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

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。

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,hematoxylineosin staining and Masson staining were used to evaluate pulmonary fibrosis in the rats.Fibronectin 1(FN1),matrix metalloproteinase 2(MMP2),andα-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 factorβ(TGF-β),interleukin 6,tumor necrosis factorα(TNF-α),and interferonγ(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 andα-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-αlevels 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-γwas 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.