乌梅喷雾剂促进唾液腺细胞自噬防治放射性口干症的机制研究

发布时间：2018-05-24 03:29

本文选题：乌梅喷雾剂 + 放射性口干症　；参考：《广州中医药大学》2017年硕士论文

【摘要】：目的:本研究拟通过放射性唾液腺损伤大鼠模型,观察乌梅喷雾剂对放射性唾液腺损伤大鼠体重、唾液分泌功能的影响,分析活体组织的病理形态,研究乌梅喷雾剂对颌下腺腺泡细胞和对腺泡细胞自噬相关基因及蛋白表达的影响,探讨乌梅喷雾剂对放射性损伤后唾液腺功能、结构、分子机制的影响,为临床应用开发奠定基础。方法:经动物伦理委员会同意选取140只健康SPF级雄性6～7周Wistar大鼠,体重约170～210g。随机分为对照组、模型组、阴性对照组、阳性对照组、实验组,每组28只。单一剂量18Gy颌下腺区照射,建立放射性唾液腺损伤大鼠模型,以生理盐水喷雾剂为阴性对照,以匹罗卡品喷雾剂为阳性对照,乌梅喷雾剂为实验组,于照射后每天三次口腔喷雾干预处理,对照组正常喂养外不作干预,模型组照射后正常喂养外不作干预。于照射后第1、7、14、28天,每天每组取7只,记录大鼠体重,探讨乌梅喷雾剂对放射性唾液腺损伤大鼠体重的影响;收集大鼠唾液,检测乌梅喷雾剂对放射性唾液腺损伤大鼠唾液分泌的影响;摘取颌下腺,采用组织切片苏木精-伊红(HE)染色,光镜观察颌下腺组织病理变化;应用逆转录-聚合酶链反应(RT-PCR)及蛋白质印迹法(Westernblot)技术检测自噬相关因子Beclin-1、Atg5的mRNA及蛋白表达水平,从分子生物学水平检测细胞自噬水平的变化。结果:(1)照射后第7、14、28天,对照组体重差增加明显高于其余四组,差异具有统计学意义(P0.05);照射后第14、28天,实验组体重差高于模型组,差异具有统计学意义(P0.05),但与阳性对照组相比,差异无统计学意义(P0.05)。(2)照射后第1天,各照射组唾液流率均低于对照组,但差异无统计学意义;照射后第7天,实验组与对照组、阳性对照组间差异无统计学意义,模型组与对照组、阳性对照组、实验组间唾液流率差异有统计学意义(P0.05)。(3)HE染色结果显示,照射后第1天,实验组、模型组、阴性对照组、阳性对照组大鼠腺体均出现轻微的萎缩,腺体细胞体积有些变小,部分发生坏死,核固缩明显,间质中可见明显的炎性细胞浸润,腺体间隔轻微的增宽等的病理改变;照射后第7、14天,实验组、阳性对照组与模型组相比,腺体细胞病理形态有所修复;照射后第28天,模型组与阴性对照组腺体存在较为严重的损伤,而实验组腺体排列规则,腺体未见萎缩,少量腺体细胞核发生固缩,腺体细胞未见明显坏死,间质中未见炎性细胞浸润,间质未见增宽,损伤腺体能明显修复了其细胞形态。(4)自噬相关基因Beclin1和Atg5在各组中均有表达;照射后第1、7、14天,实验组、模型组、阳性对照组的Beclin1基因相对表达量高于对照组,实验组与模型组的Beclin1基因相对表达量间差异无统计学意义;照射后第1、7天,实验组、模型组、阴性对照组、阳性对照组的Atg5基因相对表达量高于对照组,且具有显著性差异。(5)照射后第1、7天,实验组、模型组、阳性对照组的Beclin1蛋白表达比对照组高;照射后第1、7、14天,实验组、模型组、阴性对照组、阳性对照组的Atg5蛋白表达比对照组高,且第1、7天,实验组与模型组间Atg5蛋白表达无明显差异。结论:(1)乌梅喷雾剂可改善放射性唾液腺损伤大鼠的食欲,尤其在照射后第2到4周时有效提高放射性唾液腺损伤大鼠体重。(2)乌梅喷雾剂能有效修复大鼠颌下腺放射性损伤后的细胞形态,但就目前研究数据并未能表明乌梅喷雾剂对放射性唾液腺损伤细胞自噬活性存在影响。(3)自噬活性可能与放射性唾液腺损伤发生早期有关。
[Abstract]:Objective: To observe the effects of mume spray on the body weight and saliva secretion of rats with radioactive salivary glands, and to analyze the pathological morphology of the living tissue, and to investigate the effects of mume spray on the acinar cells of the submandibular gland and the expression of autophagy related genes and proteins in the acinus cells. The effects of Wu Mei spray on the function, structure and molecular mechanism of salivary glands after radiation injury lay the foundation for clinical application. Methods: 140 healthy SPF grade male Wistar rats were selected by the animal ethics committee. The weight of the Wistar rats was randomly divided into the control group, the model group, the negative control group, the positive control group, the experimental group, and the control group. 28 rats in each group were irradiated with a single dose of 18Gy submandibular gland area to establish a rat model of radioactive salivary gland injury, with normal saline spray as negative control, pilocarpine spray as positive control, black plum spray as the experimental group, three times of oral spray intervention after irradiation, and no intervention in the control group after normal feeding, and the model group was irradiated. After 1,7,14,28 day after irradiation, 7 rats in each group were taken every day to record the body weight of rats, and to explore the effect of dark plum spray on the weight of rats injured by radioactive salivary glands, and to collect the saliva of rats, to detect the effect of mume spray on the salivary salivary gland injury in rats, and to extract submandibular gland and slice. Hematoxylin eosin (HE) staining and optical microscopy were used to observe the histopathological changes of the submandibular glands; reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (Westernblot) were used to detect autophagy related factors Beclin-1, mRNA and protein expression levels of Atg5, and the changes of autophagy levels were detected from the molecular biology level. Results: (1) 7,14 after irradiation. On the 28 day, the weight difference of the control group was significantly higher than the other four