单剂量照射制备大鼠放射性肝纤维化模型的建立及评价

发布时间：2018-07-17 03:30

【摘要】：目的： 单次剂量照射制备大鼠放射性肝纤维化实验动物模型，观察造模后各个时期大鼠肝功能、肝脏标本病理学形态、血清学细胞因子以及肝脏免疫组化指标等改变。并初步探讨大鼠造模后相关细胞因子及TGF-β_1/Smads信号传导通路在放射性肝纤维化中的作用，揭示放射性肝纤维化的发病机制，为临床预防和治疗放射性肝纤维化提供理论基础及实验依据。 方法： 将60只实验大鼠随机分为正常对照组（A组）10只、照射组（B组）50只。除正常对照组外，，B组大鼠用10%水合氯醛0.3ml/100g全麻后，美国VARIN21-EX直线加速器照射大鼠右半肝，源皮距为100cm，剂量率600cGy/min，30Gy/单次，照射野2.5cm×2.5cm，每只动物右侧胸腔及胃肠均用铅块遮挡，照射后等待大鼠自然苏醒，A组大鼠只麻醉不进行照射。 于照射后第2、4、8、12、26周末分批分别剖杀。10%水合氯醛腹腔麻醉（0.3ml/100g体重）后，称重并记录，自腹主动脉取血，室温静置30min，在3000r/min的条件下离心10min，收集上清液置入冻存管中，在-80℃条件下冻存备用，然后检测大鼠血清中AST、ALT的活性，ELISA法测血清中TGF-β_1、TNF-α、IL-6、HA、PC-Ⅲ、LN含量，碱水解法检测大鼠肝脏组织中Hyp含量；然后立即处死大鼠，迅速切开腹腔。A组10只大鼠于第4周末处死做对照。取右肝置于10%中性甲醛溶液中固定，常规石蜡包埋，行HE染色和Masson染色及免疫组化检测肝脏组织TGF-β_1、Smad3/4/7、PDGF-BB、TIMP-1、MMP13蛋白表达情况。 结果： 与对照组（A组）比较，B组受照射肝组织表现出放射性肝炎及肝纤维化，照射组大鼠血清中AST，ALT酶活性和TGF-β_1水平自第4周升明显升高（P＜0.05或P＜0.01），于第12周末达到高峰；照射组大鼠血清中HA、PC-Ⅲ、LN含量和肝脏组织中Hyp含量于照射后第4周显著升高（P＜0.05或P＜0.01），一直呈上升趋势，照射后26周值最高；病理结果显示，大鼠肝脏病变呈慢性进行性肝纤维化改变。免疫组化结果显示，照射组大鼠肝脏组织中TGF-β_1、Smad3/4蛋白表达于照射后第2周开始升高（P＜0.05），照射后第12周达到高峰，26周开始下降；Smad7蛋白表达于照射后第2周明显减少（P＜0.05），照射后第12周达最低水平。另外，照射组大鼠肝脏组织中PDGF-BB和TIMP-1蛋白表达于照射2周升高，照射后26周最高，MMP-13蛋白表达在照射后降低，26周时表达最少。 结论： 1.单次单剂量（30Gy）照射建立大鼠右肝放射性肝纤维化模型稳定可靠，动物死亡率较低（15%左右），便于动态观察放射性肝纤维化的变化规律，为防治放射性肝纤维化的实验研究提供一种科学、可行、实用的动物模型； 2.放射性肝纤维化与TGF-β_1/Smads信号传导通路存在内在联系，TGF-β_1/Smads信号传导通路改变可能是引起放射性肝纤维化的重要机制之一。
[Abstract]:Objective: to establish an experimental animal model of radiation hepatic fibrosis in rats by single dose irradiation, and observe the liver function and pathological morphology of liver samples in each period after the establishment of the model. Changes in serum cytokines and liver immunohistochemical markers. The role of related cytokines and TGF- 尾 1 / Smads signal transduction pathway in radiation-induced liver fibrosis in rats was also studied, and the pathogenesis of radiation hepatic fibrosis was revealed. To provide theoretical basis and experimental basis for clinical prevention and treatment of radioactive liver fibrosis. Methods: 60 experimental rats were randomly divided into normal control group (group A, n = 10) and irradiation group (group B, n = 50). In addition to the normal control group, rats in group B were treated with 10% chloral hydrate 0.3ml/100g general anesthesia. The right half liver was irradiated by VARIN21-EX linear accelerator in the United States. The skin distance was 100 cm, the dose rate was 600cGy 路min ~ (30) Gy / time, 2.5cm 脳 2.5 cm, and the right thorax and gastrointestinal tract of each animal were occluded by lead. Rats in group A were anesthetized and not irradiated after irradiation. At the end of the week, the abdominal anaesthesia (0.3ml/100g body weight) of 0.10% chloral hydrate was dissected in batches, weighing and recording. Blood was taken from the abdominal aorta, rest at room temperature for 30 min, centrifuged for 10 min under the condition of 3000r/min, and the supernatant was collected and placed in the frozen tube. After cryopreservation at -80 鈩

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