphocytes were measured at different time points after irradiation with 6 Cy 纬 rays. Then the molecular mechanism of apoptosis regulated by Bcl-X_L was studied by using transgenic cell model. Results: (1) after different doses of 纬 -ray irradiation, lymph node tissues were severely damaged and lymphocytes were significantly decreased. In 6-12 Gy, apoptosis was the main mode of lymphocyte death, and apoptosis and necrosis were dominant in 15-20 Gy; (2) after 6 Gy 纬 -ray irradiation, a large number of apoptosis occurred in lymph node lymphocytes, the expression of anti-apoptotic gene and its protein Bcl-2 decreased, and the expression of pro-apoptotic gene and its protein Bax increased; (3) A large number of apoptosis was observed in AHH-1 cells after irradiation with 6 Gy 纬 rays, and the level of reactive oxygen species increased. (4) the fluorescence intensity of Rh123, which was used to detect mitochondrial transmembrane potential, did not change at the early stage of apoptosis. (5) Bcl-X_L could effectively inhibit the production of reactive oxygen species (Ros) and the cleavage of Caspase 3 in T lymphocytes induced by radiation. Conclusion: (1) apoptosis is the main cause of lymphocytopenia induced by moderate or higher doses of radiation. (2) the apoptosis of lymphocytes induced by radiation is related to the increase of Bcl-2 expression, the decrease of Bax expression and the increase of reactive oxygen species level. (3) the fluorescence intensity of Rh123 used to detect mitochondrial transmembrane potential did not change at the early stage of apoptosis. (4) overexpression of Bcl-X_L can effectively inhibit apoptosis by inhibiting the production of reactive oxygen species and the cleavage and activation of apoptotic executive protein Caspase 3.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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