groups, and the difference was statistically significant (P0.05). The weight difference of the experimental group was higher than that of the model group at the 14,28 day after irradiation (P0.05), but the difference was not statistically significant (P0.05) compared with the positive control group. (2) the saliva flow rate of each group was lower than that of the control group after first days. There was no statistical significance in the group, but on seventh days after irradiation, there was no significant difference between the experimental group and the control group. The difference of saliva flow rate between the model group and the control group, the positive control group and the experimental group was statistically significant (P0.05). (3) the results of HE staining were shown, the experimental group, the model group, the negative control group, the positive control group were positive, and the positive control group was positive. The glands of the rats were slightly atrophied, the volume of the gland cells became smaller, some of the gland was necrotic, the nuclear condensation was obvious, the interstitial infiltration of inflammatory cells and the slight widening of the glandular space were seen in the interstitium; the pathological morphology of the glandular cells was repaired on day 7,14 after exposure to the model group compared with the model group. On the twenty-eighth day after irradiation, the glands in the model group and the negative control group had more serious damage, but the glands in the experimental group were arranged regularly, the glands were not atrophied, the nucleus of the glands were shrinking, the gland cells were not necrotic, there was no inflammatory cell infiltration in the interstitium, the interstitium was not broadened, and the damaged gland could obviously repair the cell morphology. (4) the cell morphology was restored. The related genes Beclin1 and Atg5 were expressed in each group. The relative expression of Beclin1 gene in the experimental group, the model group and the positive control group was higher than that of the control group on the 1,7,14 day after irradiation. The relative expression of the Beclin1 gene in the experimental group and the model group was not statistically significant, and the experimental group, the model group, the negative control group were positive after the irradiation, and the positive control group was positive. The relative expression of Atg5 gene in the control group was higher than that in the control group. (5) the expression of Beclin1 protein in the experimental group, the model group and the positive control group was higher than the control group after 1,7 day after irradiation. The expression of Atg5 protein in the experimental group, the model group, the negative control group and the positive control group was higher than the control group after the irradiation, and the positive control group was higher than the control group, and the real time was 1,7 days. There was no significant difference in the expression of Atg5 protein between the test group and the model group. Conclusion: (1) the black plum spray can improve the appetite of the radioactive salivary gland injury rats, especially at second to 4 weeks after the irradiation. (2) the black plum spray can effectively repair the cell morphology after the radiation injury of the submandibular gland of the rat. The previous research data did not show the effect of mume spray on the autophagy activity of radioactive salivary gland damaged cells. (3) the autophagy may be related to the early occurrence of radioactive salivary gland injury.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.55

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