PI3K/Akt/mTOR信号通路调控T细胞自噬在健脾补肾方抗辐射损伤中的作用和机制研究

Chinese medicine believes that "Jianpi Bushen " closely associated with body's hematopoietic and immune function,this law is the rule to formate the "Jianpi Bushen Prescription(JBP)" . The Wnt signaling pathway associated with radiation-damage hematopoiesis was screened by gene microarray on the basis of preliminary study, which up-regulated the expression of CyclinD1, inhibited PPARγ and promoted the proliferation of bone marrow hematopoietic stem cells by regulating the transduction of Wnt pathway. The prescription can reduce the injury of the spleen and enhance the immune function as well, but its mechanism is not clear. Immunity dysfunction of T cells after radiation is a key link in the induction of disease. Autophagy plays an important role in the immune process of the body, and T cell autophagy defects can affect the function of T cells, leading to the immune dysfunction of the body. On the basis of the previous work, we used transmission electron microscope to observe autophagosome and laser confocal microscopy to trace autophagy. RT-PCR, nuclease protection test, Western blotting and serum containing serum were used to observe the effect of "Jianpi Bushen Prescription" on radiation-damage mice and T cell autophagy and Changes in cell structure and function and the regulation mechanism of PI3K/AKT/ mTOR signaling pathway in this process. Exploring the molecular mechanism of anti-radiation injury of Jianpi Bushen Prescription, and providing theoretical basis for the attenuated and synergistic effect of anti-radiation therapy at the same time.

中医认为“健脾补肾”与机体的造血和免疫功能关系密切，以此为法组建“健脾补肾方”。前期研究采用基因芯片筛选出与辐射损伤造血相关的Wnt信号通路，该方通过上调Wnt通路，促进骨髓造血干细胞的增殖，抑制其凋亡；该方亦能减轻脾脏的损伤，增强免疫功能，但其作用机制尚不明确。辐射后T细胞介导的免疫功能损伤是诱发疾病的关键环节。自噬在机体的免疫过程中发挥着重要作用，T细胞自噬缺陷，会影响T细胞的功能，进而导致机体免疫功能紊乱。本课题拟在前期工作基础上，利用电镜观察自噬体的形成、激光共聚焦荧光显微镜来监控自噬进程，利用RT-PCR、核酸酶保护实验、Western印迹及含药血清等技术方法，探讨健脾补肾方对辐射损伤小鼠脾脏、胸腺及T细胞自噬和细胞结构、功能的影响，以及PI3K/AKT/mTOR信号通路在该过程中的调节机制，深入探讨健脾补肾方抗辐射损伤的具体机制，为该方在抗辐射治疗中的减毒增效作用提供理论依据。

辐射对免疫功能的损伤存在程度重、难恢复的特点，所以免疫功能损伤是辐射病治疗中的棘手问题。自噬在机体的免疫调节过程中发挥着重要作用，PI3K/Akt/mTOR信号通路是调控自噬的主要通路。因此，PI3K/Akt/mTOR信号通路介导的细胞自噬可能是药物干预辐射损伤的有效靶点。我们前期研究表明健脾补肾方能够减轻脾脏损伤，提高免疫功能，但其具体机制尚不明确；前期基因芯片检测结果显示健脾补肾方干预辐射损伤的靶基因在PI3K/Akt信号通路富集。.本项目在前期工作基础上，以动物模型和分离培养的T细胞为研究对象，进一步深入研究健脾补肾方对PI3K/Akt/mTOR信号通路介导的细胞自噬的作用和调节机制。首先通过检测辐射损伤小鼠外周血、免疫器官指数、脾脏病理变化、脾脏组织原位凋亡情况、脾脏组织自噬水平，细胞因子IFN-γ、IL-2、TNF-α的分泌水平及信号通路分子（PI3K、Akt、mTOR）、自噬相关分子（Beclin1、Atg5、Atg12、p62、LC3-II/LC3-I）的表达，并与健脾补肾方干预后进行对比，初步明确了PI3K/Akt/mTOR信号通路介导的细胞自噬与辐射后免疫功能损伤的关系，干预PI3K/Akt/mTOR信号通路介导的细胞自噬过度活化是健脾补肾方抗辐射免疫损伤的作用靶点。通过体外细胞试验进一步探明了健脾补肾法通过促进PI3K/Akt/mTOR信号通路转录与翻译，抑制自噬过度活化，抑制细胞凋亡，调节T细胞亚群及免疫细胞因子平衡，减轻脾脏结构损伤，从而加速辐射后免疫功能的恢复。 .本项目系统地从PI3K/Akt/mTOR信号通路调控细胞自噬角度阐明健脾补肾方抗辐射免疫功能损伤的作用机理与环节，为该方在临床的应用提供实验依据，为中医药防治辐射损伤提供崭新的思路。