本文選題：烏梅噴霧劑 + 放射性口干癥　； 參考：《廣州中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:本研究擬通過放射性唾液腺損傷大鼠模型,觀察烏梅噴霧劑對放射性唾液腺損傷大鼠體重、唾液分泌功能的影響,分析活體組織的病理形態(tài),研究烏梅噴霧劑對頜下腺腺泡細(xì)胞和對腺泡細(xì)胞自噬相關(guān)基因及蛋白表達(dá)的影響,探討烏梅噴霧劑對放射性損傷后唾液腺功能、結(jié)構(gòu)、分子機(jī)制的影響,為臨床應(yīng)用開發(fā)奠定基礎(chǔ)。方法:經(jīng)動物倫理委員會同意選取140只健康SPF級雄性6～7周Wistar大鼠,體重約170～210g。隨機(jī)分為對照組、模型組、陰性對照組、陽性對照組、實(shí)驗(yàn)組,每組28只。單一劑量18Gy頜下腺區(qū)照射,建立放射性唾液腺損傷大鼠模型,以生理鹽水噴霧劑為陰性對照,以匹羅卡品噴霧劑為陽性對照,烏梅噴霧劑為實(shí)驗(yàn)組,于照射后每天三次口腔噴霧干預(yù)處理,對照組正常喂養(yǎng)外不作干預(yù),模型組照射后正常喂養(yǎng)外不作干預(yù)。于照射后第1、7、14、28天,每天每組取7只,記錄大鼠體重,探討烏梅噴霧劑對放射性唾液腺損傷大鼠體重的影響;收集大鼠唾液,檢測烏梅噴霧劑對放射性唾液腺損傷大鼠唾液分泌的影響;摘取頜下腺,采用組織切片蘇木精-伊紅(HE)染色,光鏡觀察頜下腺組織病理變化;應(yīng)用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)及蛋白質(zhì)印跡法(Westernblot)技術(shù)檢測自噬相關(guān)因子Beclin-1、Atg5的mRNA及蛋白表達(dá)水平,從分子生物學(xué)水平檢測細(xì)胞自噬水平的變化。結(jié)果:(1)照射后第7、14、28天,對照組體重差增加明顯高于其余四組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);照射后第14、28天,實(shí)驗(yàn)組體重差高于模型組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),但與陽性對照組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(2)照射后第1天,各照射組唾液流率均低于對照組,但差異無統(tǒng)計(jì)學(xué)意義;照射后第7天,實(shí)驗(yàn)組與對照組、陽性對照組間差異無統(tǒng)計(jì)學(xué)意義,模型組與對照組、陽性對照組、實(shí)驗(yàn)組間唾液流率差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)HE染色結(jié)果顯示,照射后第1天,實(shí)驗(yàn)組、模型組、陰性對照組、陽性對照組大鼠腺體均出現(xiàn)輕微的萎縮,腺體細(xì)胞體積有些變小,部分發(fā)生壞死,核固縮明顯,間質(zhì)中可見明顯的炎性細(xì)胞浸潤,腺體間隔輕微的增寬等的病理改變;照射后第7、14天,實(shí)驗(yàn)組、陽性對照組與模型組相比,腺體細(xì)胞病理形態(tài)有所修復(fù);照射后第28天,模型組與陰性對照組腺體存在較為嚴(yán)重的損傷,而實(shí)驗(yàn)組腺體排列規(guī)則,腺體未見萎縮,少量腺體細(xì)胞核發(fā)生固縮,腺體細(xì)胞未見明顯壞死,間質(zhì)中未見炎性細(xì)胞浸潤,間質(zhì)未見增寬,損傷腺體能明顯修復(fù)了其細(xì)胞形態(tài)。(4)自噬相關(guān)基因Beclin1和Atg5在各組中均有表達(dá);照射后第1、7、14天,實(shí)驗(yàn)組、模型組、陽性對照組的Beclin1基因相對表達(dá)量高于對照組,實(shí)驗(yàn)組與模型組的Beclin1基因相對表達(dá)量間差異無統(tǒng)計(jì)學(xué)意義;照射后第1、7天,實(shí)驗(yàn)組、模型組、陰性對照組、陽性對照組的Atg5基因相對表達(dá)量高于對照組,且具有顯著性差異。(5)照射后第1、7天,實(shí)驗(yàn)組、模型組、陽性對照組的Beclin1蛋白表達(dá)比對照組高;照射后第1、7、14天,實(shí)驗(yàn)組、模型組、陰性對照組、陽性對照組的Atg5蛋白表達(dá)比對照組高,且第1、7天,實(shí)驗(yàn)組與模型組間Atg5蛋白表達(dá)無明顯差異。結(jié)論:(1)烏梅噴霧劑可改善放射性唾液腺損傷大鼠的食欲,尤其在照射后第2到4周時(shí)有效提高放射性唾液腺損傷大鼠體重。(2)烏梅噴霧劑能有效修復(fù)大鼠頜下腺放射性損傷后的細(xì)胞形態(tài),但就目前研究數(shù)據(jù)并未能表明烏梅噴霧劑對放射性唾液腺損傷細(xì)胞自噬活性存在影響。(3)自噬活性可能與放射性唾液腺損傷發(fā)生早期有關(guān)。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.55

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