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腸型放射病小鼠模型中小腸細胞死亡類型的鑒定和修飾性p53蛋白的表達研究

發(fā)布時間:2019-02-25 13:50
【摘要】:目的:放射治療是腫瘤治療的三大主要手段之一,其中70%的惡性腫瘤可以進行放射治療。在許多腹腔惡性腫瘤放療過程中病人會出現(xiàn)腸道癥狀,如腹瀉、消化不良等,這是放療對腸道正常細胞的損傷引起的副作用。這種副作用的存在極大地影響了放療的進行和病人的康復。再者,目前沒有有效的藥物救治核泄漏事故中的腸型放射病。通過對照射后腸道細胞死亡形式及其機制的研究,尋找相應的救治措施,提高對正常組織的保護,以期提高放射治療的療效和救治腸型放射病。 方法:小鼠接受15Gy全身照射后,觀察記錄小鼠的生存,,繪制生存曲線;在照后指定的時間點處死小鼠,取出回腸,觀察結腸糞便形成、HE染色以及透射電鏡等形態(tài)學指標;免疫熒光法分析活性氧(ROS)的水平;ELISA法測定谷胱甘肽(GSH)、一氧化氮(NO)、過氧化氫(H2O2)以及超氧化物歧化酶(SOD)的水平;免疫印跡法分析Caspase-3,LC3B以及p53的磷酸化、乙酰化水平;免疫熒光法分析HMGB1;酶聯(lián)免疫法測定血清中的IL-6水平。 結果:照后小鼠回腸中活性氧明顯增加,而氧化指標(GSH, NO, H2O2, SOD)卻沒有明顯改變。通過形態(tài)學以及生化指標反映:在15Gy全身照射后的小鼠回腸中均顯示出凋亡、壞死以及自噬的特征。剪切的Caspase-3(凋亡標志物)的出現(xiàn),LC3-Ⅰ向LC3-Ⅱ(自噬標志物)的轉換,血清IL-6的分泌以及HMGB1(壞死標志物)的表達。此外,在15Gy照后出現(xiàn)p53基因379位賴氨酸的乙酰化以及第6、20、392位的絲氨酸的磷酸化,這種現(xiàn)象在照后2小時后消失。 結論:在照射后的腸道細胞的死亡不應被視為單一的死亡形式,而應該是一種“混合型死亡”。放療中的同步治療藥物如能促進p53基因379位賴氨酸的乙;约暗6、20、392位的絲氨酸的磷酸化,則可能降低放療后腸道細胞的死亡。
[Abstract]:Objective: radiotherapy is one of the three main methods of tumor therapy, 70% of which can be treated by radiotherapy. Intestinal symptoms such as diarrhea, dyspepsia and so on will occur in many patients with abdominal cancer during radiotherapy, which is a side effect of radiation therapy on normal intestinal cells. The presence of this side effect has greatly affected radiotherapy and patient recovery. Furthermore, there are no effective drugs available for the treatment of intestinal radiation sickness in the event of nuclear leakage. Through the study of the death form and mechanism of intestinal cells after irradiation, the corresponding treatment measures were found to improve the protection of normal tissues, so as to improve the curative effect of radiation therapy and the treatment of intestinal radiation sickness. Methods: after the mice were irradiated with 15Gy, the survival of the mice was observed, the survival curve was drawn, the mice were killed at the designated time point after irradiation, the ileum was taken out, and the colonic feces formation, HE staining and transmission electron microscopy were observed. The levels of reactive oxygen species (ROS), glutathione (GSH), (GSH), nitric oxide (NO), hydrogen peroxide (H2O2) and superoxide dismutase (SOD) were determined by immunofluorescence assay. The levels of phosphorylation and acetylation of Caspase-3,LC3B and p53 were detected by immunoblotting assay, and the levels of IL-6 in serum were determined by immunofluorescence assay for HMGB1; enzyme linked immunosorbent assay. Results: after irradiation, the active oxygen species (Ros) in the ileum increased significantly, but the oxidation index (GSH, NO, H2O2 (SOD) did not change significantly. Morphological and biochemical results showed that apoptosis, necrosis and autophagy were observed in the ileum of mice irradiated with 15Gy. The appearance of shearing Caspase-3 (apoptotic marker), the conversion of LC3- 鈪

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