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放射治疗(简称放疗)在治疗胸部恶性肿瘤(包括食道癌、乳腺癌和非小细胞肺癌)中起着重要作用[1]。为了尽量减少放疗后放射性肺纤维化(radioactive pulmonary fibrosis,RIPF)的风险,行放疗时的辐射剂量通常受到限制。放射性肺损伤的表现包括急性放射性肺炎和慢性RIPF。虽然放疗技术的进步显著降低了不良反应,但仍有近15%的胸部放疗患者出现放射性肺炎和慢性RIPF[2]。
诱导RIPF形成的方式随照射仪器、射线种类、照射方式以及物种的不同而差异显著[3]。例如,在C57BL/6小鼠模型中,行胸部照射6个月后(剂量约为15 Gy),小鼠会出现明显的RIPF[4]。采用20 Gy照射2个月龄SD大鼠的右肺,发现受照射的肺体积萎缩、质地硬韧,6个月后出现对侧肺代偿性肥大。雄性SD大鼠单侧胸部照射20 Gy后12周,I型和II型上皮细胞和内皮细胞数量减少了50%~70%,同时毛细血管内血液总量减少了75%,间质细胞的数量增加了3倍。照射后26周,肺间质中肥大细胞数目增长了540倍,其他渗出液和浆细胞数目增长了180倍[5-6]。
相对于小鼠的RIPF模型,大鼠肺部体积更大,更适于RIPF的功能研究,而且从进化上来说,大鼠要早于小鼠[7]。因此,本研究拟采用X射线辐照仪对SD大鼠行单侧右肺照射,以建立SD大鼠RIPF模型,并探寻实验中纤维化蛋白、细胞因子等作为组织纤维化程度评价指标的可行性,为RIPF的研究提供基础。
SD大鼠放射性肺纤维化模型的建立及评价
Establishment and evaluation of radioactive pulmonary fibrosis model in SD rats
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摘要:
目的 建立SD大鼠放射性肺纤维化模型,探寻实验中纤维化蛋白、细胞因子等作为组织纤维化程度评价指标的可行性,为放射性肺纤维化的研究提供基础。 方法 选取体重为180~200 g的SD雄性大鼠37只,完全随机分为对照组(5只)和照射组(32只)。照射组采用X射线行右肺单次照射,照射剂量分别为13 Gy(10只)、15 Gy(10只)和17 Gy(12只),分别于照后4个月和6个月,采用苏木精-伊红染色和马松三色染色评价大鼠肺组织纤维化程度;采用Western blot检测纤维黏连蛋白1(FN1)、基质金属蛋白酶2(MMP2)、α-平滑肌肌动蛋白(α-SMA)在肺组织中的表达;测定肺组织中羟脯氨酸含量以评价肺组织中胶原蛋白表达水平。酶联免疫吸附测定法测定支气管肺泡灌洗液中转化生长因子β(TGF-β)、白细胞介素6、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)的变化。组间比较采用独立样本 t 检验。 结果 与对照组相比,照射组4~6个月后均出现肺纤维化,纤维化程度随照射剂量的增大和照射时间的增长而增加;照射组大鼠肺组织中FN1、α-SMA的蛋白表达水平较对照组升高,MMP2的蛋白表达水平较对照组降低;6个月照射组羟脯氨酸含量较对照组升高,分别从(514.19 ±282.20)μg/mg 增加至(886.13 ±145.01)、(1188.70 ±273.84)、(1700.70 ±590.95)μg/mg,且差异均有统计学意义(t=2.621、3.609、4.004,均P<0.05);支气管肺泡灌洗液中TGF-β、白细胞介素6、TNF-α分泌量较对照组上调,差异有统计学意义(t=4.030~12.780,均P<0.05),IFN-γ较对照组下调,差异有统计学意义(t=2.498~4.303,均P<0.05)。 结论 SD大鼠放射性肺纤维化模型构建成功。大鼠肺组织纤维蛋白含量未能反映肺组织纤维化程度,肺组织羟脯氨酸含量与支气管肺泡灌洗液中细胞因子在重度纤维化时可作为评价指标。 Abstract:Objective A radioactive pulmonary fibrosis model of SD rats was established to explore the application of fibrosis-related proteins and cytokines as the evaluation degree of tissue fibrosis and serve as the basis for studies on radioactive pulmonary fibrosis. Method Thirty-seven male SD rats weighing 180-200 g were randomly divided into the control (n=5) and irradiation groups (n=32). The irradiation groups were irradiated by an X-ray line characterized by a single exposure dose of 13 Gy (n=10), 15 Gy (n=10), and 17 Gy (n=12). After exposure for 4 and 6 months, hematoxylin-eosin staining and Masson staining were used to evaluate pulmonary fibrosis in the rats. Fibronectin 1 (FN1), matrix metalloproteinase 2 (MMP2), and α-smooth muscle actin (α-SMA) expression levels in lung tissues were detected by Western blot analysis. Hydroxyproline content in the lung tissue were evaluated the lung tissue collagen protein expression levels. ELISA was employed to detect the change of cytokines, such as the transforming growth factor β (TGF-β), interleukin 6, tumor necrosis factor α (TNF-α), and interferon γ (INF-γ), in the bronchoalveolar lavage fluid. Independent sample t-test was used for the intergroup comparison. Results Compared with the control group, the model group developed pulmonary fibrosis after 4-6 months, and the degree of fibrosis increased with the irradiation dose and time. The protein expression level of FN1 and α-SMA in the lung tissues of the model group was higher than that of the control group, and the protein expression level of MMP2 was lower than that of the control group. The hydroxyproline content in the 6-month model group was higher than that of the control group, increased from (514.19±282.20) μg/mg to (886.13±145.01), (1188.70±273.84), (1700.70±590.95) μg/mg respectively (t=2.621, 3.609, 4.004, all P<0.05). The TGF-β, interleukin 6, and TNF-α levels in the bronchoalveolar lavage fluid of the model group were up-regulated compared with those in the normal control group (t=4.030–12.780, all P<0.05), whereas the IFN-γ was down-regulated (t=2.498–4.303, all P<0.05). Conclusions The SD rat model of radiation pulmonary fibrosis was successfully constructed. The fibrin content of the rat lung tissues failed to reflect the degree of lung tissue fibrosis. The hydroxyproline content in the lung tissue and the cytokines in the bronchoalveolar lavage fluid can be used as evaluation indexes in severe fibrosis. -
Key words:
- Radioactive pulmonary fibrosis /
- Model building /
- Evaluation
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