Intraperitoneal injection of bleomycin induces pulmonary fibrosis in mice: a long-term stability evaluation

doi:10.3969/j.issn.2095-4344.2017.04.004

Abstract

Abstract:

Abstract
BACKGROUND: There is no effective drug for idiopathic pulmonary fibrosis (IPF), because of a lack of the animal model imitating the complete pathogenesis of human IPF. Therefore, it is critical to establish an ideal animal IPF model used for investigating the underlying pathogenesis and developing a kind of effective drug.
OBJECTIVE: To establish an animal model that can mimic more characters of human IPF.
METHODS: Seventy male C57BL/6 mice were randomly divided into two groups, followed by subjected to the intraperitoneal injection of bleomycin (35 mg/kg) on days 1, 4, 8, 11, 15, 18, 22, and 25, twice (group A) or once (group B) a week. Mice were sacrificed at 2, 4, 6, 8, and 10 weeks after the eighth injection, and the lung tissues were moved used for hematoxylin-eosin, Masson and immunohistochemical stainings.
RESULTS AND CONCLUSION: There were various degrees of alveolitis and pulmonary fibrosis in the two groups at different time points after the last injection. The scores of alveolitis and pulmonary fibrosis in the group A began to gradually increase from the 2nd week and reached the highest level at the 6th-8th weeks until the 10th week. In contrast, the scores of alveolitis and pulmonary fibrosis in the group B peaked at the 2nd week, then fluctuately decreased, and were significantly lower than those in the group A at the 6th week (P < 0.05). Immunohistochemistry showed that type I collagen deposition was mainly distributed in the subpleural region, peri-vascular region and alveolar septa, which was consistent with Masson staining findings. The expression levels of transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in the regions developing alveolitis and pulmonary fibrosis were significantly increased. In the group A, the expression levels of type I collagen, TGF-β1, α-SMA, and the hydroxyproline content in the lung tissues reached the peak level at 6-8 weeks. However, in the group B, all above indicators reached the highest level at the 2nd week, but gradually decreased thereafter. At the 4th week, the expression Levels of TGF-β1 and α-SMA in the group B were significantly lower than those in the group A (P < 0.05). At the 6th week, the hydroxyproline and type I collagen levels in the group B were significantly lower than those in the group A (P < 0.05). In conclusion, the mouse model of pulmonary fibrosis induced by intraperitoneal injection of 35 mg/kg bleomycin twice weekly can be used to mimic the repetitive wound healing process, pathological morphology and cytokine changes of human IPF, which is prone to administration, with better stability and repeatability. This model is of great significance for the study on IPF.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Disease Models, Animal, Pulmonary Fibrosis, Bleomycin

CLC Number:

R318

Su Min-hong1, Jiang Ning2, Li Hong-tao3, Wang Zhen-guo1, Xie Yu-fen1, Zheng Xiao-bin1, Tu Chang-li1,Huang Jin1. Intraperitoneal injection of bleomycin induces pulmonary fibrosis in mice: a long-term stability evaluation[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(4): 512-519.

Figures/Tables 3

2.3 动物取材及解剖情况通过眼球取血法收集小鼠血液标本处死小鼠，开胸取肺，剖腹观察有无腹水、腹部脏器情况。两组小鼠肺组织肉眼下可见散在出血点，全肺挛缩变硬、颜色偏灰白色，肺组织表面凹凸不平，局部牵拉凹陷变形，两组小鼠均存在不同程度的腹水，腹水为淡黄色。 2.4 肺组织苏木精-伊红染色和Masson染色 A组造模后2-10周肺组织苏木精-伊红染色显示血管周围、肺泡腔内、肺泡间隔、胸膜下均有不同程度的炎性细胞浸润，以淋巴细胞为主，肺泡间隔增厚，肺泡腔内有大量胶原纤维渗出，部分肺泡腔扩大，肺泡壁变薄，部分肺泡结构被破坏消失，甚至出现大片融合的肺实质；Masson染色可见肺泡间隔大量胶原蛋白沉积，胶原沉积部位主要位于胸膜下或血管周围肺间质，呈点灶样、团片样分布，与周围正常的肺组织或病变较轻的肺组织相邻(空间异质性)，观察至造模后10周肺纤维化病灶及肺泡炎轻度仍无明显缓解倾向。B组造模后2周肺组织苏木精-伊红染色和Masson染色与A组相似，但从造模后4周开始，纤维化病灶逐渐吸收，至造模后8周只可见单个成团纤维化病灶和散在炎症细胞浸润，造模后10周只见轻微肺泡间隔增厚和血管周围胶原纤维渗出，未见明显炎性细胞浸润(图2， 3)。肺泡炎和肺纤维化评分显示：A组小鼠于造模后2周至造模后10周肺泡炎和肺纤维化评分均较高，于造模后6-8周达峰值；B组小鼠评分于造模后2周最高，与A组该时间点分值相近(P=0.580)，随后B组评分逐渐降低，于造模后4周开始B组评分明显低于A组(P < 0.05)，造模后10周B组评分降至最低(图4)。 2.5 肺组织羟脯氨酸测定两组小鼠肺组织羟脯氨酸含量于造模后2、4周均较高，且差异无显著性意义，P值分别为0.732、0.825。随时间延长，A组小鼠肺组织羟脯氨酸含量逐渐升高，但B组逐渐下降，于造模后6周开始A组羟脯氨酸含量明显高于B组(P=0.027)(图4)。 2.6 肺组织I型胶原蛋白、转化生长因子β1、α-SMA的表达 Ⅰ型胶原蛋白(图5)在胸膜下肺组织、肺泡间隔及血管周围过度沉积；转化生长因子β1(图6)和α-SMA(图7)主要表达于细胞浆内和细胞膜上，表达阳性的细胞多成团聚集于纤维化严重部位，与Ⅰ型胶原蛋白表达部位一致。两组小鼠肺组织Ⅰ型胶原蛋白、转化生长因子β1和α-SMA表达均在造模后2周明显升高，A组于造模后6-8周达高峰，造模后10周仍无明显降低；而B组于造模后4周开始逐渐降低，于造模后10周降至最低(图8)。"

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